Department of Orthopaedics and
1 Institute for Surgical Research, Ludwig Maximilians University of Munich, Munich, Germany
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Abstract |
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Methods. Clinical, histological and microcirculatory parameters (measured by intravital fluorescence microscopy) were assessed in the knee joint during acute and chronic AiA after inhibition of iNOS with L-N6-(1-iminoethyl)lysine (L-NIL). Plasma concentrations of and
were evaluated by the Griess reaction and the expression of iNOS, P- and E-selectin, intercellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) by immunohistochemistry.
Results. In both stages of the disease, plasma concentrations of and
were increased and iNOS was expressed. In the acute phase, swelling, leucocyte adhesion, leucocyte infiltration and expression of adhesion molecules were increased in arthritic animals treated with L-NIL in comparison with untreated arthritic animals. In the chronic phase, no change in the disease parameters could be detected after L-NIL treatment.
Conclusion. Increased NO production induced by iNOS during the acute phase of AiA can be regarded as a protective response in the prevention of further leucocytic infiltration and joint destruction, whereas it seems to play a subordinate role in chronic AiA.
KEY WORDS: Rheumatoid arthritis, Nitric oxide, iNOS, Synovial microcirculation, L-NIL.
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Introduction |
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The aim of our study was to investigate the effect of selective inhibition of iNOS in the acute and chronic phases of antigen-induced arthritis (AiA). We characterized the clinical course, analysed the synovial microcirculation in vivo using intravital fluorescence microscopy, and evaluated the expression of adhesion molecules in AiA after blockade of iNOS with the selective iNOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL).
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Materials and methods |
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Induction and monitoring of arthritis
Arthritis was induced as described previously by Brackertz et al. [21]. On days -21 and -14, the animals were immunized by subcutaneous injections of 100 µg methylated bovine serum albumin (mBSA) (Sigma, Deisenhofen, Germany) dissolved in 50 µl saline. The mixture was supplemented with complete Freund's adjuvant (Sigma) and 2 mg/ml of heat-killed Mycobacterium tuberculosis strain H37RA (Difco, Augsburg, Germany). Additionally, the protocol called for simultaneous intraperitoneal injections of 2x109 heat-killed Bordetella pertussis (Institute of Microbiology, Berlin, Germany). After immunization, arthritis was induced by injection on day 0 of 100 µg mBSA dissolved in 50 µl saline into the left knee joint. The control group underwent the same procedure, using equal volumes of saline for the injections. The severity of AiA was determined by the use of established histological and clinical scoring systems as described by Brackertz et al. [21] and Veihelmann et al. [22] respectively. Joint swelling was determined by measuring the transverse diameter of the knee joint, using a calliper, in units of 0.1 mm, and was expressed as the percentage change between the day of AiA induction and the day of the intravital microscopic measurements. The clinical evaluation started with the preimmunization of the animals and continued until or up to day 7 after AiA induction to represent the acute phase and day 63 to represent the chronic phase of the disease. The time points used to represent the acute and chronic phases were chosen according to the protocols of Brackertz et al. [21] and Veihelmann et al. [22].
Experimental protocol
The animals were allocated to nine groups. There were three groups in which arthritis was not induced, which were examined as follows: one group (n=6) was treated with saline and two groups (n=6 for each group) were treated with the selective iNOS inhibitor L-NIL (Sigma-Aldrich Chemie, Steinheim, Germany) either by intra-articular injection (100 mM) or systemically through the drinking water (10 mM). Intra-articular administration was chosen to mimic the potential clinical use of the inhibitor.
For the assessment of the acute and chronic phases of AiA, six groups of mice were allocated as follows. Two groups with AiA (day 7, acute AiA, n=8; day 63, chronic AiA, n=6) were not treated with L-NIL. In each phase of the disease, two further groups with AiA were treated with L-NIL. The L-NIL was given either systemically in the drinking water (10 mM) from day 2 (acute phase, n=6) or day 49 (chronic phase, n=6) after AiA induction or locally by intra-articular injections (100 mM) on days 2 and 5 (acute phase, n=6) or days 49, 53, 57 and 61 (chronic phase, n=8) after induction of AiA.
