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Urocortin Expression in Synovium of Patients with Rheumatoid Arthritis and Osteoarthritis: Relation to Inflammatory Activity

First Department of Internal Medicine (Ma.K., Yu.K., Y.T., A.H., R.Y., K.-I.I., Mo.K., H.S.) and Department of Orthopedics (Yo.K., T.K.), Kyoto Prefectural University of Medicine, Kyoto 602-8566, Japan; Arthritis and Rheumatism Branch (I.J.E.), National Institute of Arthritis and Musculoskeletal and Skin Diseases, Bethesda, Maryland 20892; and Developmental Endocrinology Branch (G.P.C.), National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892

Address all correspondence and requests for reprints to: Hajime Sano, M.D., Ph.D., Kyoto Perfectural University of Medicine, First Department of Internal Medicine, 465 Kajii-Cho, Kawaramachi-Hirokoji, Kamigyo-Ku, Kyoto, Japan 602-8566. E-mail: hsano{at}koto.kpu-m.ac.jp

Abstract

Peripherally produced CRH acts as a local auto/paracrine proinflammatory agent. Urocortin is a new member of the CRH family that acts through the family of CRH receptors. In this study, we demonstrated that the expression of urocortin mRNA in synovia of patients with rheumatoid arthritis was greater than that of patients with osteoarthritis. Also, we detected urocortin and CRH receptor immunoreactivity in the synovial lining cell layer, subsynovial stromal cells, blood vessel endothelial cells, and mononuclear inflammatory cells from the joints of rheumatoid arthritis and osteoarthritis patients. The expression of immunoreactive urocortin was significantly greater in rheumatoid arthritis than osteoarthritis (P < 0.0001) and correlated with the extent of inflammatory infiltrate. CRH receptor immunoreactivity was strong in mononuclear inflammatory cells of rheumatoid arthritis synovia. Urocortin stimulated IL-1ß and IL-6 secretion by human peripheral blood mononuclear cells in vitro. These findings suggest that, like CRH, urocortin is present in peripheral inflammatory sites, such as rheumatoid synovium, and acts as an immune-inflammatory mediator.

CRH, A 41 AMINO ACID HYPOTHALAMIC neuropeptide, is the principal regulator of the hypothalamic-pituitary-adrenal (HPA) axis; CRH stimulates anterior pituitary secretion of ACTH and hence production of glucocorticoids by the adrenal cortex (1, 2). By activating glucocorticoid and catecholamine secretion, CRH participates in the immunomodulatory effects of stress on the immune/inflammatory response (2, 3). Products of an activated immune system, such as IL-1, IL-6, and TNF-, stimulate hypothalamic CRH secretion (3, 4, 5, 6). Also, CRH itself stimulates IL-1ß and IL-6 production by peripheral blood mononuclear cells (7, 8, 9). These inflammatory cytokines are produced by synoviocytes and may stimulate the production of CRH in synovial tissues. CRH is produced in peripheral inflammatory sites in which, in contrast to its systemic indirect immunosuppressive effects, it acts as an autocrine or paracrine inflammatory cytokine (10). Indeed, we recently reported the local production of immune CRH in different inflammatory sites, including carrageenin-induced granulomatous tissues (10) and streptococcal cell wall- and adjuvant-induced arthritic joints of rats (11), and in the joints of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) (12).

Urocortin, a recently identified 40-amino acid peptide, shares 45% sequence homology with CRH (13). Like CRH, urocortin is expressed in the human central nervous system and periphery (14). Urocortin has been detected in the human placenta (15), fetal membranes (15), circulating leukocytes (16), and skin (17). Exogenous urocortin has cardiac actions (18, 19, 20) and central effects similar to those of CRH, including stimulation of ACTH secretion and appetite suppression (21); all these effects are thought to be exerted via members of the CRH family of receptors. The positive relation between immune CRH and inflammatory activity was reported earlier (7, 8, 9, 22, 23). Also, it was reported that urocortin could influence the immune system in a corticosterone-dependent and -independent manner (24, 25, 26); however, the presence and function of urocortin in peripheral inflammatory sites have not been examined as yet.

