**4.2.4 Collagen volume in infrapatella fat pads treated with Adrenomedullin**

To observe the effect of ADM on fibrosis of the infrapatellar fat pads harvested on day 21, we examined the collagen volume ratio of the infrapatellar fat pad histologically using Mallory–Azan staining. The collagen volume ratio was significantly increased in ADMtreated knees by 39% and 31% at 0.1 μg and 3 μg ADM, respectively, compared with control knees (Fig. 13 and 14). The effects of ADM on these pathological tissue changes, however, were not observed in knees treated with low-dose ADM.

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 187

The collagen volume ratio was increased in ADM-treated knees by 39% and 31% at 0.1 μg and 3 μg ADM, respectively. Data expressed as the mean ± standard error of the mean. \**P* <

To elucidate the mechanism of the anti-inflammatory effects of AM in inflamed joints, we investigated the effect of AM on cytokine mRNA expression linked to AIA. Treatment with AM reduced TNFα mRNA expression in a dose-dependent manner. Daily intra-articular injections of 3 μg AM significantly suppressed the TNFα mRNA level by 21% and 49% at day 8 and day 21, respectively, compared with controls (Fig. 15a). In contrast, AM dosedependently increased IL-6 mRNA expression. Daily intra-articular injections of 3 μg AM significantly increased the IL-6 mRNA level by 45% and 121% at day 8 and day 21, respectively, compared with controls (Fig. 15b). Although the VEGF mRNA level was suppressed by 10% at 3 μg AM on day 8, we did not observe a dose-dependent effect of AM on VEGF mRNA expression (Fig. 15d). AM treatment did not significantly alter the TGFβ

Fig. 15. Effect of adrenomedullin on cytokine mRNA expression linked to antigen-induced

0.05, compared with contralateral knees.

**4.2.5 Cytokines** 

mRNA level (Fig. 15c).

arthritis

Fig. 13. Histological analysis of infrapatellar fat-pad sections stained with Mallory – Azan from rabbits with antigen-induced arthritis

The tissues were sectioned longitudinally perpendicular to the patella ligament in the middle of the tissue, and were stained with Mallory – Azan. **(a)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg ADM for 20 days. **(b)** AIA rabbit knee was treated with daily intra-articular injections of 1 ng ADM for 20 days. **(c)** The contralateral knee of (a) was treated with daily intra-articular injections of saline for 20 days. Photographs taken at ×40 magnification. Bar = 500 μm.

Fig. 14. Quantitative evaluation of collagen volume in the infrapatellar fat pad

The collagen volume ratio was increased in ADM-treated knees by 39% and 31% at 0.1 μg and 3 μg ADM, respectively. Data expressed as the mean ± standard error of the mean. \**P* < 0.05, compared with contralateral knees.

#### **4.2.5 Cytokines**

186 Rheumatoid Arthritis – Treatment

Fig. 13. Histological analysis of infrapatellar fat-pad sections stained with Mallory – Azan

Fig. 14. Quantitative evaluation of collagen volume in the infrapatellar fat pad

The tissues were sectioned longitudinally perpendicular to the patella ligament in the middle of the tissue, and were stained with Mallory – Azan. **(a)** AIA rabbit knee was treated with daily intra-articular injections of 3 μg ADM for 20 days. **(b)** AIA rabbit knee was treated with daily intra-articular injections of 1 ng ADM for 20 days. **(c)** The contralateral knee of (a) was treated with daily intra-articular injections of saline for 20 days. Photographs

from rabbits with antigen-induced arthritis

taken at ×40 magnification. Bar = 500 μm.

To elucidate the mechanism of the anti-inflammatory effects of AM in inflamed joints, we investigated the effect of AM on cytokine mRNA expression linked to AIA. Treatment with AM reduced TNFα mRNA expression in a dose-dependent manner. Daily intra-articular injections of 3 μg AM significantly suppressed the TNFα mRNA level by 21% and 49% at day 8 and day 21, respectively, compared with controls (Fig. 15a). In contrast, AM dosedependently increased IL-6 mRNA expression. Daily intra-articular injections of 3 μg AM significantly increased the IL-6 mRNA level by 45% and 121% at day 8 and day 21, respectively, compared with controls (Fig. 15b). Although the VEGF mRNA level was suppressed by 10% at 3 μg AM on day 8, we did not observe a dose-dependent effect of AM on VEGF mRNA expression (Fig. 15d). AM treatment did not significantly alter the TGFβ mRNA level (Fig. 15c).