The dosage had been evaluated in prior experiments [22] and after personal communication with C. Bogdan [23]. For the intra-articular injections, we used microcannulae (gauge 33; Fine Science Tools, Heidelberg, Germany). All microvasculature parameters were assessed at day 7 and blood and tissue samples were obtained at day 63.
To exclude the possibility that local infections might have occurred after the intra-articular injections, microbiological analysis of synovial tissue samples was performed in three animals.
All experimental procedures were performed according to German legislation on the protection of animals.
Microsurgical procedure
Mice were anaesthetized by inhalation of isoflurane 1.2% (Forene; Abbott, Wiesbaden, Germany) and a mixture of oxygen and nitrous oxide. To prevent changes in body temperature, animals were kept on a heating pad controlled by a rectal probe. Arterial and venous catheters were implanted into the tail. The mean arterial blood pressure was assessed with an arterial catheter connected to a pressure transducer. Saline was infused continuously at 0.2 ml/h through a venous catheter with an infusion pump (B. Braun, Melsungen, Germany) to maintain stable macrohaemodynamics. The left hindlimb was placed on a specially designed stage with slight flexion of the knee joint, and was immobilized in silicone (Classic M, Munich, Germany). After partial skin resection, visualization of the intra-articular subsynovial fatty tissue was made possible by careful mobilization and partial resection of the patellar tendon. A glass coverslip was placed on the knee capsule after superfusion with 3 ml sterile saline and the intravital microscope was directed to the synovial tissue [24]. At the conclusion of the experiment, blood was drawn and tissue samples were removed, and the animal was killed with 10 mg pentobarbital (Nembutal; Sanofi, Hanover, Germany).
Intravital fluorescence microscopy
The microscopic setup has been described in detail previously [25]. Ax20 water immersion objective (total magnificationx432; Zeiss, Jena, Germany) was used to select two or three regions of interest in each animal. These regions contained either postcapillary venules (1840 µm in diameter) or capillary areas for the measurement of functional capillary density (FCD) or both. Vessel diameter and FCD were measured after a bolus injection of the in vivo fluorescent plasma marker fluorescein isothiocyanate (FITC)-dextran (molecular mass 150 kDa; 15 mg/kg body weight intravenously) (Sigma). For the measurements with FITC-dextran, we used a variable 12 V, 100 W halogen light source and Zeiss filter set 09 [band pass (BP) 450490, Farbteiler (FT) 510, long pass (LP) 520]. 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 body weight immediately before each measurement. Rhodamine epi-illumination was achieved with a 150 W variable HBO mercury lamp (Zeiss) in conjunction with Zeiss filter set 15 (BP 546/12, FT 580, LP 590). This double-fluorescence technique allowed subsequent investigation of microhaemodynamics and leucocyteendothelial cell interaction in the vessel segments. The microscopic images were captured with a CCD camera (Pieper, Gera, Germany) and recorded on S-VHS videotape using both filter blocks consecutively. Data were analysed off-line with a computer-assisted microcirculation analysis system (CAP-Image; Dr Zeintl, Heidelberg, Germany) [26].
Microcirculatory parameters
FCD was defined as the length of erythrocyte-perfused capillaries within the observation area (expressed as cm/cm2) [26]. Leucocytes interacting with the endothelium were classified as either rolling or adherent. Rolling leucocytes were defined as cells that interacted intermittently with the endothelial surface and passed through the vessel visibly more slowly than cells in the centre stream. They were quantified as the fraction of all leucocytes passing through a defined vessel segment within an observation period of 30 s (calculated as rolling cells/rolling cells+non-adherent cells). Adherent leucocytes were defined as leucocytes that remained stationary at the same location on the endothelial surface for the entire observation period of 30 s (expressed as cells/mm2 of endothelial surface). The endothelial cell surface was calculated from the diameter and length (200 µm) of the vessel segment under investigation.
