Skip Navigation

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Turesson, C.
Right arrow Articles by Lundberg, I. E.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Turesson, C.
Right arrow Articles by Lundberg, I. E.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Rheumatology 2001; 40: 1346-1354
© 2001 British Society for Rheumatology

Original Papers

Increased endothelial expression of HLA-DQ and interleukin 1{alpha} in extra-articular rheumatoid arthritis. Results from immunohistochemical studies of skeletal muscle

C. Turesson, P. Englund1, L. T. H. Jacobsson, G. Sturfelt2, L. Truedsson3, I. Nennesmo4 and I. E. Lundberg1

Department of Rheumatology, Malmö University Hospital,
1 Unit of Rheumatology, Department of Medicine, Karolinska Hospital, Stockholm,
2 Department of Rheumatology, Lund University Hospital,
3 Department of Laboratory Medicine, Section of MIG, Lund University Hospital and
4 Department of Microbiology, Pathology and Immunology, Division of Pathology, Huddinge Hospital, Stockholm, Sweden


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
Objective. To investigate markers of endothelial activation in muscle biopsies from rheumatoid arthritis (RA) patients with and without extra-articular manifestations (ExRA).

Patients and methods. Nine consecutive ExRA patients were compared with nine RA controls without ExRA, matched for age, sex and duration of RA. Muscle biopsies were obtained from the lateral vastus or anterior tibial muscle. Macrophage and lymphocyte CD markers, HLA molecules, cytokines and adhesion molecules were investigated using immunohistochemistry, and stainings were evaluated using computer image analysis and conventional microscopy. Serum concentrations of soluble adhesion molecules, tumour necrosis factor {alpha} (TNF-{alpha}) and rheumatoid factor (RF) were determined using immunoassays.

Results. The number of HLA-DQ-positive capillaries (P=0.039) and the expression of interleukin 1{alpha} (IL-1{alpha}) in endothelial cells (mean pairwise difference 0.26%; 95% confidence interval 0–0.52) were increased in ExRA patients compared with non-ExRA controls. There were no signs of inflammatory cell infiltrates or fibre degeneration. Serum levels of TNF-{alpha}, the soluble form of intercellular adhesion molecule 1, the soluble form of vascular cell adhesion molecule 1 and IgM RF were increased in the ExRA group.

Conclusion. The increased expression of HLA-DQ and IL-1{alpha} may indicate systemic endothelial activation in extra-articular RA, which could be of importance for cardiovascular comorbidity and mortality in such patients.

KEY WORDS: Rheumatoid arthritis, Extra-articular manifestations, Endothelial activation, Interleukin-1{alpha}, HLA-DQ, Cardiovascular disease, Vasculitis.


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Rheumatoid arthritis (RA) is a chronic disease characterized by inflammatory polyarthritis, which in some cases is complicated by extra-articular manifestations (ExRA). The latter range from mild signs of serositis, which may only be detectable at autopsy [1], to life-threatening necrotizing vasculitis involving internal organs [2]. In a well-defined RA population, the majority of clinically evident ExRA is made up of cases of pleuritis, pericarditis and cutaneous vasculitis [3]. The systemic nature of RA is further underlined by the presence of fever and weight loss in severe cases [4].

RA has consistently been found to be associated with an increased mortality rate compared with the general population [5–7], and most of the excess mortality is due to cardiovascular deaths [7, 8]. The mortality among patients with ExRA has been found to be even higher than among non-extra-articular RA patients [3, 9, 10]. A subset of ExRA patients with a poor prognosis has been defined [3, 11], and cardiovascular mortality appears to be particularly increased in this subgroup [3].

The mechanism behind the increased risk of cardiovascular death in RA and in ExRA in particular is not known, but chronic inflammation has been suggested as a risk factor for cardiovascular death in RA and ExRA; this is probably related to atherosclerotic vascular disease [8]. Soluble forms of intercellular adhesion molecule 1 (ICAM-1) and other cellular adhesion molecules have been detected in serum in increased amounts in patients with RA [12] as well as in other chronic diseases with established blood vessel involvement, such as systemic vasculitis [13] and systemic sclerosis [14]. In a comparison of RA patients with and without vasculitis, circulating ICAM-1 concentrations were significantly higher in the vasculitis subgroup [15]. In addition, in longitudinal studies of apparently healthy men [16] and women [17], the soluble form of ICAM-1 (sICAM-1) was found to be an important predictor of cardiovascular disease. These findings are compatible with generalized blood vessel involvement in at least subgroups of RA patients, and could be important in the pathogenesis of cardiovascular disease.

Muscle tissue is well suited to the investigation of blood vessels, as it is easy to biopsy and the muscle tissue from the extremities usually contains both microvessels and small arteries [18]. Vascular involvement in skeletal muscle in ExRA has been reported, including necrotizing arteritis [19] in a small number of patients with muscular symptoms. Vasculitis has also been reported in striated muscle biopsies from RA patients who clinically presented signs of vasculitis in other organs (e.g. neuropathy and cutaneous lesions) [20, 21], and a pattern of increased expression of adhesion molecules and HLA-DR in muscle tissue has been reported from such patients without histopathological evidence of vasculitis [22].

