Fibrin generated in the synovial fluid activates intimal cells from their apical surface: a sequential morphological study in antigen-induced arthritis

O. Sánchez-Pernaute, M. J. López-Armada, E. Calvo, I. Díez-Ortego, R. Largo, J. Egido and G. Herrero-Beaumont

Inflammation Research Unit, Division of Rheumatology, Fundación Jiménez Díaz, Universidad Autónoma, Madrid, Spain


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objective. Fibrin deposits adhered to the synovial surface are typical of rheumatoid joints. Since fibrin appears to have a role in arthritis perpetuation our aim was to investigate how these deposits are formed and the consequences of their adhesion to the tissue.

Methods. The appearance of fibrin aggregates either free in the synovial fluid or attached to the membrane was studied in rabbits with antigen-induced arthritis by histological techniques at different time points from challenge. In the fixed synovial membranes areas of fibrin-bound synovium were evaluated by qualitative variables to obtain a sequential profile of morphological changes.

Results. Fibrin aggregates appeared from the initial stages of the disease in the synovial effusion. Later on, they were localized on the synovial surface and progressive changes were noted at the fibrin–tissue interface, ending with the invasion of the aggregates by synovial cells and their incorporation into the tissue.

Conclusion. Fibrin aggregates generated inside the joint cavity may constitute a source of activation and acquisition of invasiveness of the synovial fibroblasts, a process to explore within the perpetuating mechanisms of rheumatoid arthritis.

KEY WORDS: Synovial membrane, Adhesion, Fibrin, Fibronectin, Fibroblast migration, Arthritis.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Fibrin is a well recognized inductor of synovial cell activation [1] and one of the extracellular matrix proteins participating in synovial tissue remodelling in rheumatoid arthritis (RA) [2]. It has actually been proposed as a perpetuating agent of synovitis [3]. In inflammatory disorders, extravascular activation of the clotting system leads to the formation of a plug that substitutes the original matrix [4]. There are several ways in which this process may contribute to perpetuate inflammation [5, 6]. In brief, the plug provokes ischaemia at the injured area and provides a provisional network for cell migration. Its organization is associated not only with development of fibrosis but also with activation of proteases in cascade. In turn, cleavage of coagulation proteins favours the generation of neoepitopes [7, 8] as well as release of growth factors [9], and many resident and invading cells have receptors for molecules associated with the clot [1012].

In RA there is an excessive local generation of fibrin [5, 13, 14]. The influx of great amounts of fibrinogen to the joint and local up-regulation of coagulation proteins is not paralleled by a similar rate of fibrinolytic activity, resulting in intra-articular fibrin polymerization [6, 15]. As a result, widespread fibrin deposits are found in the inflamed joints of patients, not only at the synovial interstitium but predominately on the synovial surface [16]. The origin of these deposits stuck to the tissue is controversial. It has been proposed that they represent degradation products resulting from necrosis and are discarded from the synovium into the cavity. Alternatively, they could arise inside the cavity, since fibrin polymerization also takes place within the rheumatoid synovial fluid during inflammatory flares [15]. Should fibrin aggregates generated inside the cavity come into contact with the lining layer, they could constitute a source of synovial cell activation different from the well-known mechanisms taking place at the synovial interstitium. In this regard it has been shown that local implantation of fibrin within healthy animal joints induces synovial inflammatory changes [16]. With regard to RA, early morphological studies suggested the tendency of the synovial membrane to invade attached fibrin aggregates [17, 18] and associated this process with hyperplasic villi growth [19]. However, the unavailability of sequential studies in rheumatoid joints has hampered confirmation of these theories. As a result, interaction of intra-cavitary fibrin polymers with the synovium has not been explored as a potential pathophysiological process in RA.

Antigen-induced arthritis (AIA) is an experimental model of RA also characterized by the local generation of fibrin [20]. As happens in RA, there is an imbalance between fibrin generation and degradation in AIA with implications for arthritis perpetuation [21, 22]. The model, therefore, provides an ideal setting to study the formation of intra-cavitary fibrin aggregates and their possible interaction with the synovial tissue. We have carried out a sequential follow-up of this experimental model looking into the appearance of fibrin aggregates and their relationship with the lining layer over time.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antigen-induced arthritis (AIA) in the rabbit knee
The experimental disease was induced as previously published [23]. The animals underwent two intra-dermal immunizations with 5 mg/ml ovalbumin (Sigma, St Louis, MO) in Freund's complete adjuvant (Difco, Detroit, MI) separated by 14 days. Five days after the second immunization 1 ml of a sterile solution of 5 mg/ml ovalbumin was injected into the right knee of the rabbits. A group of six animals was killed at each of the following times after challenge: 24 h, 36 h, 48 h, 72 h, 96 h and 1 week, for evaluation of the joints. All procedures were performed strictly in accordance with current local regulations.

