Platelet–endothelial cell interactions in murine antigen-induced arthritis

M. Schmitt-Sody1, A. Klose2, O. Gottschalk2, P. Metz2, H. Gebhard2, S. Zysk1, M. E. Eichhorn3, T. M. Hernandez-Richter3, V. Jansson1 and A. Veihelmann1,2

1 Department of Orthopedics, 2 Institute for Surgical Research and 3 Department of Surgery, Ludwig Maximilians University of Munich, Germany.

Correspondence to: M. Schmitt-Sody, Department of Orthopedics, Klinikum Großhadern, Marchioninistr. 15, Ludwig-Maximilians-University, 81377 Munich, Germany. E-mail: marcus.schmitt-sody{at}med.uni-muenchen.de


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objectives. Growing evidence supports the substantial pathophysiological impact of platelets on the development of rheumatoid arthritis. At present there are no methods for studying these cellular mechanisms in vivo. The aim of this study was to visualize and investigate platelet–endothelial cell interaction in the knee joint of mice with antigen-induced arthritis (AiA) by means of intravital microscopy.

Methods. In 14 mice (Balbc) intravital microscopic assessment was performed on day 8 after AiA induction in two groups (controls, AiA). The severity of AiA was assessed by measuring knee joint swelling and by histological scoring. Ex vivo fluorescently labelled rolling and adherent platelets and leucocyte–endothelium interactions were investigated by intravital fluorescence microscopy.

Results. Swelling of the knee joint as well as histological score was significantly enhanced in arthritic animals compared with controls. In control mice intravital microscopy revealed low baseline rolling and sticking of leucocytes and fluorescently labelled platelets. AiA induced a significant increase in the fraction of rolling leucocytes (3 times) and rolling platelets (6 times) compared to the control group. Furthermore, AiA induction resulted in a significantly enhanced number of adherent leucocytes (3-fold) and adherent platelets (12-fold) in comparison with control animals.

Conclusions. Platelet kinetics were directly analysed using intravital microscopy in the arthritic microcirculation in vivo for the first time. We provide the first evidence that platelets accumulate in arthritic vessels, indicating platelet activation due to AiA. Platelet recruitment and subsequent activation might play an important role in the pathogenesis of rheumatoid arthritis.

KEY WORDS: Platelets, Arthritis, Mouse, Intravital microscopy


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In rheumatoid arthritis (RA), the vascular endothelium is among the key targets for circulating mediators of inflammation and controls the trafficking of cells and molecules from the bloodstream towards the synovial tissue. Local blood vessel proliferation allows the pannus to develop and grow, thereby promoting destruction of cartilage and adjacent bone and remodelling of the joint, leading to severe pain and impairment of mobility. These changes are accompanied by alterations in the synovial microcirculation [1]. Whereas many of the mechanisms underlying the induced inflammatory response remain unknown, growing evidence suggests that platelets may also be involved in the inflammatory process of rheumatoid disease.

Platelets possess a cellular machinery which is in many aspects comparable to that of leucocytes. After activation they produce oxygen radicals and release pro-inflammatory mediators such as thromboxane A2, leucotrienes, serotonin, platelet factor 4 and platelet-derived growth factor [2, 3]. Furthermore, platelets have the potential to modulate leucocyte functional response [4]. Hence, the activation and adhesion of platelets to endothelial cells might contribute to further leucocyte activation and recruitment and aggravate endothelial cell damage at the site of inflammation [5]. It has been shown that secretory phospholipase A2 (PLA2) activity is significantly increased in the synovium and serum, corresponding to disease activity in patients with RA [6]. Platelet-activating factor is present in the synovial fluid of RA patients, and the administration of a platelet-activating factor antagonist significantly reduces inflammation [7]. Thus, PLA2 and platelet-activating factor are associated with RA as well as with platelet activation. However, the exact role of platelets in the pathogenesis of arthritis and the molecular mechanisms whereby platelets accumulate in synovial tissues have not been identified so far.