In this study, we demonstrated the expression of urocortin mRNA, peptide, and its receptor in the synovial tissues of patients with RA and OA. Moreover, we examined the relation between the expression of urocortin and inflammatory activity and whether urocortin could modulate the production of IL-1ß and IL-6 by human peripheral mononuclear cells, as immune urocortin, analogous to immune CRH.

Materials and Methods

Patients

All patients with RA (n = 19, 18 women and 1 man, mean age is 58.5 ± 13.5 yr) met the American College of Rheumatology 1987 revised criteria (27) for RA, and all patients with OA (n = 16, 15 women and 1 man, mean age of 57.6 ± 12.4 yr) met the standard American College of Rheumatology criteria (28) for OA.

Collection of tissues

Synovial tissue was collected from patients with RA or OA at the time of total joint replacement or traumatic synovium of healthy subjects was collected at the time of endoscopic surgery and handled in one of two ways: (1) flash frozen in liquid nitrogen for RNA extraction or (2) fixed in 10% neutral formalin, embedded in paraffin, and sectioned onto gelatin-coated slides for immunohistochemical analysis.

RT-PCR

Total RNA was extracted by the acid guanidinium thiocyanate- phenol-chloroform method (29) and quantified by UV absorption. Complementary DNA was prepared by RT of 2 µg of each RNA sample, using the SuperScript preamplification system for the first strand cDNA synthesis (Life Technologies, Inc., Gaithersburg, MD) in a 63-µl of reaction volume. PCR reactions were performed in a 50-µl of reaction volume containing 2 µl of each cDNA, 9 µl each of 10x Gene Taq universal buffer (NIPPON GENE, Toyama, Japan), 1 µl of 10 mM dNTP (NIPPON GENE), 2 µl of each 20 µM of sense and antisense primers, and 2.5 U of recombinant Taq DNA polymerase (NIPPON GENE). Primer sequences were as follows: human urocortin sense primer 5'-CAGGCGAGCGGCCGCG-3', human urocortin antisense primer 5'-CTTGCCCACCGAGTCGAAT-3', human glyceraldehyde-3-phosphate dehydrogenase (G3PDH) sense primer 5'-CCACCCATGGCAAATTCCATGGCA-3', and antisense primer 5'-TCTAGAGGGCAGGTCAGGTCCACC-3' (15). Urocortin cDNA amplification was performed in 50 thermal cycles: sample was heated at 94 C for 1 min, cooled at 70 C for 1 min, and then heated at 72 C for 3 min. G3PDH amplification was performed in 35 thermal cycles, with samples heated at 94 C for 1 min, cooled at 55 C for 1 min, and then heated at 72 C for 3 min. These programs were preceded by 5 min at 94 C and followed by 7 min at 72 C. PCR products were electrophoresed in a 4% agarose gel stained with ethidium bromide and visualized by UV light.

RT competitive PCR

The DNA competitor was designed such that the PCR product from the cDNA could be separate from that of its competitor, and was generated using reagents supplied in a commercial kit (competitive DNA construction kit, TaKaRa, Ohtsu, Japan) (30). Briefly, a 30-cycle PCR was carried out on the DNA using the relevant composite primers. The RNA competitor was made from DNA competitor by transcription using a commercial kit (competitive RNA transcription kit, TaKaRa) according to the manufacturer’s protocol. After correcting concentration of each total RNA sample by human ß-actin using a commercial kit (competitive PCR set, TaKaRa), which consists of human ß-actin DNA competitor, human ß-actin sense primer (5'-CAAGAGATGGCCACGGCTGCT-3'), and human ß-actin antisense primer (5'-CTTGCCCACCGAGTCGAAT-3'), we performed RT-competitive PCR using reagents supplied in the competitive DNA construction kit. Briefly, RT, which was set up using 1 µl of 10-fold dilutions of RNA competitor ranging from 10⁷ to 10¹⁰ copies/µl with 1 µl sample RNA, was performed in 8 µl reaction mixture containing 2 µL of 25 mM MgCl₂, 1 µl of 10 x RNA PCR buffer, 0.25 µl of 40U/µl Rnase inhibitor, 0.5 µl of 5 U/µl AVM reverse transcriptase XL, and 0.5 µl of 50 µM Ramdom 9 mer. These samples were incubated at 42 C for 50 min, and the reaction was terminated by heating at 99 C for 5 min, followed by rapid chilling on ice for 5 min. Competitive PCR reactions were performed in a 40-µl of reaction volume containing 10 µl of these products, 3 µl of 25 mM MgCl₂, 4 µl of 10 x RNA PCR buffer, 0.5 µl of each 20 µM of urocortin sense and antisense primers, and 1.25 U of recombinant Taq DNA polymerase (TaKaRa). Competitor and urocortin cDNA amplification was performed in 30 thermal cycles: sample was heated at 94 C for 1 min, cooled at 70 C for 1 min, and then heated at 72 C for 3 min. These programs were preceded by 5 min at 94 C and followed by 7 min at 72 C. PCR products were electrophoresed in a 4% agarose gel stained with ethidium bromide and visualized by UV light. Photographs of the ethidium bromide-stained gels were scanned, and band intensities were measured using a densitometer (ATTO 6900; ATTO Co., Tokyo, Japan). Quantity of urocortin message was determined by which the ratio of competitor- and urocortin-band intensities was equal to 1.

