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OBJECTIVE To examine cytokine and chemokine production during the evolution of rat adjuvant-induced arthritis (AIA), a model of rheumatoid arthritis. METHODS Clinical and laboratory assessment of the course of AIA was performed over a 47-day period. Levels of the cytokines tumor necrosis factor a (TNFalpha), interleukin-1beta (IL-1beta), and IL-6, as well as levels of the chemokines macrophage inflammatory protein 1alpha (MIP-1alpha) and JE, the murine homolog of monocyte chemoattractant protein 1, were determined by enzyme-linked immunosorbent assay in the sera and joints of AIA and control rats. Synovia from AIA rats were (immuno)histochemically analyzed. Results of cytokine and chemokine measurements were correlated with clinical and laboratory markers of inflammation and histology. RESULTS Early (before day 14 post adjuvant injection) and later phases of AIA could be distinguished. Cytokine and chemokine production was increased in AIA versus control rats. The production of TNFalpha, IL-1beta, MIP-1alpha, and, as determined earlier, epithelial neutrophil-activating peptide 78-like protein was abundant prior to and during the course of AIA, while that of IL-6 and JE was elevated in the late phase of AIA. Cytokine and chemokine levels were correlated with the clinical symptoms of arthritis and blood neutrophil counts. Joint levels of IL-1beta showed correlation with synovial lining proliferation and neutrophil ingress into AIA synovium. CONCLUSION Cytokines and chemokines are involved in the clinical, laboratory, and histologic changes underlying AIA. The production of these mediators may be temporally and spatially regulated. These findings may be important for the optimal timing of cytokine and chemokine targeting.

The chemokine, epithelial neutrophil-activating peptide-78 (ENA-78), is a potent neutrophil chemotaxin whose expression is increased in inflamed synovial tissue and fluid in human rheumatoid arthritis compared with osteoarthritis. Since ENA-78 has been implicated in the pathogenesis of RA, we examined the expression of an ENA-78-like protein during the development of rat adjuvant-induced arthritis (AIA). Using an ELISA assay, we found increased levels of antigenic ENA-78-like protein in the sera of AIA animals compared with control normal animals by day 7 postadjuvant injection. ENA-78-like protein levels continued to increase as AIA developed. ENA-78-like protein levels in joint homogenates were increased in AIA animals later in the development of the disease, by day 18 during maximal arthritis, compared with control animals. Expression of ENA-78-like protein in both the AIA serum and joint correlated with the progression of inflammation of the joints. Anti-human ENA-78 administered before disease onset modified the severity of AIA, while administration of anti-ENA-78 after clinical onset of AIA did not modify the disease. These data support a role for an ENA-78-like protein as an important chemokine in the progression and maintenance of AIA.

新しい血管を作り出す血管造成は発生、組織修復、炎症、動脈硬化症や腫瘍の進展など数多くの生理的、病理的過程で重要な役割を果たしている。サイトカイン、成長因子、その他の多くのメディエーターが血管造成に関与している。組織の血管新生の間、内皮細胞(endothelial cells、これ以降ECと呼ぶ)は細胞外マトリックス(ECM)成分や他のECsに接着するが、これは血管造成には必須の過程である。上に述べた可溶性のメディエーター以外にもECM巨大分子と細胞接着分子(CAMs)もまた血管造成因子として働く。CAMsの中ではE-セレクチン、脈管細胞接着分子(VCAM)-1、CD-31、やインテグリンが in vivo でも in vitro でも毛細管形成を促進する。CAMsや他の血管造成メディエーターは「血管造成病」の病因として働いている。このような因子のどれかを標的とすることがこれらの病気に適切で、かつ強力な治療となるかも知れない。

Interleukin-13 (IL-13) is a recently described lymphokine. Like IL-4, IL-13 induces the production of IL-1 receptor antagonists and suppresses the production of inflammatory cytokines by macrophages and monocytes. In this study, we investigated the effects of IL-13 on the migration and proliferation of human umbilical vein endothelial cells (HUVECs) and human dermal microvascular endothelial cells (HMVECs). We examined the effect of IL-13 on endothelial chemotaxis under two conditions: first, endothelial cells were exposed to a combination of IL-13 and a known chemotaxin, basic fibroblast growth factor (bFGF); second, endothelial cells were exposed to IL-13 alone. The effects of IL-13 on endothelial proliferation were also examined. IL-13 showed no inhibitory effects on bFGF-induced chemotactic activity on either HUVECs or HMVECs. IL-13 demonstrated chemotactic activity for HUVECs and HMVECs. For both cell types, peak chemotactic activity was at or above that of bFGF (60 nM). By varying concentrations of IL-13 in the upper and lower wells of the chemotaxis chambers, we found IL-13 to be chemotactic, and not chemokinetic, for HUVECs and HMVECs. IL-13 did not induce mitogenesis of either HUVECs or HMVECs. Although IL-13 has been shown to have many anti-inflammatory actions, these results suggest a novel role for IL-13 as a proinflammatory proangiogenic cytokine.