In our recently published study intravital microscopy was successfully used to quantify the leucocyte–endothelial cell interaction as well as angiogenesis in mouse antigen-induced arthritis (AiA) [8, 9]. AiA is an established animal model for the study of human RA [10]. The main advantage of AiA in comparison with other established animal models is that arthritis is induced in the knee joint which lends itself to intravital microscopy without inducing major trauma. No studies have so far been reported which directly demonstrate the in vivo characterization of platelet–endothelial cell interaction in the synovial microcirculation. Therefore, in the present study it was our aim to visualize and to quantify interactions of platelets with the endothelial lining under physiological conditions and to assess whether platelet interactions are altered in the knee joint of mice during AiA in vivo using our recently developed model for intravital microscopic analysis of the mouse synovial microvasculature [11].


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
Female inbred Balb/c mice (Charles River, Sulzfeld, Germany) weighing 18–21 g were used for the experiments. The experiments were approved and performed according to the German legislation for the protection of animals. Mice were randomly assigned to either the control group (n = 7) or the AiA group (n = 7). To assess the severity of AiA, joint swelling was determined daily until day 8 by measuring the transverse diameter of the knee joint, and histological sections were performed using a standard scoring protocol introduced by Brackertz et al. [10]. Intravital microscopy as well as tissue sampling were performed on day 8 after induction of AiA in both groups.

Antigen-induced arthritis (AiA)
On days –21 and –14 prior to the induction of arthritis mice were immunized by a subcutaneous injection in the left flank of 100 mg of methylated bovine serum albumin (mBSA) (Sigma, Deisenhofen, Germany), dissolved in 50 ml of Freund's complete adjuvant (Sigma) and supplemented with 2 mg/ml heat-killed Myobacterium tuberculosis strain H37RA (Difco, Augsburg, Germany) and an additional intraperitoneal injection of 2 x 109 heat-killed Bordetella pertussis (Institute of Microbiology, Berlin, Germany) as previously described by Brackertz et al. [10]. Finally, arthritis was induced on day 0 by injection of 100 mg mBSA in 50 ml saline into the left knee joint. Control animals underwent the same procedure but received equivalent volume of saline in the knee joint.

Surgical preparation
Mice were anaesthetized by inhalation of isoflurane 1.5% (Forence, Abbott, Wiesbaden, Germany) and a mixture of O2/N2O. Arterial and venous catheters were implanted into the tail. Through a 1 cm incision distal to the patellar tendon, the tendon was cut transversally and elevated to provide visual access to the intraarticular synovial tissue as described elsewhere [11]. At the end of the experiment, animals were killed with an intravenous injection of 10 mg pentobarbital (Nembutal, Sanofi, Hannover, Germany). The knee joints were then removed for histology.

Blood sampling and platelet preparation
For intravital fluorescence microscopy, separation and ex vivo fluorescent labelling of platelets was based on a previously described protocol [12]. One ml of blood from a syngenic donor mouse was harvested by cardiac puncture and collected in polypropylene tubes containing 0.2 ml volume of 38 mmol/l citric acid/75 mmol/l trisodium citrate/100 mmol/l dextrose, 15 µl prostaglandin E1 (PGE1) (Sigma, Taufkirchen, Germany) and 0.5 ml Dulbecco phosphate-buffered saline (D-PBS) (PAN Systems, Aidenbach, Germany). After centrifugation platelet-rich plasma was gently transferred to a fresh tube containing 1.5 ml D-PBS, 0.4 ml volume of 38 mmol/l citric acid/75 mmol/l trisodium citrate/100 mmol/l dextrose and 50 µl PGE1. The fluorescent marker aminoreactive succinimidylester carboxyfluorescein-diacetate (CFDA-SE) (MW 535, Molecular Probes, Eugene, OR) was added. The centrifuged platelet pellet was resuspended in 0.3 ml D-PBS. The purity of the sample and platelet concentration were determined before infusion using a Coulter AcT Counter (Coulter Corp, Miami, FL). A total of 100 x 106 fluorescently labelled platelets were transfused via the lateral tail vein, corresponding to approximately 10% of all circulating platelets [13]. Adequate functionality of fluorescently labelled platelets has been evaluated by flow cytometric analysis (FACS) (FACSort flow cytometer; Becton Dickinson, Heidelberg, Germany) before and following in vitro activation by phorbol-myristrate-acetate (PMA), (Sigma, Taufkirchen, Germany).