Nitrate and nitrite measurements
To eliminate the fluorescence markers, blood samples were ultracentrifuged with 10 000 r.p.m. for 60 min through 25 kDa microfilters (MicroSpin G25 Columns; Pharmacia Biotech, Freiburg, Germany). Total serum levels of nitrite () and nitrate (
), the stable end products of NO, were measured in all nine experimental groups using a colorimetric procedure based on the Griess reaction after nitrate had been reduced to nitrite by nitrate reductase [27], using a microplate assay method. Plasma was incubated with an equal volume of Griess reagent (1% sulphanilamide, 0.1% N-1-naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid). Absorbance at 550 nm was determined by means of a microplate reader. Concentrations were calculated from a standard sodium nitrite curve. All samples were analysed twice.
Immunohistochemistry
Expression of iNOS.
Paraformaldehyde-fixed paraffin sections were cleared in xylol, washed with phosphate-buffered saline (PBS) after being rehydrated, quenched with 0.5% hydrogen peroxide methanol solution, and incubated with 0.1% pronase E solution (Merck, Darmstadt, Germany) to initiate enzymic digestion. After washing with PBS, sections were incubated in 1% goat serum for 20 min to block non-specific binding. The sections were incubated with a polyclonal rabbit anti-mouse iNOS antibody (Dianova, Hamburg, Germany) and incubated with a biotinylated anti-rabbit immunoglobulin G (IgG) antibody (Vectastain; Vector Laboratories, Los Angeles, CA, USA) with 1.5% human serum. Slides were incubated with avidinbiotin complex (Vectastain). Sections then were counterstained with haemalum (Merck).
Expression of E-selectin, P-selectin, ICAM-1 and VCAM-1.
Tissue samples were embedded in Tissue-Tek (Miles, Elkhart, IN, USA), immediately snap-frozen in liquid nitrogen and stored at 80°C. Staining for E-selectin was performed according to the following protocol. To block the production of endogenous peroxidase, sections were quenched with 0.5% hydrogen peroxide methanol solution, incubated in 1.5% goat serum and then with the monoclonal rat anti-mouse CD62E antibody 10E 6.9, directed against E-selectin (Pharmingen, Hamburg, Germany) at 1:100 dilution in PBS for 60 min. After washing, the sections were incubated with biotinylated anti-rat IgG antibody (Vectastain) with 5% mouse serum and then with avidinbiotin complex (Vectastain) for 30 min. After incubation with 0.01% 3-amino-9-ethylcarbazol as substrate, sections were counterstained with haemalum (Merck), hydrated and coverslipped. Each tissue block was stained with and without the primary antibody to monitor background staining.
The procedure for P-selectin, ICAM-1 and VCAM-1 staining by immunohistochemistry was similar to that described above. For P-selectin, we used a polyclonal rabbit anti-mouse CD62P antibody (Pharmingen) at 1:200 dilution in PBS and a biotinylated anti-rabbit IgG-antibody (Vectastain) with 1.5% rabbit serum. For ICAM-1, we used a biotinylated monoclonal rat anti-mouse CD54 antibody (Pharmingen) at 1:100 dilution in PBS and incubation lasted overnight (18 h). A second biotinylated antibody was not necessary in this procedure. VCAM-1 staining was performed using a rat anti-mouse CD106 antibody (Pharmingen).
Histological assessment
To analyse the severity of arthritis, histological sections were made. After fixation in paraformaldehyde 8% at pH 7.2 for 12 h, the joints were incubated in 20% EDTA at pH 7.2 for 3 days at room temperature to decalcify the bone. Samples were washed with PBS and dehydrated with an automatic dehydrator (Shandon, Frankfurt, Germany). After embedding in paraffin, the joint was sliced into 3 µm thick sections, which were stained with haematoxylin and eosin.