The present study was undertaken in order to investigate whether there is more generalized endothelial activation in patients with extra-articular manifestations of RA than in non-extra-articular RA patients. We studied the expression of markers of endothelial activation and inflammation in skeletal muscle tissue and in serum from RA patients with and without extra-articular manifestations.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Patients
Nine patients with previously diagnosed RA, all fulfilling the 1987 American College of Rheumatology classification criteria for RA [23], who developed extra-articular manifestations according to predefined criteria [3, 11, 37] were included in the study. The patients were recruited from rheumatology out-patient clinics in Malmö (n=8) and Ängelholm (n=1). Their ExRA consisted of pleuritis (n=2), pericarditis (n=2), Felty's syndrome (n=2) or cutaneous vasculitis (n=3). Briefly, in the patients with pericarditis and pleuritis, clinically suspected exudation was verified by echocardiography and chest X-ray respectively. The patients with Felty's syndrome had splenomegaly by ultrasound measurement, neutrophils <1.8x109/l on at least two occasions, and no other probable explanation of the neutropenia. Cutaneous vasculitis was diagnosed by biopsy (n=2) or clinical judgement by a dermatologist (n=1). Arterial or venous insufficiency to the extent that could explain the lesions was not present. All cases were examined by the same physician (CT) before inclusion. RA controls without ExRA, matched for age, sex and duration of RA, were selected from a database (established in 1997) containing all known RA patients attending out-patient clinics run by rheumatologists in Malmö. Only controls fulfilling the 1987 American College of Rheumatology criteria for RA [23] were included. Previous and current absence of ExRA (according to the same criteria as the cases, in addition including the absence of rheumatoid nodules) in the controls was verified by evaluation of medical records, a detailed questionnaire and physical examination. Patients with previous or current rheumatoid nodules were excluded, as nodules have been shown to predict severe extra-articular disease [37] and inclusion of nodular patients in the non-ExRA group would put them at risk of developing ExRA. All cases and controls were examined by the same physician (CT), using a structured protocol. ExRA cases and non-ExRA controls were well matched for sex (three men and six women in each group), age [ExRA, median 61 yr; interquartile range (IQR) 58–69 yr; non-ExRA, 59 yr, 54–66 yr) and duration of RA (ExRA, median 15 yr, IQR 6–23; non-ExRA 17 yr, 7–20 yr). There was no major difference in the number of current smokers (ExRA 2, non-ExRA 1), previous smokers (ExRA 6, non-ExRA 5) or in systolic blood pressure (ExRA, median 130, IQR 120–140; non-ExRA 130, 120–145). There was no substantial difference in the number of individuals previously positive for RF (78% in both groups) or antinuclear antibodies (ExRA 38%, non-ExRA 25%). ExRA patients tended to be on glucocorticoid treatment (22 vs 56%) (P=0.33) and treatment with disease-modifying anti-rheumatic drugs (DMARDs) (56 vs 100%) (P=0.082) to a lesser extent than non-ExRA controls at the time when they developed the ExRA manifestation. The patients with ExRA tended to have a higher number of swollen joints [median 8, IQR 5–19 vs 2 (0–4)] (P=0.09), higher scores for Ritchie's index (median 16, IQR 8–19 vs 5, 2–8) (P=0.42) and higher levels of C-reactive protein (CRP) (median 31 mg/l, IQR 13–64 vs10, 0–21) (P=0.12).

None of the RA patients had muscle pain, weakness or other clinical signs of muscle disease. All patients gave their informed consent to participation and the study was approved by the ethics committee of Lund University.

Tissue samples
Muscle biopsy was performed before any changes in pharmacological treatment were made due to the extra-articular manifestations. Biopsies from the vastus lateralis or the tibialis anterior muscle were obtained from each patient under local anaesthesia using a semi-open technique with a conchotome [24]. At least two biopsy samples were taken from each patient, snap-frozen in isopentane chilled with liquid nitrogen, and stored at -70°C. Muscle tissues from five healthy subjects with normal histopathological findings and no clinical symptoms of muscle pain and muscle weakness served as controls. Sections from a patient with severe inclusion body myositis, with a known profile of expression of inflammatory cytokines and HLA-molecules, were used as a positive control.

Two slides from each patient (taken from the levels flanking the sections used for immunohistochemistry studies) were evaluated by a neuropathologist (IN) after staining with haematoxylin and eosin. All slides were coded and analysed blindly for the presence of degeneration, regeneration, atrophy, centrally located nuclei and inflammatory infiltrates.