Fig. 15. Effect of adrenomedullin on cytokine mRNA expression linked to antigen-induced arthritis

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 189

macrophages (Chu et al., 1991) it is plausible that ADM suppresses the production of TNFα

On the contrary, we found that ADM increased IL-6 mRNA expression in the synovial tissue (Fig.15b). Our results agree with previous findings on the effects of ADM on IL-6 production. ADM is reported to augment the production of IL-6 from NR8383 cells and Swiss 3T3 fibroblast cells stimulated with lipopolysaccharide or cytokines (Isumi et al., 1998). Several observations support the concept that IL-6 is an anti-inflammatory cytokine (Tilg et al., 1997). IL-6 has been shown to have a suppressive effect on TNFα and IL-1β production in peripheral blood mononuclear cells and exerts its anti-inflammatory effects in hepatitis by reducing the production of TNF (Schindler et al., 1990; Mizuhara et al., 1994). Our results therefore lead us to speculate that the mechanism involved in the antiinflammatory effects of ADM is related to suppression of TNFα in inflamed synovial tissue

Overproduction of IL-6 has been observed and is known to cause unfavorable clinical symptoms in immune-inflammatory diseases such as RA. Overproduction of IL-6 induces the production of rheumatoid factors and increases antibody levels, the platelet count, Creactive protein levels, and serum amyloid A protein levels in RA (Nishimoto et al., 2000). Treatment with a humanized anti-IL-6 receptor antibody has also been shown to reduce RA disease activity (Nishimoto et al., 2004). The effect of ADM on IL-6 production might therefore be an undesirable adverse effect in RA therapy. Plasma ADM levels have been reported to increase with RA disease activity and in the acute or flare phase of myocardial infarction and sepsis (Chosa et al., 2003; Yudoh et al., 1999; Kobayashi et al., 1996; Hirata et al., 1996). Recent studies have shown that ADM administration in the acute phase reaction of several disease models produced significant protective effects in organs against inflammation and oxidative stress (Kawai et al., 2004; Nakamura et al., 2004; Yang et al., 2002). Miyashita and colleagues reported that ADM administration to prevent ischemic brain damage in mice less than 72 hours after the ischemic event showed significant therapeutic effects, whereas ADM administration more than 72 hours after stroke onset

From these observations and our study findings, we speculate that the effects of ADM may be dependent on the tissue environment and the disease state; that is, the role and effects of ADM in inflammation may change during the inflammatory process. ADM acts as a strong anti-inflammatory agent in the acute or flare phase of inflammation, but in the chronic phase of inflammation ADM may act not only as an anti-inflammatory agent but also as a proinflammatory agent. It is therefore important to consider the time of administration, the route of administration and the dosage schedule of ADM in the

In our study, plasma ADM level was found to be elevated in patients with RA and the origin of ADM was shown to be synovial tissue. ADM may exert anti-inflammatory effects because the cultured RA synoviocytes secrete ADM, have ADM receptors, and inhibit IL-6 production. Therefore, the effects of daily intra-articular injections of ADM into the knees of rabbits with AIA were examined. The results suggest that ADM suppresses the inflammatory response in inflamed joints by inhibiting the expression of TNFα mRNA and

from activated macrophages in inflamed synovial tissue.

produced no significant therapeutic effects (Miyashita et al., 2006).

directly or through IL-6 production.

treatment of RA.

**5. Conclusions** 

increasing IL-6 mRNA level.

Expression levels of TNFα, IL-6, transforming growth factor beta (TGFβ), and vascular endothelial growth factor (VEGF) mRNA in the infrapatellar fat pads were determined by real-time quantitative PCR. **(a)** ADM treatment reduced TNFα mRNA expression in a dosedependent manner. Daily intra-articular injections of 3 μg ADM significantly suppressed the TNFα mRNA level by 21% and 49% at day 8 and day 21, respectively. **(b)** ADM increased IL-6 mRNA expression in a dose-dependent manner. Daily intra-articular injections of 3 μg AM significantly increased the IL-6 mRNA level by 45% and 121% at day 8 and day 21, respectively. **(c)** ADM treatment did not alter the TGFβ mRNA level. **(d)** Although the VEGF mRNA level was suppressed by 10% at 3 μg AM on day 8, a dose-dependent effect of ADM on VEGF mRNA expression was not observed. Open and closed columns represent the data at day 8 (*n* = 5 in each group) and day 21 (*n* = 3 in each group), respectively. Data expressed as the mean ± SEM. \**P* < 0.05 and \*\**P* < 0.01, compared with contralateral knees

