Activated leucocytes express and secrete macrophage inflammatory protein-1{alpha} upon interaction with synovial fibroblasts of rheumatoid arthritis via a ß2-integrin/ICAM-1 mechanism

M. Hanyuda, T. Kasama, T. Isozaki, M. M. Matsunawa, N. Yajima, H. Miyaoka1, H. Uchida2, Y. Kameoka3, H. Ide and M. Adachi

Division of Rheumatology and Clinical Immunology, First Department of Internal Medicine, Showa University School of Medicine, 1Department of Orthopedics, Showa University School of Medicine, 2Department of Orthopedics, Furukawabashi Hospital and 3Division of Genetic Resources, National Institute of Infectious Diseases, Tokyo, Japan.

Correspondence to: T. Kasama, Division of Rheumatology and Clinical Immunology, First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan. E-mail: tkasama{at}med.showa-u.ac.jp


    Abstract
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 Abstract
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 Materials and methods
 Results
 Discussion
 Supplementary data
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Objective. To examine the expression and regulation of chemotactic factor, macrophage inflammatory protein-1{alpha} (MIP-1{alpha}) by fibroblast-like synoviocytes (FLS), monocytes and polymorphonuclear neutrophils (PMN) isolated from the synovial fluid (SF) of rheumatoid arthritis (RA) patients.

Methods. Monocytes or PMN obtained from RA SF were co-cultured with unstimulated semiconfluent RA FLS. Culture supernatants were assayed for MIP-1{alpha} by enzyme-linked immunosorbent assay. The expression of MIP-1{alpha} mRNA and protein was also determined by Northern blot analyss and immunohistochemistry respectively.

Results. Interaction of activated leucocytes with FLS synergistically increased MIP-1{alpha} expression and secretion via a mechanism mediated by ß2-integrin/ intercellular adhesion molecule 1.

Conclusion. MIP-1{alpha} expression within inflamed joints appears to be regulated not only by inflammatory cytokines but also by the physical interaction of activated leucocytes and FLS, and plays a crucial role in the progression and maintenance of RA synovitis.

KEY WORDS: MIP-1{alpha}, Rheumatoid arthritis, Inflammatory cytokines, Fibroblast-like synoviocytes, ß2-integrin, Intercellular adhesion molecule 1, Monocytes, polymorphonuclear neutrophils, Synovitis, Chemokines.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
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Progression of rheumatoid arthritis (RA) is characterized by the appearance of inflammatory cells in both the pannus and joint fluid and by eventual tissue destruction. Various mediators, including inflammatory cytokines and adhesion molecules, have been implicated in the pathogenesis of RA [13], and specific cell–cell interactions, especially between synovial cells and invading cells, appear to play pivotal roles in the progression of RA. Chemokines, as well as other inflammatory mediators, appear to play key roles in the pathogenesis of RA, and the coordinated production of chemokines and proinflammatory cytokines is likely to be important in the orchestration of the inflammatory responses observed in patients with RA [36]. Macrophage inflammatory protein (MIP)-1{alpha}, a member of the C-C chemokine family, is detected in the synovium of human RA and also in collagen-induced arthritis, an experimental model of RA, and plays an important role in the recruitment of macrophages and certain T lymphocytes into the joint cavity [3, 5, 79]. An imbalance of T helper type 1 (Th1) and Th2 cytokines, with a predominance of Th1 cytokines, is thought to be of pathogenetic importance in RA [1012]. The Th1 phenotype expresses certain chemokine receptors, including CCR5, a specific receptor of MIP-1{alpha} [13]. Accumulation of CCR5-positive T lymphocytes is seen in the inflamed synovium and in the synovial fluid of RA, and seems to have an important role in the recruitment of Th1 lymphocytes into the joint [1417]. Thus, chemokines, including MIP-1{alpha}, and invasion of specific receptor-bearing activated T cells and macrophages seem to be crucial in the initiation and development of rheumatoid synovitis [5].