Characterization of urocortin and CRH receptor antibodies

Urocortin antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) is an affinity-purified goat polyclonal antibody raised against a peptide corresponding to amino acids 102–121 mapping at the carboxy terminus of rat urocortin precursor (the sequence of rat urocortin precursor is identical to this mouse and human sequences), which is 122-amino acid protein with a carboxy terminus including a putative 40-amino acid peptide (13, 14). CRH receptor antibody (Santa Cruz Biotechnology, Inc.) is an affinity-purified goat polyclonal antibody raised against a peptide mapping at the carboxy terminus of the CRH receptor (CRH-R) type 1 (CRH-R1) precursor of human origin. It reacts with both CRH-R1 and CRH-R2 of human origin.

Immunohistochemistry

Immunohistochemical staining was performed using the avidin- biotin peroxidase complex. Synovial tissue specimens were preserved in 10% formalin, embedded in paraffin, serially sectioned onto microscope slides at a thickness of 4 µm, and then deparaffinized. The slides were immersed for 45 min in 0.3% peroxide in methanol to deplete endogenous peroxidase activity. Nonspecific binding sites were saturated by exposure to 0.2% BSA and normal rabbit serum diluted 1:66.7 in PBS for 20 min. Polyclonal, urocortin- (25 µg/ml), or CRH receptor-specific antibody (50 µg/ml), urocortin specific antibody (25 µg/ml) preincubated with the corresponding control peptide (Santa Cruz Biotechnology, Inc., 75 µg/ml) for 30 min, or control normal goat IgG (25 µg/ml, Vector, Burlingame, CA), was applied to tissue sections and incubated in a humidified chamber at room temperature for 40 min. Then the slides were washed with PBS for 10 min. Biotinylated rabbit antigoat IgG (Vector) in 10 ml PBS was applied to tissue sections, and the slides were incubated at room temperature for 30 min. They were then washed with PBS for 10 min, followed by incubation with prepared avidin DH-biotinylated peroxidase complex (Vector) for 45 min and washed with PBS for 10 min. Finally, color was developed by immersing the sections in a peroxidase substrate solution containing 0.05% (wt/vol) 3, 3'-diaminobenzidine tetrahydrochloride, 0.04% (wt/vol) nickel chloride, and 0.01% hydrogen peroxide in 0.05 mol/l Tris (pH 7.2) for 5 min. The sections were counterstained with 0.5% light green (Sigma, St. Louis, MO). Positive staining was indicated by brownish black deposits, and background staining was light green.

For each tissue specimen, the extent and intensity of staining with anti-urocortin or anti-CRH receptor specific antibody was graded on a 0 to 4+ scale on coded slides by two blinded observers on two separate occasions, and an average score was calculated. A 4+ grade implies that all staining was maximally intense throughout the specimen, whereas 0 implies that staining was absent throughout the specimen. This methodology is identical to that used in previous studies (11, 12, 31, 32).

Mononuclear cell infiltration, one measure of the degree of inflammation in the tissue specimens, was similarly assessed on hematoxylin- and eosin-stained sections cut from the same paraffin blocks used for immunohistochemical staining. Thus, a 4+ grade implies that all tissues were intensely infiltrated with mononuclear cells, whereas 0 implies that mononuclear cell infiltration was absent throughout (12, 32).