Intravital fluorescence microscopy
The microscopic set-up has been described in detail previously [14]. CFDA-labelled platelets were visualized after intravenous injection using a variable 12 V, 100 W halogen light source. For in vivo labelling of the leucocytes, the fluorescent marker rhodamine 6G (Sigma) was injected intravenously as a single bolus of 0.15 mg/kg immediately prior to measurement. Rhodamine epillumination was achieved using a 150 W variable HBO mercury lamp. The use of different fluorescence filter sets allowed selective visualization of either CFDA-labelled platelets or leucocytes labelled by rhodamine 6G. Microscopic images were acquired and recorded on videotape. Data analysis was performed off-line using a computer-assisted analysis system (CAP-Image, Dr Zeintl, Heidelberg, Germany) [15].

Microcirculatory parameters
Platelets and leucocytes were classified according to their interaction with the endothelial cell lining as free-flowing, rolling or adherent cells. Rolling platelets or leucocytes were defined as cells crossing an imaginary perpendicular through the vessel under study at a velocity significantly lower then the centreline velocity in the microvessel. They were determined as the fraction of all platelets and leucocytes passing a pre-defined vessel segment within an observation interval of 30 s. Adherent cells were defined as cells that did not move or detach from the endothelial lining in each vessel segment within an observation period of 30 s. Adherence was quantified as number of cells per square millimetre of endothelial surface, calculated from the diameter and length of the vessel segment observed (1/mm2).

Statistical analysis
The data are expressed as mean ± S.E.M. Statistical significance was tested using a rank sum test (Mann–Whitney U-test) and a repeated measurements ANOVA on ranks (Friedman's test). P values <0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Significant swelling of the knee joint was observed from day 1 until day 8 in mice with AiA compared with the measurements in control joints. The histological score showed two or more synovial lining cells and perivascular infiltrates of leucocytes as well as hyperplasia of the synovium and pannus formation, indicating that an arthritis had been induced in comparison with the control group. No significant change of the mean arterial blood pressure occurred during the entire course of the experiment in either group. There was no significant loss of body weight in the AiA group in comparison with the control group, indicating no severe systemic reaction (data not shown).

Leucocyte–endothelial cell interaction in vivo
Rolling and adhesion of leucocytes were only rarely observed in vessels of the normal synovial tissue. In contrast, there was a significant increase in the fraction of rolling leucocytes 8 days after AiA induction (0.344 ± 0.014) compared with the control group (0.112 ± 0.007). Furthermore, the number of adherent leucocytes increased significantly (1434.8 ± 243.2/mm2) compared with that of the control animals (400.2 ± 50.1/mm2) indicating activation of leucocytes and/or endothelial cells due to AiA.

Platelet–endothelial cell interaction in vivo
Platelet interaction with the microvascular endothelium demonstrated striking parallels to leucocyte–endothelial cell interactions, but to a much greater extent. Typical intravital microscopic images of CFDA-labelled platelets in a control mouse and in mice with AiA are shown in Fig. 1. Similar to leucocytes, platelets rarely interacted with the microvascular endothelium in normal synovial tissue of control animals. In contrast, as shown in Fig. 2, AiA induced a significant increase in the fraction of rolling platelets (0.36 ± 0.01) compared with the control group (0.062 ± 0.009). In parallel, AiA induction resulted in a significantly (more than 12-fold) enhanced number of adherent platelets (1813.2 ± 431.7/mm2) at day 8 in comparison with the control animals (143.6 ± 28.7/mm2). Platelet aggregation and thrombus formation have also been observed.



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FIG. 1. Visualization of CFDA-labelled platelets within the arthritic microcirculation in vivo by intravital fluorescence microscopy (magnification x432). Platelet–endothelial cell interaction in a high-density capillary network of mice with antigen induced arthritis (A–C) and in vessels of the control group (D).

 


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FIG. 2. Fraction of rolling platelets (given as the number of rolling platelets over the sum of the rolling and non-adherent platelets) (A) and number of platelets adherent (per mm2) (B) to the endothelium in post-capillary venules in the synovium of the mouse knee joint with AiA in comparison with controls. *P < 0.05 versus control groups. Data are given as mean ± S.E.M. (n = 7). Statistical significance was tested using a rank sum test.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Whereas several distinct adhesive interactions involved in the process of arthritis-induced leucocyte accumulation have been described [8], the mechanisms that initiate platelet accumulation in the synovial microvasculature have not been identified to date. The current study, therefore, has visualized and investigated for the first time in an animal model of arthritis in vivo the interactions of platelets with the microvascular endothelium using intravital fluorescence microscopy.