The aim of the present study was to examine the regulation of MIP-1{alpha} expression in RA synovitis. In particular, we focused on the effect of interaction of fibroblast-like synoviocytes (FLS) with inflammatory synovial monocytes and polymorphonuclear neutrophils (PMN) on the expression of MIP-1{alpha}. Our results showed that the interaction of activated inflammatory leucocytes and FLS synergistically increased the expression and secretion of MIP-1{alpha}, via a mechanism mediated by ß2-integrin/intercellular adhesion molecule 1 (ICAM-1).


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
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Reagent preparation
Completed medium consisted of DMEM (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 10% heat-inactivated fetal bovine serum (FBS; Gibco Laboratories, Grand Island, NY, USA). Monoclonal and polyclonal antibodies (Abs) for MIP-1{alpha} were purchased from Austral Biologicals (San Ramon, CA, USA), and R & D Systems (Minneapolis, MN, USA) respectively. Monoclonal Abs against human CD11b and CD18 were purchased from Ancell Corporation (Bayport, MN, USA) and the monoclonal Ab against ICAM-1 was purchased from R & D Systems.

Isolation and culture of peripheral blood and synovial fluid monocytes and PMN
RA synovial fluid (SF) was obtained from knee punctures of 32 RA patients with RA. RA SF monocytes and PMN were obtained from knee punctures of 23 patients with RA. Normal peripheral blood (PB) monocytes and PMN were obtained from 10 age- and sex-matched healthy individuals. PMN were isolated by centrifugation on a Ficoll–Hypaque (Pharmacia LKB Biotechnology, Piscataway, NJ, USA) density gradient, and were resuspended at a density of 5 x 106 cells/ml in complete medium. The mononuclear cells, isolated by centrifugation on a Ficoll–Hypaque density gradient, were then separated by centrifugation on a density gradient (1.068 g/ml; Nycodenz, Nycomed, Oslo, Norway), as described previously [18]. The final cell preparations contained more than 75–80% monocytes, based on their morphology and non-specific esterase staining; their viability was greater than 98%. The recovered monocytes were resuspended at a density of 1 x 106 cells/ml in complete medium.

All human experiments were performed in accordance with protocols approved by the Human Subjects Research Committee at Showa University, and informed consent was obtained from all patients and volunteers.

Preparation of FLS
Synovial tissues were obtained from patients with RA and patients with osteoarthritis (OA), all of whom underwent joint replacement surgery. Synovial membrane cell suspension cultures were prepared by collagenase and DNase digestion of minced membranes, as described previously [18, 19]. Isolated FLS were cultured in completed medium in 25-mm2 tissue culture flasks (Becton Dickinson, Bedford, MA, USA). The cells were used from passages 3–10, when they morphologically resembled fibroblast-like synoviocytes and were Mo-1- and MHC class II-negative, indicating the absence of type A or ‘macrophage-like’ synoviocytes.

Co-culture of synovial fluid monocytes or neutrophils with FLS
SF monocytes or PMN were layered onto unstimulated semiconfluent FLS monolayers in 48-well plates (NalgeNunc International, Tokyo, Japan), and culture supernatants were collected at selected times thereafter. In some experiments, a transwell membrane (pore size 0.45 µm; Becton Dickinson) was used to separate the two cell groups, while in others anti-integrin Abs or adhesion molecules were added to the co-cultures.

MIP-1{alpha} levels assayed by specific ELISA
MIP-1{alpha} was quantified specifically using the double ligand enzyme-linked immunosorbent assay (ELISA) method, in a modification of a previously published assay [20]. Monoclonal murine anti-human MIP-1{alpha} (1 µg/ml) served as the primary Ab, and biotinylated polyclonal goat anti-MIP-1{alpha} (0.1 µg/ml) served as the secondary Ab. The detection limit for the MIP-1{alpha} ELISA was ~50 pg/ml.

Immunohistochemistry
Cell-associated MIP-1{alpha} was visualized immunohistochemically in a previously published assay [20]. Briefly, FLS were grown to near confluence in an eight-well Lab-Tek chamber slide (NalgeNunc International), and then incubated for 24 h with or without either monocytes or PMN. After incubation, the slides were fixed, stained with polyclonal rabbit anti-MIP-1{alpha} Ab (1:200 dilution), purchased from PeproTech EC (London, UK) or in pre-immune rabbit IgG, as first antibodies, and with biotinylated goat anti-rabbit IgG (1:20; Biogenex Laboratories, Burlingame, CA, USA) as a second antibody. The substrate for the red colour reaction was 3-amino-9-ethylcarbazole in N,N-dimethylformamide.