Cytokine assay

Venous blood was drawn by venipuncture into a syringe containing approximately 20 U/ml sodium heparin. The blood donors were all healthy adult humans (n = 5, mean age 32.4 ± 8.5 yr). None was taking any prescription medication or showed any evidence of clinical disease. For the separation of mononuclear cells (MNC), heparinized blood was subjected to a density-gradient centrifugation method in Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) according to the manufacturer’s suggested protocol. The final MNC pellet were seeded in 24-well flat-bottom microtiter plates (Nunc, Roskilde, Denmark) at a density of 5 x 10⁵ cells/well in a total volume of 400 µl of RPMI 1640 medium (Nissui Pharmaceutical, Tokyo, Japan) containing 10% FBS (BioWhittaker, Inc., Walkersville, MD), 100 U/ml penicillin and 100 µg/ml streptomycin (Life Technologies, Inc./Life Technologies Inc., Rockville, MD). Duplicate cultures were then prepared by adding the medium containing urocortin at concentrations of 0, 1, 10, or 100 nM. After incubation at 37 C in a humidified atmosphere of 5% CO₂/95% air for 24 h, the culture supernatants were recovered and stored at -80 C until cytokine measurements. Concentrations of IL-1ß and IL-6 were measured using respective commercial ELISA kits (Biosource International Inc., Camarillo, CA).

Statistical analysis

The graded scores of the extent and intensity of immunostaining with anti-urocortin or anti-CRH receptor antibody or mononuclear cell infiltration of synovial tissue sections were analyzed with the Mann-Whitney U test and Spearman’s rank correlation. The results of RT-competitive PCR were analyzed with the Mann-Whitney U test. The results of bioassay are given as the mean ± SD of the percent change in IL-1ß or IL-6 production and analyzed for significant changes between treatments of urocortin by paired t test. Differences were considered significant when P < 0.05.

Results

RT-PCR

Amplification of cDNA with urocortin primers used in this study predicted a fragment of 145 bp in length. The predicted size band was detected in all patients with RA (n = 6) and OA (n = 3) after amplification of reverse-transcribed mRNA. Four samples obtained from individual RA patients (lane 14) and two samples from individual OA patients (lanes 5 and 6) are shown in Fig. 1A. Positive control performed with total RNA of human placenta (OriGene Technologies, Rockville, MD) also showed the expected band (145 bp) (Fig. 1A, lane 7). Negative control performed with no cDNA added to the PCR reaction yielded no detectable band (Fig. 1A, lane 8). Primers specific for G3PDH generated the expected 598-nucleotide band in all cases (Fig. 1B, lane 17).

View larger version (15K): [in this window] [in a new window]   Figure 1. RT-PCR analysis of urocortin mRNA expression. Four samples obtained from individual RA patients (lanes 1–4) and two samples from individual OA patients (lanes 5 and 6) are shown. A, Primers specific for human urocortin generated the expected 145-bp band (lanes 1–7). B, Primers specific for human G3PDH generated the expected 598-bp band (lanes 1–7). Negative control reactions yielded no detectable bands (lane 8). Lanes 1–4 = RA; 5, 6 = OA; 7 = human placenta (positive control), 8 = no cDNA (negative control).

Competitive PCR demonstrated that the expression of urocortin mRNA in synovia of patients with RA (n = 4) was greater than those of patients with OA (n = 4) (P = 0.0209). It demonstrated that about a 10-fold increase in urocortin mRNA transcripts was detected in the synovial tissue from patients with RA than OA synovium (Fig. 2).

View larger version (46K): [in this window] [in a new window]   Figure 2. RT-Competitive PCR analysis of urocortin mRNA expression. A, Typical image obtained from RT-competitive PCR analysis of urocortin mRNA extracted from RA synovium. B, Typical image obtained from RT-competitive PCR analysis of urocortin mRNA extracted from OA synovium. Competitive PCR demonstrated that about a 10-fold increase in urocortin mRNA transcripts was detected in the synovial tissue from patients with RA than OA synovium.