Objective. Angiogenesis is an integral component of the vasculoproliferative phase of rheumatoid arthritis (RA). Recently, a heparin-binding cytokine termed hepatocyte growth factor (HGF), or scatter factor (due to its ability to disperse cohesive epithelial colonies), was described. We conducted this study to investigate the hypothesis that this cytokine was present in the milieu of the inflamed joint, and that it contributed to the chemotaxis of endothelial cells in the synovial tissue. Methods. We examined synovial fluid, synovial tissue, and peripheral blood from 91 patients with RA and other arthritides. We used 83 total samples in an enzyme-linked immunosorbent assay to quantitate the HGF in synovial fluids and peripheral blood. To determine whether the HGF was biologically active, an epithelial scatter factor assay was performed. Immunohistochemical analysis was used to determine localization in synovial tissues. To define a function for synovial HGF, we preincubated rheumatoid synovial fluids with neutralizing anti-HGF and measured the ability of these synovial fluids to induce endothelial chemotaxis. Results. Synovial fluid from patients with RA contained a mean ± SEM HGF concentration of 2.0 ± 0.3 ng/ml, while synovial fluid from patients with other arthritides (including inflammatory arthritis) contained 2.4 ± 0.7 ng/ml HGF. Osteoarthritis (OA) patient samples contained the smallest quantities of synovial fluid HGF at 0.9 ± 0.1 ng/ml. RA synovial fluid contained significantly more HGF than did RA peripheral blood (1.1 ± 0.2 ng/ml) (P < 0.05). Rheumatoid synovial fluids induced more scattering of cells than did OA synovial fluids, suggesting a role for this cytokine in rheumatoid joint destruction. Interleukin-1β induced expression of rheumatoid synovial tissue fibroblast antigenic HGF and scatter factor activity. Immunohistochemically, HGF, as well as the HGF receptor (the met gene product), localized to significantly more rheumatoid synovial tissue lining cells than normal lining cells (P < 0.05). Both HGF and its receptor immunolocalized to subsynovial macrophages as well. Levels of synovial tissue immunoreactive HGF correlated positively with the number of synovial tissue blood vessels. Anti-HGF neutralized a mean of 24% of the chemotactic activity for endothelial cells found in 10 rheumatoid synovial fluid samples. Conclusion. These results indicate that synovial HGF may contribute to the vasculoproliferative phase of inflammatory arthritides such as RA, by inducing HGF-mediated synovial neovascularization. These findings point to a newly described role for HGF in the fibroproliferative phase of RA-associated synovitis.

Objective. To examine adhesion molecule expression during the progression of inflammation in a rheumatoid arthritis model of adjuvant-induced arthritis (AIA) in rats. Methods. Immunohistochemical analysis was used to determine the distribution of the following adhesion molecules: lymphocyte function–associated antigen 1 (LFA-1; CD11a/CD18), Mac-1 and p150/95 (CD11bc/CD18), intercellular adhesion molecule 1 (ICAM-1), and CD44 in tissue sections from the ankle joints of rats with AIA. Control animals and those with AIA were killed at intervals over a 54-day period after injection with mineral oil and Mycobacterium butyricum, respectively. Results. CD44 and LFA-1 were expressed on lymphocytes, macrophages, and synovial (ST) lining cells. CD44 expression on macrophages was found to be increased compared with control animals by day 18, and was significantly increased by day 41. CD44 expression on lymphocytes significantly increased earlier, on days 11–18. Increased LFA-1 expression on macrophages occurred late, on day 41. LFA-1 expression on lymphocytes was significantly increased on days 25, 47, and 54. ST lining cells exhibited two distinct periods of increased expression, one early, on days 11–25 and one later, on days 41–54. CD11b/c was expressed on macrophages and ST lining cells, showing a significant increase on AIA rat ST lining cells compared with control animals on day 4. No differences in ICAM-1 expression on endothelial cells between rats with AIA and controls were found on any of the days examined. Conclusion. CD44 expression is up-regulated on macrophages and lymphocytes during the early development of AIA, while LFA-1 expression is up-regulated later in the development of AIA. The up-regulation of CD44 and LFA-1 at different times in the development of AIA suggests an important role for these adhesion molecules in establishing and sustaining an inflammatory response in the AIA joint.