Isolation of total RNA and Northern blot analysis
Isolations of total cellular RNA and Northern blot were performed as described previously [20]. RNA was separated using 1% agarose gels, transblotted to nitrocellulose, and hybridized with a 32P-5' end-labelled oligonucleotide probe specific for human MIP-1{alpha} [21] or ß-actin [22]. Specific mRNA was quantified using imaging analysis software (NIH Image 1.61; National Institutes of Health Public Software, Bethesda, MD, USA).

Statistical analysis
Data were analysed on a Power Macintosh computer using a statistical software package (Statview 4.5; Abacus Concepts, Berkeley, CA, USA) and expressed as the mean ± S.E.M. Group data were compared by analysis of variance; the means of groups whose variances were determined to be significantly different were then compared by Student's t-test. A P value <0.05 was considered significant.


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Production of MIP-1{alpha} through the interaction of FLS and leucocytes
To examine the profiles of MIP-1{alpha} expression, we first conducted a series of experiments to assess the induction of MIP-1{alpha} expression by unstimulated FLS or leucocytes, as well as by the interaction of FLS with leucocytes. As shown in Fig. 1A, no MIP-1{alpha} secretion was seen in unstimulated RA FLS. On the other hand, RA SF leucocytes (monocytes and PMN) secreted relatively little MIP-1{alpha} (Fig. 1A). In contrast, the amount of MIP-1{alpha} secreted into the supernatant significantly increased when unstimulated FLS were co-cultured with RA SF monocytes or PMN, representing a synergistic effect. In addition, to determine whether the augmented production of MIP-1{alpha} was specific to leucocytes in the RA SF, we tested the capacity of FLS and either PB monocytes or PMN obtained from healthy individuals to produce MIP-1{alpha}. Although increased production of MIP-1a was observed in RA FLS-PB monocyte co-cultures, the increase was less pronounced than in RA FLS-SF leucocyte co-cultures (Fig. 1A).



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FIG. 1. Secretion of MIP-1{alpha} mediated by the interaction of FLS and SF leucocytes. Monocytes (5 x 105/0.5 ml/well) or PMN (2.5 x 106/0.5 ml/well) obtained from SF or PB (control) were layered onto unstimulated semiconfluent FLS monolayers obtained from RA and OA patients in 48-well plates. In addition, either FLS or leucocytes were fixed with 1% paraformaldehyde (10 min) and were co-cultured. (A) Co-cultures of SF or control leucocytes with RA FLS. (B) Co-cultures of SF leucocytes with RA or OA FLS. (C) Either RA FLS or RA SF leucocytes were fixed with 1% paraformaldehyde (10 min) and were co-cultured. Supernatants were collected 24 h after co-culture and MIP-1{alpha} was measured by ELISA. Data represent the mean (pg/ml) ± S.E.M. of five independent experiments performed using three different RA fibroblasts and five different RA SF leucocytes. *P < 0.05 vs monocytes, PMN, FLS alone or fixed cells.

 
We next tested the capacity of FLS obtained from OA patients to produce MIP-1{alpha}. Although synergistically enhanced secretion of MIP-1{alpha} was observed in OA FLS–RA SF leucocyte co-cultures, the enhancement was less pronounced than in RA FLS–SF leucocyte co-cultures (Fig. 1B). Moreover, to determine the cell populations responsible for MIP-1{alpha} production, one of the cell populations was fixed with 1% paraformaldehyde (PFA) prior to co-culture (Fig. 1C). In these experiments, the fixation of FLS completely inhibited MIP-1{alpha} secretion by PMN co-cultures and decreased secretion by monocyte co-cultures (51.5%) compared with the co-cultures with non-fixed FLS. Additionally, MIP-1{alpha} secretion by fixed leucocytes co-cultured with FLS was not seen. Although PFA treatment may decrease the adhesion of either type of leucocyte to fixed FLS [18], these data indicate that the major cellular source of MIP-1{alpha} is leucocytes during co-culture with FLS. Confirmation was provided by immunohistochemical studies; up-regulation of MIP-1{alpha} expression was noted in co-cultures either of monocytes or PMN with FLS (Fig. 2). Although small amounts of MIP-1{alpha} antigen were present in unstimulated RA FLS and leucocytes (not shown in Fig. 2), markedly greater amounts were seen in co-cultures of both monocytes with FLS and PMN with FLS.