Immunohistochemistry studies
Cryostat sections 7 µm thick were mounted on gelatin-coated glass slides (Cell-line Associates, Newfield, NJ, USA) and air-dried for 30 min at room temperature. The sections were stored unfixed at -70°C until stained. A standard immunohistochemistry protocol [25] was used to stain for cell type-specific antigens and HLA molecules. Briefly, the sections were acetone-fixed and endogenous peroxidase was blocked with 1% hydrogen peroxide and 2% sodium nitride. Antibodies to lymphocyte functioning-associated antigen 1 (LFA-1{alpha}) or CD11a (clone G52.2) were kindly provided by Manuel Patarroyo, MTC, Stockholm, Sweden. Antibodies to CD3 (clone SK7), CD4 (clone SK3), CD8 (clone SK1), CD20 (clone L27), HLA-DQ (clone SK10) and HLA-DR (clone L243) were purchased from Becton Dickinson, San Jose, CA, USA. Antibodies to CD19 (clone HD37), CD68 (clone PGM1), CD163 (clone Ber-MAC3) and HLA-ABC (clone W6/32) were purchased from Dako, Glostrup, Denmark. Antibodies to ICAM-1 and CD54 (clone 84H10) were purchased from Serotec, Oxford, UK. Antibodies to vascular cell adhesion molecule-1 (VCAM-1) or CD106 (clone 51–10C9) were purchased from Pharmingen, San Diego, CA, USA. Antibodies to very late activation antigen 4{alpha} (VLA-4{alpha}) and CD49d (clone HP2/1) were purchased from Immunotech, Marseille, France. Antibodies to IL-1{alpha} (clones 1277-89-7, 1277-82-29 and 1279-143-4) were purchased from Towbin Heusser, Basel, Switzerland. Normal horse serum (1%) was added in order to reduce background caused by interaction of tissue IgG and Fc receptors with the Fc region of the secondary antibodies. A biotinylated secondary antibody was used [biotin–horse anti-mouse IgG (Vector, Burlingame, CA, USA)] and, after addition of avidin–biotin peroxidase complex, the stain was developed using 3-amino-9-ethylcarbazole. Staining for IL-1{alpha} was performed as described previously [18], using a modified immunohistochemistry protocol for intracellular staining with saponin as detergent [26].

Quantitation of stained cells
The tissue sections were analysed with a Reichert–Jung Polyvar microscope. Quantitation was performed both by conventional microscopy and by computer-assisted imaging analysis. For the conventional reading, the numbers of positively stained mononuclear cells and endothelial cells of the whole tissue section were estimated. The origin of the muscle biopsies was concealed to the investigators during the quantitation. The sections were read on different occasions by two of the authors (CT and IEL) with concordant results. Mean values of these readings were divided by the total tissue area of the biopsy, measured by computer image analysis (see below).

The numbers of mononuclear cells per unit area of cryocut section stained positively for the markers CD3, CD4, CD8, CD68 and CD163 in combination, CD19 and CD20 in combination, LFA-1 and VLA-4 were determined in the same way. The number of cells and vessels stained positively for VCAM-1, ICAM-1 and HLA-DQ were scored separately; only the number of positive vessels is given in the results as no extra information was derived from this subdivision. For HLA-ABC and HLA-DR staining, a 5-point scale was used to score the percentage of positively stained muscle fibre area relative to the total section area: 0=positive staining in endothelial cells, negative muscle fibres; 1+=1–10% of the fibre area; 2+=11–25%; 3+=26–50%; 4+=51–100%.

Computer image analysis
For stainings that featured a large number of positive vessels or large positive areas, computer image analysis was used to quantify the stained areas. IL-1{alpha} and HLA-DQ staining was analysed by image analysis. The images were assessed in a Quantimet 600 (Q600) image analyser (Leica Cambridge, Cambridge, UK). The image processor was controlled by a computer (PC). Special software was written for this application, in a high-level language (QUIPS). The results of the image analysis showed the positively stained area as a percentage of the total tissue section area. Image analysis was also used to assess the total biopsy specimen area in mm2. Mean values of three measured sections from each biopsy were used.

Circulating cytokines, adhesion molecules and rheumatoid factors
Blood was drawn immediately after the muscle biopsy was taken. Serum was stored at -20°C and was available from seven ExRA patients and nine RA controls for the analysis of circulating cytokines, soluble adhesion molecules and RF isotypes, which was performed by a blinded investigator (LT). The ExRA patients subject to serum analysis suffered from pleuritis (n=2), cutaneous vasculitis (n=3), pericarditis (n=1) and Felty's syndrome (n=1). Tumour necrosis factor {alpha} (TNF-{alpha}) was measured with an automated immunoassay system, Immulite (DPC, Los Angeles, CA, USA). sICAM-1, soluble VCAM-1 (sVCAM-1), soluble E-selectin and soluble L-selectin were measured by enzyme-linked immunosorbent assay (ELISA) using kits from R&D Systems (Minneapolis, MN, USA). IgM, IgG and IgA RF isotypes were analysed by ELISA as described previously [27], which was calibrated against the WHO RF reference preparation.