The model presented is the first to enable simultaneous quantification of macrohaemodynamics, microhaemodynamics and leucocyte and platelet kinetics in the synovial microcirculature. Since no fluorescent dye may exclusively stain platelets after in vivo injection, we have chosen an ex vivo labelling method for platelets which has been proven to be valid and useful for investigations of platelet kinetics and mechanisms of platelet–endothelial cell interaction in tumour angiogenesis [12]. The influence of the separation and labelling procedure on platelet functionality was examined by FACS analysis following stimulation by PMA. FACS analysis demonstrated that platelets were not activated due to the separation and labelling procedure, and fluorescently labelled platelets still possessed the ability to form aggregates.

We observed that arthritis induced transient interaction, rolling and permanent adhesion of platelets to the arthritic endothelium. In normal synovial vessels intravital microscopy revealed low baseline rolling and sticking of fluorescently labelled platelets along the endothelial lining. Within the arthritic microvasculature we observed a 6-fold enhanced number of rolling platelets and a more than 12-fold enhanced number of adherent platelets, whereas the number of rolling and adherent leucocytes increased only 3 times after induction of AiA.

Platelets are the first and most numerous cellular corpuscles that accumulate at any site of damage to the vascular endothelium. Upon stimulation, platelets express receptors for adhesive proteins, they generate reactive oxygen species and nitric oxide as well as release pro-inflammatory mediators, growth factors, cytokines and cytotoxic proteases [2, 16]. Thereby, platelets can initiate and regulate the processes of inflammation, host defence and tissue repair. They are integrated in a complex regulatory network which exists between leucocytes, endothelial cells, smooth muscle cells and fibroblasts in the course of inflammation [17]. Many inflammatory diseases are characterized by signs of platelet activation, but it seems difficult to establish a causal relationship between platelet activation and inflammation in most cases. However, irrespective of the primary events that induce the inflammatory process, several facts suggest that platelets contribute to the severity of diseases like inflammatory bowel disease, asthma, atherosclerosis and RA [18–20].

The presence of intense platelet–endothelial cell interaction and platelet aggregation, as we have shown, suggests that the luminal surface of arthritic microvessels provides enhanced adhesive structures for platelets and platelets become activated within the microcirculation of arthritic synovial tissue. Firm adherence of circulating leucocytes to inflamed vascular endothelium is an essential component of a cascade that results in the adhesion and eventual emigration of leucocytes through the vessel wall. The primary molecules responsible for initiation of leucocyte and platelet adhesion, von Willebrand factor and P-selectin, respectively, are stored in the same storage granules—Weibel–Palade bodies [21, 22]—indicating that platelets might contribute significantly to arthritis-induced inflammatory responses. By adhesion of platelets to endothelial cells or subendothelial structures, they might occupy a position analogous to endothelium with respect to leucocyte accumulation and emigration.

Treatment of human RA with platelet-activating factor antagonist BN 50730 significantly improves clinical indicators of disease activity during the treatment period, with a progressive return to baseline values during the follow-up period [7]. Platelet-derived microparticles are significantly increased in patients with RA compared with healthy controls and correlate with disease activity [23]. Furthermore a significant increase in platelet–leucocyte complexes was found in RA patients compared with the control group [24]. These results confirm the hypothesis that platelets are involved in the inflammatory process of RA. However, investigations rest on histological and clinical parameters such as joint swelling or systemic markers of joint inflammation, such as CRP. The assessment of platelet accumulation in the synovial tissue ex vivo does not provide information about the dynamic character of platelet–endothelial cell interaction.

In conclusion, our present study has shown that ex vivo labelling and recirculation of autologous platelets enables us to investigate platelet dynamics on the synovial microcirculation by means of intravital microscopy. We have demonstrated that platelets, similarly to leucocytes, roll along and firmly adhere to microvasculature endothelium in AiA. Our findings provide a platelet-dependent mechanism that, by amplifying and sustaining inflammation, could contribute to the pathogenesis of RA. Mechanisms for the participation of platelets in RA have not been fully examined yet. Further investigation of these molecular mechanisms and pathophysiological consequences may lead to new approaches to curb the development and progression of RA.


    Acknowledgments
 
This study was supported by grant no 328 from the ‘Förderung für Forschung und Lehre’ (FoeFoLe), Ludwig-Maximilians-University of Munich.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Submitted 17 October 2004; revised version accepted 7 March 2005.



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