Endothelial cells (ECs) are key participants in angiogenic processes that characterize tumor growth, wound repair, and inflammatory diseases, such as human rheumatoid arthritis (RA). We and others have shown that EC molecules, such as soluble E-selectin, mediate angiogenesis. Here we describe an EC molecule, Lewisy-6/H-5-2 glycoconjugate (Ley/H), that shares some structural features with the soluble E-selectin ligand, sialyl Lewisx (sialyl Lex). One of the main previously recognized functions of Lewisy is as a blood group glycoconjugate. Here we show that Ley/H is rapidly cytokine inducible, up-regulated in RA synovial tissue, where it is cell-bound, and up-regulated in the soluble form in angiogenic RA compared with nonangiogenic osteoarthritic joint fluid. Soluble Ley/H also has a novel function, for it is a potent angiogenic mediator in both in vitro and in vivo bioassays. These results suggest a novel paradigm of soluble blood group Ags as mediators of angiogenic responses and suggest new targets for therapy of diseases, such as RA, that are characterized by persistent neovascularization.

A hallmark of both adjuvant-induced arthritis (AIA) and rheumatoid arthritis is chronic joint inflammation characterized by ingress of leukocytes into the inflamed synovial tissue. The timing of expression of adhesion molecules, which govern the ingress of leukocytes, is important in the orchestration of an inflammatory response. We examined the expression of vascular cell adhesion molecule-1 (VCAM-1), sialo adhesin, platelet and endothelial cell adhesion molecule-1 (PECAM-1), and leukosialin (CD43) in AIA, starting at adjuvant injection (day 0), through the peak of inflammation (day 18 postadjuvant injection), until day 54. VCAM-1 is constitutively expressed on the lining layer and ECs and its expression levels do not change throughout the progression of AIA. Sialoadhesin synovial tissue lining cell expression is decreased after adjuvant injection. In contrast, PECAM-1 expression is increased on synovial tissue lining cells on day 7 and is elevated through day 54 (peaking on day 54 with six-fold more cells expressing PECAM-1). PECAM-1 expression on endothelial cells peaks on day 7 with three-fold more cells expressing it, while on macrophages expression maximizes on day 25 with six-fold more cells expressing PECAM-1. CD43 expression is increased on synovial tissue lining cells, macrophages, neutrophils, and lymphocytes on days 18 and 25, before going back to basal levels. The increased expression of PECAM-1 and CD43 on leukocytes at the height of inflammation in AIA suggests important roles for these adhesion molecules in potentially binding their EC ligands resulting in leukocyte ingress into the synovial tissue.

Nutritional deprivation in the early postnatal period severely inhibits cerebellar growth and development, which is related in part to reduced levels of growth factors. Cyclin D1 encodes a growth factor-inducible regulatory subunit of a serine/threonine kinase that is capable of phosphorylating the tumor suppressor pRB, thereby allowing normal progression through the G 1 phase of the cell-cycle. Because the abundance of cyclin D1 is rate limiting in this progression, we examined the regulation of cyclin D1 expression in vivo, using a model of nutritional deprivation. Cyclin Dl expression in cerebella of fed control rats was detected in the external granular layer and was associated with cellular proliferation within this layer. Nutritional deprivation of rats reduced cerebellar weight, as well as the thickness of the molecular layer that largely consists of cells migrating from the external granular layer. Refeeding partially restored cerebellar weight, molecular layer thickness and increased external granular layer cyclin D1 immunostaining. Since nutritional deprivation is accompanied by lower levels of circulating insulin-like growth factor-I (IGF-I), we determined whether IGF-I directly stimulated the cyclin D1 promoter. The human cyclin D1 promoter linked to the luciferase reporter gene was stably integrated into PC12 cells. IGF-I stimulated cyclin Dl promoter activity 4- to 6-fold at 6 hours (h). These findings are consistent with the notion that nutritional deprivation may affect proliferative growth by altering expression of cyclin D1 in the germinal cell layer and that regulation of cyclin Dl expression by growth factors may contribute to normal neonatal cerebellar development. The reduction in cyclin D1 expression as cells differentiate in the cerebellum is consistent with a potential role for cyclin D1 in this process.

Adjuvant-induced arthritis (AIA) in rats is a relevant model for human rheumatoid arthritis (RA). In this study, th expression of the cytokines TNF-alpha, IL-1 and IL-6, as well as the chemokines MIP-1-alpha, MCP-1 and ENA-78 in the sera and joint homogenates of AIA and control, sham-injected rats was studied over a 47-day period. All of these cytokines and chemokines showed increased production in AIA. In addition, TNF-alpha, IL-1, ENA-78 and MIP-1-alpha could be termed as "early" mediators, as their production increased in the first 14-21 days and it correlated with early events in synovitis, such as neutrophil ingress, joint swelling and general symptoms. TNF-alpha may have mostly systemic, while IL-1 mainly local synovial effects. IL-6 and MCP-1 were found to be "late" inflammatory mediators, as their secretion was up-regulated after 2 weeks post-adjuvant injection and remained high during the observation period. Also, significant correlation was found between the production of TNF-alpha and that of chemokines. In conclusion, the differential expression of "early" and "late" cytokines and chemokines may account for various events underlying synovitis in AIA.