Synovial tissue from patients with RA revealed urocortin immunostaining in the synovial lining cell layer, synovial fibroblast-like cells, blood vessel endothelial cells, and infiltrating mononuclear inflammatory cells (Fig. 3, A and B). Adjacent sections stained with urocortin-specific antibodies depleted with urocortin control peptide were completely negative (Fig. 3C). Similarly adjacent sections stained with normal goat IgG were completely negative (Fig. 3D). Synovium from patients with OA revealed low-grade urocortin expression in the synovial lining cell layer, blood vessel endothelial cells, and scattered infiltrating mononuclear immunoreactive inflammatory cells (Fig. 3E). Synovium from healthy subjects revealed less grade urocortin expression (Fig. 3F).

View larger version (180K): [in this window] [in a new window]   Figure 3. Urocortin immunostaining in synovium from patients with RA or OA. A, B, RA synovium stained with anti-urocortin antibody (25 µg/ml). C, RA synovium stained with anti-urocortin antibody (25 µg/ml) preincubated with control peptide (75 µg/ml) for 30 min. D, RA synovium stained with normal goat IgG (25 µg/ml). E, OA synovium stained with anti-urocortin antibody (25 µg/ml). F, Normal synovium stained with anti-urocortin antibody (25 µg/ml). Positive staining is indicated by black deposits; the background is light green on original sections. SL, Synovial lining cell layer; BV, blood vessels. (Original magnification x 25 in A, C, D; x 100 in B, E, F.)

View larger version (101K): [in this window] [in a new window]   Figure 4. CRH receptor immunostaining in synovium from patients with RA or OA. A, RA synovium stained with anti-CRH receptor antibody (50 µg/ml). B, RA synovium stained with normal goat IgG (50 µg/ml). C. OA synovium stained with anti-CRH receptor antibody (50 µg/ml). Positive CRH receptor immunostaining is indicated by black deposits (the background is light green on original sections). SL, synovial lining cell layer; BV, blood vessels. (Original magnification, x100 in A, B; x25 in C.)

View larger version (13K): [in this window] [in a new window]   Figure 5. Statistical analysis of urocortin immunostaining in RA vs. OA patients. The extent and intensity of staining with anti-urocortin antibody in the synovium from patients with RA and OA was graded 0–4+ by blinded observers on synovial specimens from 19 RA and 16 OA patients. Urocortin immunostaining was more intense and diffuse in synovium of RA patients than that of OA patients (P < 0.0001; Mann-Whitney U test).

View larger version (17K): [in this window] [in a new window]   Figure 6. Statistical analysis of urocortin immunostaining vs. inflammatory activity. The correlation between immunoreactive urocortin expression and mononuclear cell infiltration in the synovium from patients with RA and OA was examined. The extent and intensity of mononuclear cell infiltration was graded 0–4+ on hematoxylin and eosin sections by blinded observers. The extent and intensity of staining with anti-urocortin antibody on synovium from 19 RA and 6 OA patients were significantly correlated (r = 0.926; P < 0.0001; Spearman’s rank correlation) to the extent and intensity of MNC infiltration.

View larger version (12K): [in this window] [in a new window]   Figure 7. Statistical analysis of CRH receptor immunostaining in RA vs. OA patients. The extent and intensity of staining with anti-CRH receptor antibody in the synovium from patients with RA and OA was graded 0–4+ by blinded observers on synovial specimens from 10 RA and 12 OA patients. CRH-R immunostaining was more intense and diffuse in synovium of RA patients than that of OA patients (P < 0.001; Mann-Whitney U test).

As shown in Fig. 8, the addition of urocortin into the cultures of peripheral blood mononuclear cells caused increased production of IL-1ß and IL-6. The urocortin-induced stimulation of IL-1ß was significant at 1 (P = 0.0470), 10 (P = 0.0240), and 100 nM (P = 0.0298). The urocortin-induced stimulation of IL-6 was significant at 10 (P = 0.0043) and 100 nM (P = 0.0491).