To evaluate the severity of the arthritis, the histological scoring system introduced by Brackertz et al. [21] was used: 0=normal knee joint; 1=normal synovium with occasional mononuclear cells; 2=two or more synovial lining cells and perivascular infiltration of leucocytes; 3=hyperplasia of synovium and dense infiltration; 4=synovitis, pannus formation and cartilage/subchondral bone erosions.
Statistical analysis
The data are expressed as mean±S.E.M. Statistical significance was determined by repeated measurements analysis of variance on ranks (Friedman's test) and Dunn's follow-up test. P<0.05 was considered significant.
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Results |
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Acute phase of AiA
The functional capillary density (332±15 cm/cm2), the fraction of rolling leucocytes (0.49±0.03) and the number of leucocytes adherent to the endothelium (437±53/mm2) were significantly increased in the acute phase of AiA compared with control values (245±17 cm/cm2, 0.29±0.02 and 162±36/mm2). The diameter of the arthritic knee joints increased significantly (by 5.7±0.4%) in comparison with controls (0.4±0.4%) (Table 1). In the histological sections, dense leucocytic infiltration was evident in the acute phase and the clinical score was 1, indicating evidence of slight systemic effects in acute AiA (Table 2
). Furthermore, iNOS expression could be detected in chondrocytes and endothelial cells as well as in synovial fibroblasts and cells infiltrating the synovium, whereas in the controls no staining was detectable (Table 3
). Correspondingly, the plasma concentration of
+
was significantly elevated in the arthritic animals (108±18 µmol/l) in comparison with the control group (43±6 µmol/l; Fig. 1a
). E-selectin and P-selectin were strongly expressed, whereas ICAM-1 and VCAM-1 were expressed only moderately in the synovium during acute AiA.
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Inhibition of iNOS in acute AiA
There was no change in the functional capillary density or fraction of rolling leucocytes after inhibition of iNOS by L-NIL. However, the number of leucocytes adherent to the endothelium and the diameter of the arthritic knee joints were significantly greater in L-NIL-treated arthritic animals than in untreated arthritic animals (Table 1). This difference was more pronounced when L-NIL was given locally into the knee joint (Table 1
). There was no change in the clinical score (Table 2
), whereas leucocyte infiltration and pannus formation were increased after L-NIL treatment (Table 2
). Expression of iNOS was moderate in cells of the synovium, and the plasma concentration of
+
was significantly reduced to almost control values after either local or systemic application of L-NIL in acute AiA, indicating effective inhibition of NO production by L-NIL (Fig. 1a
). All adhesion molecules were strongly expressed in acute AiA after L-NIL treatment (Table 3
).
Chronic phase of AiA
There were no differences in FCD (301±16 cm/cm2) or in the fraction of rolling leucocytes (0.18±0.03) between mice with chronic AiA and the control groups (Table 1). However, as in the acute phase, the number of leucocytes adherent to the endothelium was elevated in the chronic phase of AiA (395±78/mm2) in comparison with the control. Leucocyte infiltration appeared less pronounced in the chronic phase but was associated with distinct pannus formation and destruction of cartilage and bone, although there was no evidence of general clinical symptoms, as indicated by the clinical score of 0 in these animals (Table 2
). As we found in the acute phase of AiA, the diameter of the arthritic knee joints increased significantly (by 6.3±0.4%). Expression of iNOS could be detected in chondrocytes, endothelial cells, synovial fibroblasts and cells infiltrating the synovium. Correspondingly, the plasma concentration of
and
was significantly elevated in the arthritic animals (75±4 µmol/l) in comparison with the control group. In contrast to the acute phase, the chronic phase showed weak expression of iNOS and adhesion molecules in the synovium of animals with chronic AiA (Table 3
).