#### **4.3 Therapeutic efficacy of ADM injection to the knee in AIA in rabbits**

In the present study we have shown that daily injections of ADM into the knee joint spaces of rabbits with AIA ameliorated the inflammatory response associated with the disease. Treatment with ADM reduced joint swelling, and reduced the expression of TNFα mRNA, edematous changes and the number of infiltrating inflammatory cells in the synovial tissue. We observed that ADM suppressed joint swelling (Fig. 9 and 10). Histologically, ADM treatment reduced edematous changes and increased the ratio of connective tissue in the infrapatellar fat pad (Fig. 13 and 14). A previous study showed that TNFα induced cytoskeletal reorganization of endothelial cells and increased endothelial permeability by stimulating TNF receptors 1 and 2 (Ferrero et al., 2001). In addition, TNFα facilitates the ability of VEGF to promote excessive vascular permeability (Clauss et al., 2001). TNFα also suppresses the expression of matrix genes and the induction of connective tissue growth factor by TGFβ during the wound healing response (Leask & Abraham, 2004). TNFα therefore aggravates edematous changes and suppresses the fibrotic response of the tissue. Moreover, ADM was shown to reduce endothelial hyperpermeability induced by hydrogen peroxide, thrombin, and *Escherichia coli* hemolysin (Hippenstiel et al., 2002).

Two research groups reported recently that ADM signaling deficiency in mice resulted in midgestation death and massive edema. The cause of this edema was shown to be a result of fragility and hyperpermeability of blood vessels in one group and to be a failure of lymphatic vessel growth in the other (Ichikawa et al., 2008; Fritz et al., 2008). The evidence from these studies suggests that ADM plays an important role in preventing edema. From these observations, we speculate that ADM not only suppresses the production of TNFα, but also directly and indirectly inhibits edematous changes in the inflamed joint.

Although RA is a chronic and systemic inflammatory disorder of unknown etiology, TNFα has been shown to play a central role in the pathogenesis of RA (Moreland et al., 1997; Elliott et al., 1993; Arend et al., 1995). TNFα stimulates the proliferation of synovial cells and the production of matrix metalloproteinases by chondrocytes and synovial cells, and induces the release of other proinflammatory cytokines, leading to joint destruction (Arend et al., 1995; Nishimoto et al., 2000). We have shown that daily injections of ADM into the knee joint spaces of rabbits with AIA suppressed the expression of TNFα mRNA in the synovial tissue in a dose-dependent manner (Fig. 15a). It has been reported that ADM suppressed the secretion of TNFα from lipopolysaccharide-stimulated RAW 264.7 macrophages and NR8383 macrophages (Wong et al., 2005; Kubo et al., 1998; Wu et al., 2003). Because the major source of TNFα in inflamed synovial tissue of RA is due to

Expression levels of TNFα, IL-6, transforming growth factor beta (TGFβ), and vascular endothelial growth factor (VEGF) mRNA in the infrapatellar fat pads were determined by real-time quantitative PCR. **(a)** ADM treatment reduced TNFα mRNA expression in a dosedependent manner. Daily intra-articular injections of 3 μg ADM significantly suppressed the TNFα mRNA level by 21% and 49% at day 8 and day 21, respectively. **(b)** ADM increased IL-6 mRNA expression in a dose-dependent manner. Daily intra-articular injections of 3 μg AM significantly increased the IL-6 mRNA level by 45% and 121% at day 8 and day 21, respectively. **(c)** ADM treatment did not alter the TGFβ mRNA level. **(d)** Although the VEGF mRNA level was suppressed by 10% at 3 μg AM on day 8, a dose-dependent effect of ADM on VEGF mRNA expression was not observed. Open and closed columns represent the data at day 8 (*n* = 5 in each group) and day 21 (*n* = 3 in each group), respectively. Data expressed

In the present study we have shown that daily injections of ADM into the knee joint spaces of rabbits with AIA ameliorated the inflammatory response associated with the disease. Treatment with ADM reduced joint swelling, and reduced the expression of TNFα mRNA, edematous changes and the number of infiltrating inflammatory cells in the synovial tissue. We observed that ADM suppressed joint swelling (Fig. 9 and 10). Histologically, ADM treatment reduced edematous changes and increased the ratio of connective tissue in the infrapatellar fat pad (Fig. 13 and 14). A previous study showed that TNFα induced cytoskeletal reorganization of endothelial cells and increased endothelial permeability by stimulating TNF receptors 1 and 2 (Ferrero et al., 2001). In addition, TNFα facilitates the ability of VEGF to promote excessive vascular permeability (Clauss et al., 2001). TNFα also suppresses the expression of matrix genes and the induction of connective tissue growth factor by TGFβ during the wound healing response (Leask & Abraham, 2004). TNFα therefore aggravates edematous changes and suppresses the fibrotic response of the tissue. Moreover, ADM was shown to reduce endothelial hyperpermeability induced by hydrogen