Statistical analysis
Fisher's exact test was used to assess differences between the ExRA and non-ExRA groups regarding previous RF and antinuclear antibody status and current treatment with DMARDs and corticosteroids. In comparisons of other clinical parameters and the results of serum analysis and immunohistochemistry studies, the paired t-test was used for parameters with a normal distribution (i.e. pairwise differences between cases and controls in areas positive for IL-1{alpha} by computer image analysis and pairwise differences for IgM RF titres). The results are presented as mean pairwise differences and 95% confidence intervals. For parameters without a normal distribution, the Wilcoxon signed rank test was used, and the results are presented as median pairwise differences and P values for between-group differences. Correlations between clinical, serological and immunohistochemical variables were investigated using Spearman's correlation test.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Histopathological assesment
In haematoxylin/eosin-stained sections, single, small, perivascular, cellular infiltrates were noted in one ExRA patient and one non-ExRA patient. There were, however, no inflammatory cell infiltrates surrounding or invading the muscle fibres and no signs of fibre degeneration. Specific staining showed scattered cells featuring CD3, CD4, CD8, CD68/163, CD19/20, LFA-1{alpha} and VLA-4, without differences between the groups.

Interleukin-1{alpha} and adhesion molecules in muscle tissue
IL-1{alpha} was expressed in endothelial cells of arterioles, venules and capillaries. The patients with ExRA had more extensive staining for IL-1{alpha}, especially in the arterioles (Fig. 1Go). The total area stained for IL-1{alpha} (by computer image analysis) was greater in ExRA patients than in non-ExRA controls with RA [mean pairwise difference 0.26%, 95% confidence interval (95% CI): 0–0.52]. The area stained in non-ExRA patients did not differ substantially from that in the healthy controls (Fig. 3Go). There was no difference in IL-1{alpha} expression between patients on glucocorticosteroid treatment and non-glucocorticoid-treated patients (P=1.0). The size of the positive area correlated with the number of swollen joints [Spearman's correlation coefficient (r)=0.51; P=0.03] in the total group studied, but not in the ExRA (r=0.14; P=0.71) or non-ExRA group (r=00.42; P=0.23) when studied separately. There was no correlation between IL-1{alpha} expression and CRP or Ritchie's index.



View larger version (136K):
[in this window]
[in a new window]
  		FIG. 1. (a) Prominent IL-1{alpha} staining in muscle tissue arterioles of a patient with extra-articular RA. Immunohistochemical staining, magnification x250. (b) Weak IL-1{alpha} staining in muscle tissue arterioles of an RA patient with no extra-articular manifestations. Immunohistochemical staining, magnification x250.

 


View larger version (6K):
[in this window]
[in a new window]
  		FIG. 3. Increased IL-1{alpha} staining shown by computer image analysis in ExRA patients compared with non-ExRA patients (mean pairwise difference 0.26%, 95% CI 0–0.52).

 
ICAM-1 and VCAM-1 expression in endothelium and infiltrating cells was limited, without major differences between the groups (Table 1Go).


View this table:
[in this window]
[in a new window]
  		TABLE 1. Stained vessels quantified by conventional microscopy

 

MHC class I and II expression
HLA-ABC and -DR antigens were expressed in the endothelium of most blood vessels in ExRA patients, non-ExRA patients and healthy controls, without obvious differences between the groups. Expression of HLA-DQ, on the contrary, was highly variable, from being almost absent in some samples to being present in hundreds of capillaries in some. The endothelium of the capillaries of some extensively stained sections from ExRA patients had a thickened appearance when stained for HLA-DQ (Fig. 2aGo), resembling the high endothelial venules of lymph nodes. The number of stained capillaries (by manual scoring) was significantly increased in ExRA patients (median 22.5/mm2, IQR 15.9–74.6 vs 7.9, 5.9–15.5) (P=0.039). There was also a trend towards a larger HLA-DQ-positive tissue area, as assessed by computer image analysis, in ExRA patients (median 0.28%, IQR 0.086–1.03 vs 0.10, 0.058–0.12) (P=0.074) than in non-ExRA patients. There was a correlation between the number of HLA-DQ-positive capillaries and the total DQ-positive area in the total group studied (r=0.75, P=0.0003) as well as in the ExRA and non-ExRA subgroups separately. The HLA-DQ staining pattern in non-ExRA patients did not differ from that in the healthy controls (Fig. 4Go). There was no correlation between HLA-DQ expression and disease activity parameters (number of swollen joints, Ritchie's index and CRP) in RA patients; this applied to the analysis of all the RA patients together and of the ExRA and non-ExRA groups studied separately.



