**3.2 IL-6 secretion was inhibited in cultured FLS after addition of ADM**

The present study demonstrated that cultured RA synoviocytes expressed the mRNA for ADM and also actively secreted ADM (Fig. 5). The active secretion of ADM by RA synoviocytes seems to be similar to its active secretion by endothelial cells (Hojo et al., 2001; Uemura et al., 2002). ADM in the culture medium seemed to be actual AM because reversephase HPLC revealed that most of the ADM secreted into the medium emerged at the

Fig. 5. Time course curve for secretion of AM and intracellular ADM

The cells were incubated for the indicated time-periods in serum free medium, and the ADM was determined by RIA. Intracellular ADM was too low to be determined.

We observed that plasma ADM concentration in patients with RA was higher than that of healthy controls, and plasma ADM and plasma CRP levels were found to be well correlated. Our data suggest that plasma ADM levels increase with the activity of RA. We conclude that ADM probably plays a part in the regulation of the inflammatory process of RA, and its plasma and/or joint fluid levels could be used as an index of the degree of RA. Therefore, we investigated the role of ADM in inflammation in cultured RA synoviocytes (fibroblast-

Synovial tissue specimens were collected under sterile conditions from 6 patients with RA. Specimen collection occurred during total knee arthroplasty in all patients with RA. All RA patients fulfilled 1987 American College of Rheumatology criteria for RA. All of them were treated with only NSAIDs, DMARDs and hyaluronic acid injection into the joint before samples were obtained. The methods of FLS culture was described previously

To examine the molecular forms of ADM, extracts of cells cultured on a 100-mm dish and 15 ml of the conditioned medium (1%FBS) were analyzed by reverse-phase high-performance liquid chromatography (HPLC) with a column of TSK ODS 120 A (Tosoh, Tokyo, Japan). A linear gradient of 10% to 60% acetonitrile was run in 0.1% trifluoroacetic acid for 60 minutes and the ir-AM level in each fraction was measured by RIA. The recovery of ir-ADM from

The present study demonstrated that cultured RA synoviocytes expressed the mRNA for ADM and also actively secreted ADM (Fig. 5). The active secretion of ADM by RA synoviocytes seems to be similar to its active secretion by endothelial cells (Hojo et al., 2001; Uemura et al., 2002). ADM in the culture medium seemed to be actual AM because reversephase HPLC revealed that most of the ADM secreted into the medium emerged at the

The cells were incubated for the indicated time-periods in serum free medium, and the

ADM was determined by RIA. Intracellular ADM was too low to be determined.

**3.2 IL-6 secretion was inhibited in cultured FLS after addition of ADM** 

Fig. 5. Time course curve for secretion of AM and intracellular ADM

**3. Effiency of ADM in cultured synovia (fibroblast-like synoviocytes)** 

like synovium) in a subsequent investigation.

**3.1 Methods of ADM study in cultured FLS 3.1.1 Fibroblast-like synoviocytes (FLS) culture** 

**3.1.2 Characterization of secreted ADM** 

this HPLC system was greater than 80%.

(Nanki, et al., 2001).

identical elution position to that of authentic human ADM (1-52), which is the full-length human ADM peptide (Fig. 6). The minor peak that was eluted earlier was thought to be oxidized ADM containing methione sulfoxide. The intracellular ADM concentration remained extremely low in RA synoviocytes (Fig. 5), suggesting that these cells constitutively secrete ADM. Thus, RA synoviocytes seem to secrete ADM rapidly after its synthesis, with little intracellular storage.

Fig. 6. Analysis by reverse-phase HPLC of immunoreactive adrenomedullin (ir-ADM) secreted into the media

A linear gradient of acetonitrile of 10% to 60% was made in 0.1% trifluoroacetic acid for 60 minutes at a flow rate of 1.0mL/min. The arrow indicates the elution position of synthetic human AM. The specificity of the RIA was clarified.

Fig. 7. Inhibitory effect of ADM about the IL-6 secretion into the media in cultured FLS

Effects of ADM 22-52 10-6M (ADM blocker) on control level of IL-6 secretion in cultured fibroblast like synoviocytes (FLS) donated from RA patients. Serum-starved FLS were incubated 24 hours. Values are the means±SEM of six wells examined. Each was compared with the cells incubated with 1%FBS media (control). Each set of experiments was repeated three times and identical results were obtained.

When the synoviocytes reached confluence, ADM (10-8 M) was added to the culture media. After 24 hours, the level of IL-6 in culture media significantly decreased. Moreover, ADM (22-52), which is an ADM antagonist, elevated the level of IL-6 in culture media (Fig. 7).

Role of Adrenomedullin in Patients with Rheumatoid Arthritis 181

Inflammatory cells, including lymphocytes and plasma cells, were counted in the superficial and deep portions of the infrapatellar fat pads (three fields under ×200 magnification in each portion) in H & E-stained specimens. The inflammatory cell count

To measure the collagen volume, the images of sections with Mallory–Azan stain were projected onto a color imaging analysis system (Mac SCOPE version 2.3.2; Mitani, Fukui, Japan). In each section, 10 separate sites were analyzed at ×40 magnification. The collagen volume fraction was obtained by calculating the mean ratio of connective tissue to the total

Total RNA was extracted from the infrapatellar fat pad with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's protocol and was then reversetranscribed into cDNA with the SuperScript First-Strand Synthesis System for RT-PCR kit (Invitrogen). To measure rabbit TNFα, IL-6, vascular endothelial growth factor (VEGF), transforming growth factor beta (TGFβ), and β-actin mRNA levels, we used the quantitative

We measured the plasma ADM concentration before and 15, 30, 60 and 120 minutes after intra-articular injection of 3 μg ADM (*n* = 6). No significant change, however, was observed in the plasma concentration of ADM (Figure (Figure1).1). The intra-articular injection of

Fig. 8. Sequential concentrations of plasma ADM following intra-articular ADM injection in

Whole-blood samples (total 1 ml) were taken from a peripheral artery in the rabbit ear using a 22-gauge needle before and 15, 30, 60 and 120 minutes after intra-articular injection of 3 μg

was performed by two independent observers.

**4.1.5 Measurement of cytokine mRNA** 

RT-PCR method of real-time quantitative PCR.

rabbits with antigen-induced arthritis.

**4.2 Result after joint injection of ADM in AIA rabbit 4.2.1 Adrenomedullin concentration in plasma** 

ADM did not therefore increase the level of ADM in plasma.

tissue area.

These observations lead us to conclude that ADM inhibited the secretion of IL-6 and regulated the IL-6 production in the cultured RA synoviocytes.

Besides the findings in our study, a similar report stated that ADM reduced constitutive production of IL6 from RA synoviocytes in a dose-dependent manner. A high concentration of ADM (>10-8 mmol/l) significantly reduced constitutive production of IL6 compared with a low concentration of ADM (<10-9 mmol/l) (p=0.0029) (Nanke et al., 2003). Then, we tried to inject ADM into the knee in antigen-induced arthritis in rabbits.