Histological studies and analysis of the events
At the time of death, both synovial fluid (SF) and membrane from the arthritic joints were obtained to examine the occurrence of microscopic fibrinous aggregates. Synovial fluid was aspirated and smears were prepared on glass slides, air dried and fixed with cold acetone. The infra-patellar synovium was dissected, washed and fixed as previously described [23]. Dehydrated tissues were embedded in paraffin, serially cut at 7 µm and mounted on poly-L-lysine precoated glass slides. Morphological evaluation was carried out on 12 slides per animal (each fifth to a total width of 420 µm) after haematoxylin and eosin (HE) staining. On each slide the number of fibrin aggregates stuck to the luminal side of the intima was counted. When an adhered aggregate was identified, adjacent slides were explored to obtain a three-dimensional approximation of the entire area of contact. Morphological features were evaluated by categorical variables and their occurrence or absence in each aggregate was expressed as a percentage with regard to the number of adhered aggregates. Variations between percentages were studied over time.

Immunohistochemistry
Tissue samples were rehydrated and incubated with the antibodies listed in Table 1Go in the conditions described there. As previously reported [24], endogenous peroxidase was inhibited with 1% hydrogen peroxide-methanol and enzymatic digestion was performed for 30 min at 37°C in a humid chamber with 0.1% trypsin or 0.5% pepsin. Incubation with a blocking solution (6% bovine serum albumin in phosphate-buffered saline with 3% secondary host serum) preceded the addition of specific antibodies. Colour was developed with diaminobenzidine and tissues were counterstained with haematoxylin, dehydrated and mounted in DPX medium. Controls were performed with pre-immune IgG.


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TABLE 1. Host, specificity, source and dilution of the antibodies employed in the study

 
Synovial fluid smears were stained with the anti-fibrinogen antibody diluted 1:1000 in buffered medium and examined under fluorescence microscopy. A blind, semi-quantitative 0 to 3 point scale scoring was employed to assess the abundance of the aggregates in the smears.

Statistics
Data were expressed as mean±S.E. Time-dependent analysis of the data was carried out with Kruskal–Wallis and Mann–Whitney non-parametric tests, employing SPSS 8.0 software. Linear regression analyses were performed using the least squares method. Differences between groups were considered to be significant where P < 0.05.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Fibrin aggregates appeared in the SF in the initial stages of AIA and adhered to the tissue subsequently (Fig. 1Go)
Although fibrin deposits have been described in AIA, there is little information regarding how they are produced or when they appear. In our study, a sequential follow-up from the initial stages of inflammation was designed to look for fibrin aggregates appearing at the SF or attached to the synovial membrane. By immunofluorescence, fibrin aggregates were observed in the smears from SF as bright networks with trapped leucocytes (Fig. 1AGo). The fixed synovial membranes were stained and the presence of fibrin deposits on the luminal side of the intima was evaluated. The deposits were identified by their typical morphological aspect, clearly differing from the synovial interstitium underneath [4, 25] (Fig. 1BGo). Immunofluorescence to fibrinogen confirmed the nature of the attached aggregates (Fig. 1CGo). Although the inflamed synovial tissue had perivascular and subintimal collections which bound the anti-fibrinogen antibody, the tissue was less immunoreactive than the adhered aggregates, and there was a sharp limit between them.



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FIG. 1. Characterization of fibrin aggregates in antigen-induced arthritis. (A) A photomicrograph of fibrin aggregates in a synovial fluid (SF) smear after direct immunofluorescence to fibrinogen (x100). (B) A synovial membrane stained with HE showing an amorphous eosinophilic mass bound to the synovial intima (x200). Immunofluorescence to fibrinogen shows in (C) an aggregate adhered to a synovial fold (x200). The graph (D) shows the variation on time (x axis) of SF volume (principal y axis), semi-quantitative scoring of free aggregates, and average number of adhered aggregates per tissue (secondary y axis). Both SF volume and SF aggregates decreased in a time-dependent fashion: R2=0.67, P<0.001; R'2=0.53; P'<0.001. Adhesion of aggregates to the intimal layer was a rare event at 24 h, and consistently found at every other point of evaluation (*P < 0.05; **P<0.01), peaking at 48 h and declining at 1 week (#P<0.05 vs 48 h).