Two research groups reported recently that ADM signaling deficiency in mice resulted in midgestation death and massive edema. The cause of this edema was shown to be a result of fragility and hyperpermeability of blood vessels in one group and to be a failure of lymphatic vessel growth in the other (Ichikawa et al., 2008; Fritz et al., 2008). The evidence from these studies suggests that ADM plays an important role in preventing edema. From these observations, we speculate that ADM not only suppresses the production of TNFα, but

Although RA is a chronic and systemic inflammatory disorder of unknown etiology, TNFα has been shown to play a central role in the pathogenesis of RA (Moreland et al., 1997; Elliott et al., 1993; Arend et al., 1995). TNFα stimulates the proliferation of synovial cells and the production of matrix metalloproteinases by chondrocytes and synovial cells, and induces the release of other proinflammatory cytokines, leading to joint destruction (Arend et al., 1995; Nishimoto et al., 2000). We have shown that daily injections of ADM into the knee joint spaces of rabbits with AIA suppressed the expression of TNFα mRNA in the synovial tissue in a dose-dependent manner (Fig. 15a). It has been reported that ADM suppressed the secretion of TNFα from lipopolysaccharide-stimulated RAW 264.7 macrophages and NR8383 macrophages (Wong et al., 2005; Kubo et al., 1998; Wu et al., 2003). Because the major source of TNFα in inflamed synovial tissue of RA is due to

as the mean ± SEM. \**P* < 0.05 and \*\**P* < 0.01, compared with contralateral knees

**4.3 Therapeutic efficacy of ADM injection to the knee in AIA in rabbits** 

peroxide, thrombin, and *Escherichia coli* hemolysin (Hippenstiel et al., 2002).

also directly and indirectly inhibits edematous changes in the inflamed joint.

macrophages (Chu et al., 1991) it is plausible that ADM suppresses the production of TNFα from activated macrophages in inflamed synovial tissue.

On the contrary, we found that ADM increased IL-6 mRNA expression in the synovial tissue (Fig.15b). Our results agree with previous findings on the effects of ADM on IL-6 production. ADM is reported to augment the production of IL-6 from NR8383 cells and Swiss 3T3 fibroblast cells stimulated with lipopolysaccharide or cytokines (Isumi et al., 1998). Several observations support the concept that IL-6 is an anti-inflammatory cytokine (Tilg et al., 1997). IL-6 has been shown to have a suppressive effect on TNFα and IL-1β production in peripheral blood mononuclear cells and exerts its anti-inflammatory effects in hepatitis by reducing the production of TNF (Schindler et al., 1990; Mizuhara et al., 1994). Our results therefore lead us to speculate that the mechanism involved in the antiinflammatory effects of ADM is related to suppression of TNFα in inflamed synovial tissue directly or through IL-6 production.

Overproduction of IL-6 has been observed and is known to cause unfavorable clinical symptoms in immune-inflammatory diseases such as RA. Overproduction of IL-6 induces the production of rheumatoid factors and increases antibody levels, the platelet count, Creactive protein levels, and serum amyloid A protein levels in RA (Nishimoto et al., 2000). Treatment with a humanized anti-IL-6 receptor antibody has also been shown to reduce RA disease activity (Nishimoto et al., 2004). The effect of ADM on IL-6 production might therefore be an undesirable adverse effect in RA therapy. Plasma ADM levels have been reported to increase with RA disease activity and in the acute or flare phase of myocardial infarction and sepsis (Chosa et al., 2003; Yudoh et al., 1999; Kobayashi et al., 1996; Hirata et al., 1996). Recent studies have shown that ADM administration in the acute phase reaction of several disease models produced significant protective effects in organs against inflammation and oxidative stress (Kawai et al., 2004; Nakamura et al., 2004; Yang et al., 2002). Miyashita and colleagues reported that ADM administration to prevent ischemic brain damage in mice less than 72 hours after the ischemic event showed significant therapeutic effects, whereas ADM administration more than 72 hours after stroke onset produced no significant therapeutic effects (Miyashita et al., 2006).

From these observations and our study findings, we speculate that the effects of ADM may be dependent on the tissue environment and the disease state; that is, the role and effects of ADM in inflammation may change during the inflammatory process. ADM acts as a strong anti-inflammatory agent in the acute or flare phase of inflammation, but in the chronic phase of inflammation ADM may act not only as an anti-inflammatory agent but also as a proinflammatory agent. It is therefore important to consider the time of administration, the route of administration and the dosage schedule of ADM in the treatment of RA.