View larger version (138K):
[in this window]
[in a new window]
  		FIG. 2. (a) Skeletal muscle with capillaries with endothelium positive for HLA-DQ in a muscle biopsy from a patient with extra-articular RA. Immunohistochemical staining, magnification x250. (b) Skeletal muscle capillaries with weak or negative HLA-DQ-staining in an RA patient with no extra-articular manifestations. Immunohistochemical staining, magnification x250.

 


View larger version (6K):
[in this window]
[in a new window]
  		FIG. 4. Increased number of HLA-DQ-expressing capillaries in ExRA patients compared with non-ExRA patients (P=0.039).

 
HLA-ABC expression in muscle fibres was detected in five ExRA patients, five non-ExRA RA patients and one healthy control. In five sections, more than 10% of the muscle fibres (by visual scoring) were HLA-ABC-positive. Four of these had ExRA (one with Felty's syndrome, two with pericarditis and one with cutaneous vasculitis) and one was a control RA patient.

Muscle fibres stained for HLA-DR were present in two ExRA patients, both of whom had presented with pericarditis. A relatively small number of fibres (<10%) were stained in both cases.

Serum analysis
Serum concentrations of TNF-{alpha}, sICAM-1, sVCAM-1 and IgM RF were significantly higher in the ExRA group (Table 2Go). The concentrations of TNF-{alpha} and sICAM-1 were highly correlated (r=0.68, P=0.003). TNF-{alpha} and sICAM-1 correlated with the number of swollen joints when all the RA patients were analysed as one group, but not in the two groups when studied separately. IgM RF levels were higher in the ExRA group (mean pairwise difference 938 IU/ml, 95% CI 182–1694), and IgM RF levels correlated with the number of HLA DQ-positive capillaries (r=0.56, P=0.02).


View this table:
[in this window]
[in a new window]
  		TABLE 2. Circulating factors determined by ELISA

 


   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
In this systematic immunohistochemical study of muscle tissue, the expression of HLA-DQ and IL-1{alpha} was increased in the endothelium of blood vessels in skeletal muscle tissue of RA patients with ExRA compared with those without extra-articular manifestations. This occurred in the absence of clinical and histopathological signs of localized inflammation in the muscle tissue studied, indicating that the abnormal IL-1{alpha} and HLA-DQ expression may be a general feature of the vascular system of ExRA patients. Elevated serum levels of sICAM-1, sVCAM-1 and TNF-{alpha} were also found in ExRA patients compared with the non-ExRA controls. Concentrations of RF, in particular IgM RF, were also increased in ExRA. Moreover, IgM RF levels correlated with the number of capillaries expressing HLA-DQ in muscle tissue.

The ExRA cases included in this study manifested a spectrum of different systemic features of RA. They fulfilled the Malmö criteria for extra-articular disease, which have been demonstrated to identify patient groups with an increased mortality rate, especially in vascular disease, compared to RA patients in general [3, 11]. The present findings may thus be of relevance to the comorbidity and mortality associated with extra-articular RA.

The immunohistochemical protocols used in this study have previously been used extensively in studies of inflammatory myopathies [18, 26, 28]. We used computer image analysis to assess the staining of relatively small numbers of large areas (as in the case of the arteriolar staining for IL-1{alpha}), as the total area stained was considered to reflect the amount of cytokines produced rather than the number of positive arterioles. We relied on conventional microscopy when the stained areas (cells and microvessels) were small and the visual counting of numbers instead of the percentage stained area was considered to be more accurate (e.g. when staining for lymphocyte and macrophage CD markers, adhesion molecules). In the case of HLA-DQ expression, for which the biopsies varied both in the number of stained vessels and in the area stained in each vessel, both methods were used, and we demonstrated a correlation between the number of stained capillaries (assessed by conventional microscopy) and the stained area (measured by image analysis).

The increased serum concentrations of TNF-{alpha} and sICAM-1 in the ExRA group, which did not correlate with the immunohistochemical findings, illustrate that several different inflammatory pathways may be involved in systemic RA. TNF-{alpha} is recognized as an important factor in the pathogenesis of RA [29]. Treatment with TNF-blocking agents reduces the serum level of sICAM-1 [30] and decreases clinical and laboratory signs of disease activity [31, 32], although the specific effect on extra-articular manifestations has not been evaluated.

In a recent study of rheumatoid vasculitis, increased expression of the adhesion molecules ICAM-1 and VCAM-1 and of HLA-DR was observed in muscle biopsy specimens from vasculitis patients with and without perivascular infiltrates, compared with controls with non-vasculitic RA and osteoarthritis [22]. Expression of HLA-DQ and IL-1{alpha} was, however, not evaluated in that study. As the case definitions were different from the criteria for ExRA used in the present series, the results are not directly comparable.

The staining patterns observed in microvessels of muscle tissue in the present study resemble the findings described previously in endothelial cells of small blood vessels in the inflamed synovium of RA patients, where increased expression of IL-1{alpha} [33] and HLA-DQ [34, 35] was reported. On the contrary, synovial HLA-DQ expression in endothelial cells was not found in a group of patients with reactive arthritis [35], suggesting that this finding is specific for microvessels of RA patients.