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FIG. 2. Representative photomicrographs showing the immunohistochemical localization of antigenic MIP-1{alpha} within interacting leucocytes and FLS. After incubation for 24 h, the cells were labelled with anti-MIP-1{alpha} Abs. RA FLS plus RA SF monocytes (A) or PMN (B). (A and B) Staining with anti-MIP-1{alpha} antibody, demonstrating a significant presence of cell-associated MIP-1{alpha} antigen (arrows) in leucocytes, but not in fibroblasts (arrowheads). There was no non-specific staining in co-cultures incubated with control IgG (not shown). Original magnification x 500). [See Supplementary figure 2 available at Rheumatology Online.]

 
We then examined the dynamics of MIP-1{alpha} secretion. When RA SF monocytes and PMN were cultured separately for 24 h, each secreted a small amount of MIP-1{alpha} (data not shown). In contrast, MIP-1{alpha} was detectable in the supernatants of RA FLS monocyte co-cultures within 4 h, and levels were markedly higher after 24 h. Similarly, FLS PMN co-cultures also yielded significantly elevated levels of MIP-1{alpha} within 24 h (Fig. 3A). In addition, the magnitude of the response was dependent on the number of leucocytes plated in the co-cultures (Fig. 3B). These results indicate that MIP-1{alpha} secretion is dependent on both the duration of culture of FLS and leucocytes and on the number of leucocytes present in the culture.




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FIG. 3. Effects of time (A) and leucocyte number (B) on MIP-1{alpha} secretion. (A) RA SF monocytes (5 x 105/0.5 ml/well) or PMN (2.5 x 106/0.5 ml/well) were layered onto RA FLS monolayers, and supernatants were collected 2, 4, 12 and 24 h later. Varying numbers of SF monocytes or PMN (B) were layered onto RA FLS monolayers, and supernatants were collected 24 h later. Each point or bar represents the mean (pg/ml) ± S.E.M. of three independent experiments performed using three different RA fibroblasts and three different RA SF leucocytes.

 
Because FLS–lymphocyte interactions have been demonstrated to induce chemical mediators and matrix metalloproteinase [23], it was important to rule out contaminated lymphocytes as the major source of MIP-1{alpha} in FLS–leucocyte co-cultures. For this purpose, we examined the effect of mononuclear lymphocytes on MIP-1{alpha} secretion by co-cultures of FLS lymphocytes. Monocytes were depleted from mononuclear cell suspension by adhesion to a plastic dish for 2 h, and then monocyte-depleted non-adherent lymphocytes (1 x 106/ml; monocyte contamination <=5%) were added to FLS cultures. Although a synergistic effect of lymphocytes on MIP-1{alpha} secretion was also observed (FLS, 8.6 ± 0.6 pg/ml; monocyte-depleted lymphocytes, 349.2 ± 27.8 pg/ml, FLS plus monocyte-depleted lymphocytes, 1366.6 ± 573.1 pg/ml), the effects of lymphocytes on MIP-1{alpha} secretion were limited compared with FLS–monocyte co-cultures (FLS plus monocytes, 24 189.0 ± 523.5 pg/ml), suggesting that contaminated lymphocytes may not be potentially involved in increased MIP-1{alpha} secretion.

Next, we examined the steady-state expression of MIP-1{alpha} mRNA in co-cultures using Northern blot analysis. Consistent with the expression of MIP-1{alpha} protein, Northern blot analysis revealed that the steady-state expression of MIP-1{alpha} mRNA was significantly up-regulated in monocytes/PMN co-cultured with FLS (Fig. 4).