Inhibition of iNOS in chronic AiA
There was no change in FCD, the number of leucocytes adherent to the endothelium and knee joint diameter in the chronic phase of the disease after L-NIL treatment (Table 1 and Fig. 2
). However, the fraction of rolling leucocytes was significantly increased after both systemic and local L-NIL application (Table 1
). As in acute AiA, the plasma concentration of
+
was significantly reduced to control values after either systemic or local administration of L-NIL in comparison with chronic AiA (Fig. 1b
), whereas iNOS expression was unchanged (Table 3
). Clinical score and histological sections revealed no difference in clinical symptoms and leucocytic infiltration of the synovium after iNOS inhibition (Table 2
) compared with untreated animals with chronic AiA. However, immunohistochemistry showed that there was strong expression of E- and P-selectin, ICAM-1 and VCAM-1 in chronic AiA after L-NIL treatment, in contrast to untreated animals with chronic AiA (Table 3
).
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Discussion |
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After treatment with L-NIL in acute AiA, we observed increased joint swelling and further enhancement of leucocyte adherence in synovial microvessels and subsequent infiltration into synovial tissue. These findings are in line with our recently published study in which joint inflammation was found to be exacerbated in iNOS-deficient mice presenting with acute AiA [20]. Swelling of the knee joint and leucocyte infiltration were enhanced in the iNOS-/- arthritic animals in comparison to iNOS+/+ with AiA. Moreover, there was a significant increase in the number of leucocytes adhering to the endothelium and strong expression of P-selectin and VCAM-1 only in iNOS-/- mice with AiA [20]. As this was also true after L-NIL treatment of arthritic animals with acute AiA, the induction of NO production by iNOS can be regarded as a protective response reducing leucocyte adhesion and infiltration in acute murine AiA.
However, McCartney-Francis et al. and similar Ialenti et al. reported suppression of streptococcal cell wall (SCW)-induced arthritis, which is another animal model for rheumatoid arthritis, after non-selective NOS-inhibition in rats [16, 28]. Thus, potential toxic effects of NO as a result of peroxynitrite formation were implicated in the pathogenesis of joint inflammation. The same authors recently found unexpected exacerbation of disease when they used L-NIL in SCW-induced arthritis, which is similar to our results in AiA [29]. As they observed concomitant expression of eNOS and nNOS as well as iNOS in cells of the synovium, the authors concluded that either iNOS-derived NO was protective or eNOS and/or nNOS were also perpetrators of joint damage. However, selective inhibitors of iNOS, which are able to minimize effects on tissue perfusion [30], here demonstrated potential suppression of tissue damage in rat adjuvant arthritis after prophylactic administration; in contrast, there was no amelioration of the disease state when the inhibitors were administered therapeutically during established disease. It has been emphasized that the crucial time for iNOS involvement in the development of arthritis in this model is during the early stage of immunization after adjuvant injection [17, 18, 31]. A possible explanation is that the cytotoxic products of NO formation, such as peroxynitrite, have accumulated sufficiently before therapeutic administration of L-NIL to initiate the subsequent irreversible joint destruction. Thus, it is likely that the effects of increased NO production in murine AiA are stage-dependent and that increased NO production acts as a double-edged sword, as both toxic and anti-inflammatory effects are exhibited with regard to the regulation of leucocyte adhesion and emigration.
From the clinical point of view, it is suggested that local application of a therapeutic agent is more practical and might be able to minimize unwanted side-effects of the drug. Therefore, we also investigated the local administration of L-NIL directly into the arthritic knee joint. The plasma concentration of +
was reduced whether the administration of L-NIL was systemic or local. The increases in leucocyte adherence and joint diameter were more pronounced in arthritic animals treated locally with L-NIL. This indicates that the increased plasma concentration of
+
was related mainly to cells of the synovium of the arthritic joint, and also that the inflammatory reaction in the knee joint is dependent on
and
concentrations within the joint.