Increased endothelial IL-1{alpha} staining in muscle tissue has also been found in patients with inflammatory myopathies [18, 26], but in the myositis patients IL-1{alpha} was expressed in the capillaries as well as in the arterioles, whereas in the present series the staining was more prominent in the arterioles. HLA-DQ expression in the endothelial cells of myositis patients was less prominent than in ExRA patients (unpublished data, own observations). This pattern may thus be characteristic of the rheumatoid disease process, and disseminated endothelial IL-1{alpha} and HLA-DQ expression could be important in the pathogenesis of ExRA. Felty's syndrome and rheumatoid vasculitis have been reported to be associated with the genetic subtype HLA-DQ7 [36], and in a series of ExRA patients with a range of manifestations similar to those in the present patients, a trend towards an association with HLA-DQ7 was noted [37]. In the present series, there was a correlation between IgM RF levels and the number of HLA-DQ-positive capillaries in muscle tissue. A possible mechanism could be the induction of endothelial HLA-DQ expression by circulating immune complexes in genetically predisposed individuals.

The major limitations of the present study are the limited number of patients in each group, the possibility of significant findings due to multiple testing, and the possible influence of factors not controlled for in the analysis. The results should therefore be confirmed in a larger study. The distribution of ExRA manifestations was similar to that found in a retrospective survey of ExRA from a well-defined RA population [3], indicating that the ExRA sample is representative of this subgroup of RA patients. The matching of control RA patients from the same catchment area for age, sex and disease duration should prevent selection bias among the controls as well as confounding by the potential risk factors for vascular disease. The non-ExRA patients all had non-nodular disease, as rheumatoid nodules are known predictors of severe extra-articular disease [37]. There was a trend towards fewer swollen joints in the non-ExRA patients at inclusion, but there was no correlation between joint count indices or CRP on one side and the expression of IL-1{alpha} or HLA-DQ on the other side, in either the ExRA or the non-ExRA group. It is, however, still possible that the increased HLA-DQ and IL-1{alpha} expression in the ExRA group may in part be explained by more widespread articular inflammation. The relative importance of this could be elucidated by stratification by the number of swollen joints in a larger series, but such an analysis was not possible for the present series. However, the vascular staining pattern described may be an important pathophysiological mechanism in systemic RA, regardless of whether it is determined in part by the extent of articular inflammation.

Endothelial activation has been defined as ‘a quantitative change in the level of expression of specific gene products (i.e. proteins), which in turn endow endothelial cells with new capabilities that cumulatively allow endothelial cells to perform new functions' [38]. IL-1{alpha} expression has been reported in arteriosclerotic lesions, but not in normal blood vessels [39]. IL-1{alpha} induces tissue factor-like procoagulant activity and plasminogen activation inhibitor synthesis in human endothelial cells in vitro [40], indicating that increased IL-1{alpha} production may have a procoagulant effect on vascular endothelium. MHC class II expression occurs in endothelial cells in some, but not all large blood vessels [41], and has been found in endothelium overlying atherosclerotic plaques [42]. IL-1{alpha} and HLA-DQ may thus be important in atherosclerotic vascular disease. Endothelial activation of this sort could be important in other systemic inflammatory conditions, although this requires further study.

In conclusion, we have demonstrated increased expression of HLA-DQ and IL-1{alpha} in the endothelial cells of skeletal muscle blood vessels in patients with extra-articular manifestations of RA compared with non-extra-articular RA controls. This may indicate systemic endothelial activation in extra-articular RA. Serum levels of IgM RF, TNF-{alpha} and circulating sICAM-1 and sVCAM-1 were also increased in extra-articular RA compared with non-ExRA patients, indicating that several inflammatory pathways may be involved in extra-articular disease. Further studies should be performed in order to investigate possible associations between the immunohistochemical findings, individual extra-articular manifestations and other clinical features, including cardiovascular disease.


   Acknowledgments
 
We thank Eva Lindroos for excellent work with the staining procedures, Associate Professor Manuel Patarroyo for providing anti-LFA-1 antibodies, Gertrud Hellmer for excellent work with the ELISA procedures, Jan-Åke Nilsson for important comments on the statistical analysis and Dr Bo Holmgren, Dr Ylva Lindroth and Dr Lida Marsal for valuable aid in patient recruitment. This work was supported by the Swedish National Association Against Rheumatism, Lund University, Karolinska Institutet, the Swedish Medical Research Council (grant 13489), King Gustaf V 80 year Fund, the Alfred Österlund Foundation, the Professor Nanna Svartz Foundation, the Börje Dahlin Foundation and the Eva and Alex Wallström Foundation.