 
As expected, there was a time-dependent diminution in the volume of SF from the arthritic joints: R2=0.67, P<0.001. A synovial effusion was present in 100% of rabbits up to 72 h after challenge. At this time, the amount of SF had begun to descend (200±60 µl vs 370±40 µl at 24 h) and after longer periods only a few drops could be aspirated from the joints. The number of fibrin aggregates observed in the SF smears was scored on a semi-quantitative 0 to 3 point scale, and found to be higher at the beginning of the disease (R2=0.53, P<0.001). From 2.6±0.4 at 24 h, the score fell to 1.2±0.5 at 72 h (P<0.05) and continued to decrease over time. Synovial fluid from animals with arthritis of 72-h to 1-week duration were usually acellular and did not show fibrinogen immunoreactivity.

In the fixed synovial membranes attached aggregates were counted as described in the Methods section. They were scarcely detected at 24 h, their frequency increasing between 48 h (P<0.05) and 72 h (P<0.05). At 1 week after challenge, fibrin polymers adhered to the tissue were still identified, but in a lower proportion (P<0.05 vs 48 h). In summary, as shown in Fig. 1DGo, aggregates were first detected in the smears and as they tended to disappear from the SF, deposits of fibrin were found attached to the tissue. The delay in the peak of ‘adhered’ with regard to ‘free, unbound’ fibrin aggregates suggested that they were generated from the SF and subsequently came into contact with the synovial membrane.

A casual contact of the aggregates with the synovial barrier might be followed by detachment or could, on the other hand, precede a firm adhesion through binding of receptors at the surface of synoviocytes. To study this possibility we examined the attached aggregates for fibronectin using immunoperoxidase techniques. Fibronectin is an adhesive protein known to cross-link with intra- and extravascular fibrin matrices [4] and its presence within the aggregates would provide binding sites for integrins. As became evident, fibronectin was a major component of all the aggregates adhered to the synovial intima, and it was particularly highly expressed at the areas of interface with the membrane (Fig. 2A, BGo). We also found that synovial cells from the intima were rich in the integrin subunit {alpha}5, which participates in the specific binding of certain domains of fibronectin [26] (Fig. 2C, DGo). This potential connection, and probably other binding pairs, would enable a firm attachment once the fibrin matrix was in contact with the tissue.



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FIG. 2. Participation of fibronectin (FN) in the anchorage of the aggregates to the synovial membrane. (A) FN immunoperoxidase of an inflamed synovial membrane (x200) in which several synovial villi are observed (unfilled stars). An aggregate is found adhered to the lining layer. The interface area (arrows) is strongly positive while inside the aggregate there is also immunoreactivity to FN, distributed in patches. (B) shows a x1000 magnification of the lining cells from (A) forming a row between fibrin deposit and synovium. (C) Immunoperoxidase to the {alpha}5 integrin subunit in a rabbit with AIA (x150); despite the strong infiltration by mononuclear cells, immunoreactivity to the FN-binding integrin is selectively restricted to the lining layer and to vessel walls. (D) shows a negative control of (C) in which the tissue was incubated with pre-immune IgG.

 

Interaction of fibrin aggregates with the underlying synovial tissue
Yet the deposits observed at the synovial surface could represent fibrin generated both in the effusion and inside the tissue. To ascertain their origin we examined the changes at the fibrin–synovium interface over time; in this way, we also explore the consequences derived from the attachment of fibrin aggregates to the underlying synovial cells.

We studied the areas of contact between synovium and fibrin at the four time points at which a higher number of adhered aggregates was found (36, 48, 72 and 96 h). At the interface we examined the integrity of the intimal layer and the development of abnormalities in the normal organization of synovial cells (Fig. 3Go). We could distinguish two kinds of alteration in the synovial fibroblasts separating the matrices. One was disarrangement of the lining layer, as defined by a loss of the normal bipolar shape of cells, which developed cytoplasmic projections and changed orientation (Fig. 3AGo). The other one was an increased cellularity at the area of contact, often associated with active mitosis (Fig. 3BGo). When these alterations were prominent, parallel immunostaining of fibrin and fibronectin was key in distinguishing the precise limit between synovium and aggregates, as illustrated in Fig. 3B and CGo. The percentage of aggregates showing interface alterations was recorded and their variation in time was studied (Fig. 3DGo). At the earlier time points, an interface between the synovial tissue and the fibrin mesh was clearly distinguished in 86, 76 and 59% of cases, at 36, 48 and 72 h, respectively, decaying to 18% at 96 h (P<0.01 vs 36 h, P<0.005 vs 48 h, P<0.05 vs 72 h). Changes in the normal bipolar morphology of intimal cells were found in approximately 20% of the cases at 36 and 48 h, and significantly increased at 72 h (76% cases; P<0.05 vs 36 h, P<0.01 vs 48 h) and at 96 h (66%). The separating lining consisted of a monolayer of cells in 95% of cases at 36 h, while it appeared hypercellular and composed of multiple rows in 42% of cases at 48 h (P<0.05 vs 36 h) and in 58% at 72 h (P<0.05 vs 36 h).