As there was no difference in the fraction of rolling leucocytes in arthritic animals after inhibition of iNOS in acute AiA, we assume that the expression of cellular adhesion molecules was already increased in early AiA, as indicated by immunohistochemistry. Thus, inhibition of iNOS did not result in further up-regulation and a subsequent increase in the fraction of rolling leucocytes. In accordance, Hersmann et al. [32] and our group [22] have demonstrated earlier, distinct expression of various cellular adhesion molecules during the acute phase of murine AiA. NOS inhibition has been shown to promote the expression of P-selectin in the mesenteric microcirculation of the rat [33] and the expression of P-selectin and ICAM-1 mRNA in hepatic ischaemiareperfusion [34].
Our findings of increased leucocyte adhesion and inflammatory reactions after selective inhibition of iNOS are consistent with results obtained in intestinal inflammation of iNOS-deficient mice, in which there was increased neutrophil infiltration of the inflamed mesentery and heavy tissue damage [35]. Furthermore, iNOS-deficient mice with endotoxaemia showed enhanced leucocyteendothelium interactions and there was greater recruitment of leucocytes isolated from these animals when they were perfused over purified E-selectin in vitro [36]. Likewise, in immunohistochemical studies we observed distinct expression of E- and P-selectin and VCAM-1 after blockade of iNOS, which may account for our findings in the microcirculation. The mechanisms by which NO influences the expression of adhesion molecules are still under debate. It has been shown recently that NO displays inhibitory effects by preventing the activation of nuclear factor B (NF-
B) and the subsequent expression of VCAM-1, ICAM-1 and E-selectin by increasing the expression and nuclear translocation of I
B
[37, 38]. These results suggest that the events elicited by the inhibition of iNOS at the microcirculatory level very likely account for the aggravation of acute murine AiA.
Clinical symptoms and leucocyte infiltration were less pronounced in chronic AiA, although increased pannus formation and cartilage destruction were obvious in the histological sections in comparison with acute AiA. This indicates similarity to human RA, in which more joint destruction and less inflammation is often evident in the late stages of the disease.
Leucocyte adherence and leucocyte infiltration and joint swelling were unaffected by iNOS inhibition in chronic AiA, in contrast to acute AiA. Similar results were obtained in models of postacute and chronic inflammation, in which therapeutic administration of iNOS inhibitors failed to ameliorate experimental arthritis [17, 18]. Furthermore, no therapeutic benefit of iNOS inhibition was observed in experimental chronic colitis [39]. The fraction of rolling leucocytes was increased after L-NIL treatment. One possible explanation is that the increased expression of the selectins that was found after inhibition of iNOS accounts for these results. While the expression of adhesion molecules was weak in untreated animals with chronic AiA, treatment with L-NIL was followed by discrete up-regulation of the expression of P-selectin, E-selectin and VCAM-1 in synovial microvessels. Interestingly, the increase in the fraction of rolling leucocytes was not followed by increased leucocytic adherence or infiltration, as observed in the acute phase of AiA. This may have been due to the different cell populations recruited during the different phases of the disease, as histological sections revealed that the subsynovial cellular infiltrate in the acute phase was dominated largely by neutrophils, whereas more mononuclear cells could be identified in the chronic phase. VCAM-1 and ICAM-1 play major roles in chronic inflammation and VCAM-1 is known to be monocyte and lymphocyte selective [40].
Thus, it can be assumed that NO release in chronic AiA (in contrast to the acute phase) should be considered to be an epiphenomenon that does not contribute essentially to the pathogenesis of chronic murine AiA. The results of this study point up the problems associated with the inhibition of iNOS as a therapeutic strategy. Further studies are needed to clarify whether there is a therapeutic window in chronic joint inflammation when the toxic effects of NO are overweighted and when the inhibition of iNOS could be a beneficial treatment regimen.
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Acknowledgments |
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Notes |
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*The first two authors contributed equally to this work.
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References |
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