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FIG. 4. Northern blot analysis of MIP-1{alpha} mRNA expression induced by the interaction of SF leucocytes and RA FLS. SF monocytes (mono) or PMN were layered onto RA FLS monolayers. Total RNA was isolated 12 h later, after which Northern blotting was performed. (A) Representative Northern blots of steady-state MIP-1{alpha} mRNA and ß-actin mRNA. MIP-1{alpha} mRNA expression was quantitated and normalized to ß-actin as the MIP-1{alpha}/ß-actin mRNA ratio (B). Data are mean ± S.E.M. of three independent experiments which were performed using three different RA fibroblasts and three different RA SF leucocytes.

 
Involvement of integrin/ICAM-1 ligand interactions in the up-regulation of MIP-1{alpha} secretion by co-cultures
To determine the mechanism of induction of MIP-1{alpha} expression in co-cultures, we cultured the two cell groups together in a chamber, but separated by a transwell membrane (pore size 0.45 µm). The membrane allowed passage of soluble factors but prevented physical contact between the cell groups. As shown in Table 1, the synergistic augmentation of MIP-1{alpha} secretion was significantly blocked by the presence of the transwell membrane, except for supernatants from the monocyte side, suggesting that direct cell–cell contact is important for augmented MIP-1{alpha} secretion.


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TABLE 1. Effects of a transwell membrane filter on MIP-1{alpha} secretion

 
Because fibroblasts are known to interact with monocytes or PMN via pathways mediated by adhesion molecules, including the ICAM-1–integrin pathway [18, 24], we next investigated the potential role of these molecules in FLS–leucocyte interactions by assessing the capacity of specific Abs to inhibit MIP-1{alpha} production by co-cultured cells. Production of MIP-1{alpha} by FLS–monocyte and FLS–PMN co-cultures was reduced by 39.6, 54.3 and 36.0% and by 58.0, 71.3 and 69.8% following the separate addition of anti-CD11b, anti-CD18 and anti-ICAM-1 mAb (5 µg/ml) respectively (Fig. 5). Addition of each antibody to the cells cultured individually or of control (mouse) IgG to co-cultures had little effect on MIP-1{alpha} production.



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FIG. 5. Effects of anti-integrin and anti-adhesion molecule neutralizing mAbs on MIP-1{alpha} secretion. SF monocytes (mono) (A) or PMN (B) were layered onto RA FLS monolayers in 48-well plates, after which monoclonal antibodies (mAbs) (5 µg/ml) against CD11b, CD18 or ICAM-1 were added. After incubation for 24 h, the supernatants were harvested and assayed by ELISA. Each bar represents the mean (pg/ml) ± S.E.M. of three independent experiments performed using three different RA fibroblasts and three different RA SF leucocytes. *P < 0.05 vs the respective co-culture in the absence of mAb.

 
Effects of neutralizing antibodies against TNF-{alpha} on MIP-1{alpha} secretion by co-cultures
Inflammatory cytokines, including tumour necrosis factor {alpha} (TNF-{alpha}) and interleukin (IL) 1 are potent inducers of MIP-1{alpha}, and they can be induced by the interaction of fibroblasts and monocytes [25]. Therefore, to determine whether the augmented production of MIP-1{alpha} was regulated via TNF-{alpha} or IL-1 that was newly synthesized by in situ cell–cell interactions, we investigated the effects of TNF-{alpha} or IL-1ß neutralization by mAb (mAb against TNF-{alpha} was purchased from PeproTech EC and mAb against IL-1ß was kindly provided by Dr Y. Hirai, Immunological Products and Development, Otsuka Pharmaceutical, Tokushima, Japan) on MIP-1{alpha} induction. Neither neutralizing antibody influenced the MIP-1{alpha} concentration in the medium of FLS–leucocyte co-cultures [anti-TNF-{alpha} + FLS + monocytes, 117.7%; anti-TNF-{alpha} + FLS + PMN, 91.8%; anti-IL-1ß + FLS + monocytes, 112.3%, anti-IL-1ß + FLS + PMN, 110.1%, relative to cultures without either antibody (100%); n = 3].