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FIG. 3. Cell changes detected at the interface area. Alteration of cell shape and disarrangement of intimal cells is shown in (A); note that synoviocytes under the deposit have changed orientation, developed cytoplasmic projections and lost cell–cell contacts (x400). (B) shows a group of aggregates in contact with a synovial membrane. There is an increased cellularity at the separating lining (x100). In this case, the parallel staining for FN in (C) helps to distinguish the edge between the two matrices. (D) The distribution over time of these morphological findings is shown in the graph: the synovial intima was perfectly formed in most cases at the initial periods and suffered a progressive distortion, being difficult to identify at 96 h (unfilled circles); in turn, disarrangement and hypercellularity significantly increased at 72 h with regard to early stages, reflecting activation of lining fibroblasts located under the fibrin deposits. *P < 0.05; **P < 0.01; #P < 0.005.

 

Invasion of fibrin aggregates by synovial fibroblasts
The phenotypic alterations shown by lining fibroblasts following the anchorage of fibrin aggregates suggested a process of fibroblast activation induced by fibrin. Additionally, fibronectin is known to promote migration of fibroblasts through fibrin matrices [27], and this protein was found to be widespread within the aggregates. These findings led us to explore whether there could be a guided invasion of the fibrin deposits by synovial fibroblasts. We examined the appearance of cells inside (Fig. 4AGo) and at the surface of the aggregates (Fig. 4B, CGo) at the different periods of evaluation. Cells were found embedded inside 50% of the aggregates at the initial periods, the percentage increasing to 83% at 96 h (P<0.05). Time-dependent changes in cell distribution at the surface of aggregates were also detected. At the earlier phases, fibrin matrices tended to have nude surfaces, in some instances showing a row of cells next to the margins connecting with the synovial intima. The percentage of aggregates showing this border cellularity increased from 38% at 36 h to 90% at 96 h (P<0.05 vs 36 h, P<0.05 vs 48 h). A total encircling of the aggregate by a layer of cells was seen in some cases (Fig. 4D, EGo). This was a late event found in 70% of cases at 96 h (P<0.01 vs 36 h, P<0.01 vs 48 h, P<0.01 vs 72 h). Encircling cells were morphologically similar to fibroblast-like synoviocytes and they expressed markers of this cell population, such as CD55 (Fig. 4FGo) and ß-actin (not shown) [28]. On the whole, these data suggested that a progressive advance of lining fibroblasts took place after the adhesion of fibrin, ending with the complete incorporation of the aggregates within the tissue by development of a new lining layer at their surface.



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FIG. 4. Cell distribution within the fibrin matrices. (A) Fibroblast-like cells were frequently found inside the aggregates, as shown in this photomicrograph (x400). (B) shows an aggregate attached to an inflamed tissue (x200). At the base of the aggregate, the synovial lining layer (arrow heads) and sublining signs of inflammation are seen. There are superficial cells laid on the aggregate at the margin of contact with the membrane. (C) Observe, in a x1000 magnification of the edge, the row of cells covering the characteristic reticular fibrin matrix. (D) shows a case with severe inflammation, consisting of intimal hyperplasia, vessel congestion, and subintimal infiltrates of mononuclear cells (x100). At the base of the synovial fold there is a rounded aggregate partially sunk into the hyperplasic intima. (E) In a x1000 magnification, cells similar to the lining synoviocytes appear inside and also encircling the matrix, which looks near to complete epithelization. (F) Immunodetection of CD55 shows expression of this surface molecule by the cells epithelizing an aggregate (x100).

 


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
There is a tendency to consider the joint cavity as a site to exude degradation products and dead cells, but it can potentially be a source of synovial cell activation. Actually, synovial intima is a thin layer separating an interstitium from a fluid-bathed cavity, and exposes a large surface susceptible to receive information from the cavity through membrane receptors. To maintain the barrier, cells are necessarily polarized, that is adhesion molecules and other signalling transducer receptors are redistributed to the apical membrane of the cell, while the basolateral membrane concentrates cytoskeletal elements to enable attachment to the substratum and sealing of the surface [29]. This conformation is particularly important in the synovium, since this epithelium-like tissue lacks tight cell–cell junctions as well as a basal membrane. Although normally strongly attached to the synovial interstitium by their fibrillar organization, we should consider the synovial lining cells as potentially entirely free for migration, shape or architecture changes and proliferation.