   Notes
 
Correspondence to: C. Turesson, Department of Rheumatology, Malmö University Hospital, S-205 02 Malmö, Sweden Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

  1. Kirk J, Cosh J. The pericarditis of rheumatoid arthritis. Q J Med1969;37:397–423.
  2. Geirsson A, Sturfelt G, Truedsson L. Clinical and serological features of severe vasculitis in rheumatoid arthritis: prognostic implications. Ann Rheum Dis1987;46:727–33.[Abstract/Free Full Text]
  3. Turesson C, Jacobsson L, Bergström U. Prevalence and mortality of extraarticular rheumatoid arthritis. Rheumatology1999;38:668–74.[Abstract/Free Full Text]
  4. Roubenoff R, Roubenoff RA, Cannon JG et al. Rheumatoid cachexia: cytokine driven hypermetabolism accompanying reduced body cell mass in chronic inflammation. J Clin Invest1994;93:2379–86.
  5. Cobb S, Anderson F, Bauer W. Length of life and cause of death in rheumatoid arthritis. N Engl J Med1953;249:553–6.
  6. Wolfe F, Mitchell DM, Sibley JT et al. The mortality of rheumatoid arthritis. Arthritis Rheum1994;37:481–94.[ISI][Medline]
  7. Jacobsson LTH, Knowler WC, Pillemer S et al. Rheumatoid arthritis and mortality. A longitudinal study in Pima Indians. Arthritis Rheum1993;36:1045–53.[ISI][Medline]
  8. Wållberg-Jonsson S, Öhman M-L, Rantapää-Dahlqvist S. Cardiovascular morbidity and mortality in patients with rheumatoid arthritis in northern Sweden. J Rheumatol1997;24:445–51.[ISI][Medline]
  9. Foster AC, Forstot SL, Wilson LA. Mortality rate in rheumatoid arthritis patients developing necrotizing scleritis or peripheral ulcerative keratitis. Ophthalmology1984;91:1253–63.[ISI][Medline]
  10. Vollertsen RS, Conn DL, Ballard DJ, Ilstrup DM, Kazmar RE, Silverfield JC. Rheumatoid vasculitis: survival and associated risk factors. Medicine1986;65:365–75.[Medline]
  11. Turesson C, O'Fallon WM, Crowson CS, Gabriel SE, Matteson EL. Occurrence of extra-articular disease manifestations is associated with excess mortality in a population based cohort of patients with rheumatoid arthritis. Arthritis Rheum2000;43:S385.
  12. Littler AJ, Buckley CD, Wordsworth P, Collins I, Martinson J, Simmons DL. A distinct profile of six adhesion molecules (ICAM-1, ICAM-3, VCAM-1, E-selectin, L-selectin and P-selectin) in rheumatoid arthritis. Br J Rheumatol1997;36:164–9.[Abstract/Free Full Text]
  13. Coll-Vincent B, Grau JM, López-Soto A et al. Circulating soluble adhesion molecules in patients with classical polyarteritis nodosa. Br J Rheumatol1997;36:1178–83.[Abstract/Free Full Text]
  14. Denton CP, Bickerstaff MCM, Shiwen X et al. Serial circulating adhesion molecule levels reflect disease severity in systemic sclerosis. Br J Rheumatol1995;34:1048–54.[Abstract/Free Full Text]
  15. Voskuyl AE, Martin S, Melchers I, Zwinderman AH, Weichselbraun I, Breedveld FC. Levels of circulating intercellular adhesion molecule-1 and -3 but not circulating endothelial leukocyte adhesion molecule are increased in patients with rheumatoid vasculitis. Br J Rheumatol1995;34:311–5.[Abstract/Free Full Text]
  16. Ridker PM, Hennekens CH, Roitman-Johnson B, Strampfer MJ, Allen J. Plasma concentrations of soluble intercellular adhesion molecule 1 and risk of future myocardial infarction in apparently healthy men. Lancet1998;351:88–92.[ISI][Medline]
  17. Ridker PM, Hennekens CH, Buring JE, Rifai N. C-reactive protein and other markers of inflammation in the prediction of cardiovascular disease in women. N Eng J Med2000;342:836–43.[Abstract/Free Full Text]
  18. Lundberg I, Ulfgren A-K, Nyberg P, Andersson U, Klareskog L. Cytokine production in muscle tissue of patients with idiopathic inflammatory myopathies. Arthritis Rheum1997;40:865–74.[ISI][Medline]
  19. Sokoloff L, Wilens SL, Bunim JJ. Arteritis of striated muscle in rheumatoid arthritis. Am J Pathol1951;27:157–73.
  20. Kraus A, Palacios A, Munoz L. Muscular involvement in systemic rheumatoid vasculitis. Br J Rheumatol1992;31:355–8.[Free Full Text]
  21. Voskuyl AE, van Duinen SG, Zwinderman AH, Breedveld FC, Hazes JMW. The diagnostic value of perivascular infiltrates in muscle biopsy specimen for the assessment of rheumatoid vasculitis. Ann Rheum Dis1998;57:114–7.[Abstract/Free Full Text]