View larger version (29K): [in this window] [in a new window]   Figure 8. Effect of urocortin on the production of IL-1ß and IL-6 by peripheral blood mononuclear cells. The results of the bioassay are given as the mean ± SD of the percent change in IL-1ß and IL-6 production (n = 5). These peripheral blood mononuclear cells were obtained from five normal individuals. A, The stimulatory effect of urocortin on IL-1ß was significant at 1 (P = 0.0470), 10 (P = 0.0240), and 100 nM (P = 0.0298) concentrations. B, The stimulatory effect of urocortin on IL-6 was significant at 10 (P = 0.0043) and 100 nM (P = 0.0491) concentrations. (, P < 0.01, *, P < 0.05)

In the present study, we demonstrated that immunoreactive urocortin and CRH-R are expressed in synovial lining cell layer, subsynovial stromal cells, blood vessel endothelial cells, and infiltrating mononuclear inflammatory cells in synovial tissues of arthritic joints. The expression of urocortin and its mRNA in synovial tissues in RA was greater than that in OA and correlated significantly with the degree of inflammation. Interestingly, CRH-R was expressed more intensively in infiltrating mononuclear inflammatory cells. Moreover, urocortin could stimulate the production of IL-1ß and IL-6 by the human peripheral mononuclear cells. Thus, urocortin may have a proinflammatory effect in the synovium of RA patients via the CRH-R.

In 1989 we demonstrated that the marked differences of the inflammatory responses between Lewis rats and their histocompatible Fischer rats were owing to an inability of the HPA axis in the former to release adequate amounts of glucocorticoids; we localized this defect in the CRH-secreting neurons of the hypothalamus (33). Subsequently, we showed that RA patients did not increase cortisol secretion following surgery, in spite of high levels of IL-1ß and IL-6. The hypoactivity of their HPA axis response to immune/inflammatory stimuli probably resided in their hypothalamus as well (34). Dissociation of ACTH and ß-endorphin secretion in response to CRH, and later impaired cortisol secretion in the presence of intact ACTH secretion were reported in RA patients, although the locus of the defect was not clear (35, 36). In a more recent study, Crofford et al. (37) detected a negative effect of cortisol on IL-6 in newly diagnosed, unmedicated patients with RA as well as a clear inability of their HPA axis to respond to inflammatory stimuli. The entire area was summarized in two recent editorials that concluded that, most likely, the locus of the HPA axis defect in RA was suprapituitary (38, 39).

CRH is present locally in carrageenin-induced rat granulomas, streptococcal cell wall–induced rat arthritis (10, 11), and human synovial fluids and tissues of patients with RA and OA (12). In addition, the expression of CRH correlated significantly with the degree of inflammation. An anti-CRH antibody and/or a CRH-R1-specific nonpeptidic antagonist called antalarmin reduced inflammation in the carrageenin-treated rat and in rats and mice with experimental uveitis (10, 23, 40). These results suggested that CRH functions peripherally as a proinflammatory factor. The expression of immune CRH is increased in animals or humans with decreased central hypothalamic CRH secretion (10, 11, 33, 37, 38, 39).

In this study, we demonstrated that mRNA and immunoreactive urocortin was localized in a significant proportion of cells from the synovial lining cell layer, synovial fibroblast-like cells, blood vessel endothelial cells, and infiltrating mononuclear inflammatory cells in the synovium of patients with RA. In contrast, synovium from healthy subjects revealed less grade urocortin expression. We cannot accurately specify which cell types secrete urocortin. CRH, substance P, and other neuropeptides possibly involved in pathophysiology of chronic inflammatory arthritis, such as RA, may be produced locally but also delivered to inflamed tissues by peripheral nerves (41, 42, 43, 44, 45, 46, 47). Thus, urocortin in the inflamed joints might also be secreted by the terminals of peripheral neurons as well as by immune cells (3).

Many studies have shown evidence that CRH has direct effects on leukocytes; for example, CRH directly stimulates production of POMC-related peptides (48, 49, 50); causes secretion of IL-1, IL-2 (7), and IL-6 (8, 9); stimulates lymphocyte proliferation; enhances the proliferative response of leukocytes to lectins; and increases the expression of the IL-2 receptor on T lymphocytes (51, 52). All these data suggest strongly that CRH might have local direct effects on the immune/inflammatory reaction. We clarified that the expression of urocortin and its mRNA in synovial tissues in RA was greater than that in OA and correlated significantly with the degree of inflammation. The expression of urocortin in RA joints was slightly lower than that of CRH but has a similar distribution (data not shown). Moreover, addition of urocortin into cultures of human peripheral blood mononuclear cells caused increased production of IL-1ß and IL-6, as did CRH (7, 8).