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In the present study, we demonstrated that substantial amounts of MIP-1{alpha} are secreted by RA SF monocytes and, to a lesser extent, by RA SF PMN co-cultured with FLS. Our results suggest that cell–cell interactions occurring in RA synovitis may be an important process that induces MIP-1{alpha} expression. The synergistic augmentation of MIP-1{alpha} production was dependent on the interaction between synovial FLS and leucocytes. Indeed, the necessity for physical contact between the cells was clearly demonstrated in studies that showed complete inhibition of MIP-1{alpha} production in the presence of a transwell membrane that separated FLS from leucocytes. The pathway governing MIP-1{alpha} production by cell–cell contact was, to a large extent, promoted by a ß2-integrin/ICAM-1-mediated mechanism (Fig. 5).

MIP-1{alpha} expression and secretion are induced by stimulation of inflammatory cytokines, including IL-1 and TNF-{alpha}, and by lipopolysaccharide, interferon {gamma}, IL-17, hypoxia, ultraviolet radiation and infection with various viruses, which play a crucial role in the recruitment of macrophages and certain T lymphocytes during immune/inflammatory reactions [5, 2628]. In particular, several studies revealed the expression of MIP-1{alpha} and its importance in the synovitis of human RA and also collagen-induced arthritis, an experimental model of RA [3, 79]. Specific cell–cell interactions, especially between synovial cells and invading mononuclear cells, play a pivotal role during the progression of RA synovitis. Our results were in agreement with those of a previous study reported by Steinhauser et al. [29], who showed that MIP-1{alpha} expression was up-regulated by interaction of monocytes and pulmonary fibroblasts, which was mediated by an ICAM-1-integrin pathway. Taken together with our present data, the interaction of RA FLS and leucocytes (monocytes or PMN) plays an important role in the inflammation of RA synovitis, through MIP-1{alpha} expression via a ß2-integrin/ICAM-1-mediated mechanism. However, this pathway cannot solely account for MIP-1{alpha} induction, and it remains to be tested whether other adhesion molecules, such as ß1-integrin [30], or inflammatory mediators, such as reactive oxygen intermediates [29], are involved in the induction of MIP-1{alpha} mediated by the interaction of RA FLS and activated leucocytes.

Th1 cells and Th1-type cytokines have an important role in the development of progressive synovitis in RA [10, 31]. CCR5, a specific receptor for MIP-1{alpha}, is expressed preferentially in Th1 compared with Th2 cells [26, 32]. Indeed, CCR5-positive cells have been demonstrated in RA synovium [17], and the interaction of CCR5 with its ligand, including MIP-1{alpha}, is crucial for the development and severity of RA, on the basis of the recent demonstration that RA activity correlates with the polymorphism of CCR5 [15, 33, 34]. The findings of these early studies, together with our present data, support the hypothesis that MIP-1{alpha} secreted by activated SF leucocytes interacting with fibroblasts might contribute to the migration of Th1 cells through CCR5 in the development of RA. Furthermore, the present data clearly demonstrate that activated PMN interacting with fibroblasts are an important cellular source of MIP-1{alpha} in RA synovitis, because most of the leucocytes infiltrating the SF of rheumatoid joints are PMN. The PMN in RA SF are in an activated state, and produce a variety of inflammatory mediators [3537]. We have previously demonstrated that neutrophils are an important cellular source of MIP-1{alpha} [38], and that infiltrating activated neutrophils in the RA SF constitutively express MIP-1{alpha} [20]. Neutrophils, the biologically active leucocyte population, almost certainly contribute significantly to the disease process during active RA.

In conclusion, our data strongly suggest that infiltrating activated leucocytes play an important role in the synovial inflammation of RA by expressing and secreting MIP-1{alpha} upon interaction with synovial fibroblasts through a ß2-integrin/ICAM-1-mediated mechanism. Our results could potentially serve as the basis for a new therapeutic approach, targeting the interaction of these two major cell groups.


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Supplementary data are available at Rheumatology Online.


    Acknowledgments
 
We thank Dr Y. Hirai, Immunological Products and Development, Otsuka Pharmaceutical, Tokushima, Japan, for supplying monoclonal anti-human IL-1ß antibody. We also thank Mrs H. T. Takeuchi for the expert technical assistance. This study was supported in part by the Uehara Memorial Foundation and the High-Technology Research Centre Project (Ministry of Education, Science, Sport, and Culture of Japan).

Conflict of interest

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Supplementary data
 References
 

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Submitted 11 February 2003; Accepted 31 March 2003





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