In this study we show that fibrin aggregates generated inside the joint cavity during inflammatory flares interact with and activate synovial cells. At the initial stages of antigen-induced arthritis, development of fibrin aggregates was a common feature. Synovial fluid was rapidly drained but the aggregates remained, our data showing that contact between aggregates and synovial membrane then took place, being maximal at 48–72 h after challenge. We could establish a dynamic pathophysiological process at the areas of contact between fibrin aggregates and synovial cells, concluding with the incorporation of fibrin into the synovial membrane. In brief, this process would involve proliferation and migration of the intimal fibroblasts, as summarized in Fig. 5Go.



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FIG. 5. Scheme of the incorporation of luminal fibrin into the inflamed tissue in antigen-induced arthritis. (A) Arising of fibrin aggregates inside the joint cavity: at the beginning of the disease, the incoming of fibrinogen and other necessary elements to activate haemostasis leads to formation of fibrin moulds in the synovial fluid. (B) Adhesion phase: casual contact of fibrin polymers with the synovial membrane, probably facilitated by clearance of the effusion, is followed by a firm attachment due to the existence of ligands to extracellular matrix elements at the apical surface of the synovial cells. Fibrin aggregates are thus transformed in deposits stuck to the intimal layer. (C) Lining disarrangement: with the binding of matrix receptors at the free surface of the epithelial layer, the typical bipolar conformation of the cell is lost. Synoviocytes change shape and prepare to migrate, showing at the same time frequent mitotic activity and increasing rows at the interface. (D) Migration: two simultaneous processes then seem to account for a guided invasion of the fibrin deposit—scatter through the matrix and marginal migration. Scatter is typical of cells performing an invasive growth programme, and although not studied in this work, it most probably requires secretion of metalloproteinases. (E) Encircling and epithelization of the aggregates: this step results from fibroblast migration and concludes with sealing of the surface. (F) Integration of fibrin aggregates into the inflamed tissue: the sequence probably continues with remodelling of the fibrin matrix inside the synovial membrane due to circulation of cells and digestion of the participants in fibrin clotting, a process on its own known to account for perpetuation of inflammation in RA (see Introduction).

 
Our study highlights the role of fibronectin in fibrin–synovium interaction. This protein not only participates in the anchorage to the synovium but also promotes disarrangement and invasiveness of fibroblasts, as has been shown in wounds and fibrin clots [27]. The interaction of fibronectin with its cell receptor, as well as the coupling of other peptides accompanying fibrin matrices, are known to activate the target cell [1, 12, 30]. Type III collagen, which was also found in the fibrin aggregates (data not shown), could be relevant in offering binding sites for adhesion molecules, disruption of intercellular contacts and loss of polarization of fibroblasts, as has been shown elsewhere [31].

Formation of fibrin aggregates in an inflamed joint cavity is probably non-specific but common to inflammatory reactions. None the less, as happens with other homeostatic pathways, activation of the clotting system during inflammation has to be strongly regulated. It is likely that formation and degradation of fibrin take place in balance in most joint pathologies and, as a result, attachment of the aggregates to the synovial intima does not acquire relevance. We propose that in RA, as happens in AIA, synovial cells may invade these aggregates. Indeed, some of the features of the rheumatoid joint, such as areas of denuded tissue recovered by fibrinoid necrosis or by fibrin–fibronectin polymers, could represent different stages of the phenomenon we describe [32]. Unlike what happens in AIA, rheumatoid joints continue to show episodes of swelling and effusion with disease progression, so this phenomenon could appear not only at the initial stages but at any time during the course of the disease.

In summary, besides the classic pathways of activation of synovial cells, we have shown in this work how cells can also be stimulated from events taking place at the joint lumen and become involved in a process of remodelling very similar to the processes of wound healing and vessel neointimal formation. Our work suggests that luminal fibrin provides an active front of invasive transformation for lining synoviocytes, most likely involved in perpetuation of synovitis and therefore a potential therapeutic target in RA.


    Acknowledgments
 
The authors would like to thank Dr Ferdinand Breedveld for his interest in our data and his advice and Dr Rosario Sánchez-Pernaute for editorial assistance.


    Notes
 
Correspondence to: O. Sánchez-Pernaute, Fundación Jiménez Díaz, Av. Reyes Católicos, 2, 28040 Madrid, Spain. E-mail: Osanchez{at}fjd.es Back


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Submitted 25 October 2001; Accepted 16 May 2002