A recent study demonstrated that CRH receptors were upregulated on immune cells such as macrophages and B and T lymphocytes (53, 54) from hindpaw infiltrates and from draining lymph nodes (55). We showed that CRH-R is expressed in the synovial lining cell layer; subsynovial stromal cells; blood vessel endothelial cells; and, more intensively, in infiltrating mononuclear inflammatory cells in the synovium of patients with RA and OA. There are two homologous molecular species of CRH-Rs that differ from each other in anatomic distribution as well as pharmacologic profile (56). CRH-R1 is a major receptor isoform regulating pituitary ACTH secretion, and CRH’s vasodilatory effects appear to be mediated by CRH-R2 (56). Studies of Webster et al. (23) suggest that most of the proinflammatory actions of CRH/urocortin are mediated by CRH-R1 rather than CRH-R2. Similarly, Theoharides et al. and others (23, 57) demonstrated that the marked mast cell-degranulating effects of CRH were inhibited by antalarmin, a CRH-R1-specific antagonist. The CRH-R antibody used in this study reacts to both CRH-R1 and CRH-R2; however, these studies suggest that the local effect of urocortin on inflammation is mediated by CRH-R1. Indeed, we demonstrated that urocortin stimulate only production of IL-1ß and IL-6 by human peripheral blood mononuclear cells, which express intense immunoreactive CRH-R, compared with other cells. However, in our preliminary studies we have showed no significant effect of urocortin on the production of IL-1ß and IL-6 by cultured fibroblast-like synoviocytes (data not shown).

In contrast, urocortin has been reported to have direct anti-inflammatory or protective effects in vivo and in vitro. Agnello et al. (25) demonstrated corticosteroid-independent inhibition of TNF- production by urocortin in vivo. However, in his study IL-1ß production was not inhibited by urocortin in a corticosteroid-independent manner, and it was rather increased when corticosterone was blocked, but serum IL-6 was not affected by urocortin. Poliac et al. (24) showed that urocortin suppressed encephalomyelitis through activation of the HPA axis and might do directly via the immune system, and Okosi et al. (26) suggested that urocortin protected cardiac myocytes from cell death induced by hypoxia through CRH-R2. Although urocortin may have a potential anti-inflammatory or cytoprotective function mediated by CRH-R2, in the arthritis its proinflammation, presumably CRH-R1-mediated effect, appears to predominate. Thus, the role of urocortin in the peripheral site may depend on types of its receptor, and further examinations are required in this field.

As another anti-inflammatory mechanism of urocortin, Turnbull et al. (58) demonstrated that the edema owing to thermal injury in rats was inhibited by systemic urocortin treatment. The pharmacologic doses of urocortin administered iv, however, had most likely produced hypotension leading to a decreased ability of the animals to mount a full peripheral inflammatory response. Recently, Torpy et al. (59) demonstrated that equivalent levels of hypotension produced by urocortin or hydralazine were associated with decreased inflammatory responses to carrageenin, and the decrement of blood pressure correlated with the decrease of the inflammatory response. Thus, we suggest that urocortin administered iv acts as vasodilator, which leads to hypotension, rather than as immune urocortin with direct antiinflammatory effects.

In conclusion, urocortin and the CRH-R are strongly expressed in synovial tissues of RA and, to a lesser extent, OA patients. The data reported here suggest that, like CRH, urocortin acts as an autocrine or paracrine proinflammatory mediator in the synovium of RA and OA patients via CRH-R1s and may be involved in the pathophysiology of RA and other inflammatory diseases.

Acknowledgments

We thank Misuzu Nakajima, Kumiko Yasui, and Yoshiko Ikenouchi for assistance in preparation of the manuscript.

Footnotes

Abbreviations: CRH-R, CRH receptor; G3PDH, human glyceraldehyde-3-phosphate dehydrogenase; HPA, hypothalamic-pituitary-adrenal; MNC, mononuclear cells; RA, rheumatoid arthritis; OA, osteoarthritis.

Received November 9, 2000.

Accepted May 11, 2001.

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