  22. Verschueren PC, Voskuyl AE, Smeets TJ, Zwinderman KH, Breedveld FC, Tak PP. Increased cellularity and expression of adhesion molecules in muscle biopsy specimen from patients with rheumatoid arthritis with clinical suspicion of vasculitis, but negative routine histology. Ann Rheum Dis2000;59:598–606.[Abstract/Free Full Text]
  23. Arnett FC, Edworthy SM, Bloch DA et al. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum1988;31:315–24.[ISI][Medline]
  24. Henriksson KG. ‘Semi-open’ muscle biopsy technique. A simple outpatient procedure. Acta Neurol Scand1979;59:317–23.[ISI][Medline]
  25. Frostegård J, Ulfgren A-K, Nyberg P et al. Cytokine expression in advanced human atherosclerotic plaques: Dominance of proinflammatory (Th1) and macrophage-stimulating cytokines. Atherosclerosis1999;145:33–43.[ISI][Medline]
  26. Nyberg P, Wikman A-L, Nennesmo I, Lundberg I. Increased expression of interleukin-1{alpha} and MHC class I in muscle tissue of patients with chronic inactive polymyositis and dermatomyositis. J Rheumatol2000;27:940–8.[Medline]
  27. Truedsson L, Sjöholm AG, Sturfelt G. Complement activating rheumatoid factors in rheumatoid arthritis studied by hemolysis in gel: relation to antibody class and response to treatment with podophyllotoxin derivatives. Clin Exp Rheumatol1985;3:29–37.[ISI][Medline]
  28. Lundberg I, Kratz A-K, Alexandersson H, Patarroyo M. Decreased expression of interleukin-1{alpha}, interleukin-1ß and cell adhesion molecules in muscle tissue following corticosteroid treatment in patients with polymyositis and dermatomyositis. Arthritis Rheum2000;42:336–48.
  29. Feldmann M, Brennan FM, Maini RN. Role of cytokines in rheumatoid arthritis. Annu Rev Immunol1996;14:397–440.[ISI][Medline]
  30. Paleolog EM, Elliott MJ, Feldmann M, Maini RN, Woody JN. Deactivation of vascular endothelium by monoclonal anti-tumor necrosis factor {alpha} antibody in rheumatoid arthritis. Arthritis Rheum1996;39:1082–91.[Medline]
  31. Moreland LW, Baumgartner SW, Schiff MH et al. Treatment of rheumatoid arthritis with a recombinant human tumor necrosis factor receptor (p75)-Fc fusion protein. N Eng J Med1997;337:141–7.[Abstract/Free Full Text]
  32. Maini RN, Breedveld FC, Kalden JR et al. Therapeutic efficacy of multiple intravenous infusions of anti-tumor necrosis factor monoclonal antibody combined with low-dose weekly methotrexate in rheumatoid arthritis. Arthritis Rheum1998;41:1552–63.[ISI][Medline]
  33. Deleuran BW, Chu CQ, Field M et al. Localization of interleukin-1{alpha}, type 1 interleukin receptor and interleukin-1 receptor antagonist in the synovial membrane and cartilage/pannus junction in rheumatoid arthritis. Br J Rheumatol1992;31:801–9.[Abstract/Free Full Text]
  34. Barkley D, Allard S, Feldmann M, Maini RN. Increased expression of HLA-DQ antigens by interstitial cells and endothelium in the synovial membrane of rheumatoid arthritis patients compared with reactive arthritis patients. Arthritis Rheum1989;32:955–63.[ISI][Medline]
  35. Klareskog L, Johnell O, Hulth A. Expression of HLA-DR and HLA-DQ antigens on cells within the cartilage–pannus junction in rheumatoid arthritis. Rheumatol Int1984;4:11–5.
  36. Hillarby MC, Clarkson R, Grennan DM et al. Immunogenetic heterogeneity in rheumatoid disease as illustrated by different MHC associations (DQ, Dw and C4) in articular and extra-articular subsets. Br J Rheumatol1991;30:5–9.[Abstract/Free Full Text]
  37. Turesson C, Jacobsson L, Bergström U, Truedsson L, Sturfelt G. Predictors of extra-articular rheumatoid arthritis. Scand J Rheumatol2000;29:358–64.[Medline]
  38. Pober JS. Cytokine mediated activation of vascular endothelium. Am J Pathol1988;133:426–33.[Abstract]
  39. Brody JI, Pickering NJ, Capuzzi DM, Fink GB, Can CA, Gomez F. Interleukin-1 as a factor in occlusive vascular disease. Am J Clin Pathol1992;97:8–13.[ISI][Medline]
  40. Dejana E, Brevario F, Erroi A et al. Modulation of endothelial cell functions by different molecular species of interleukin-1. Blood1987;69:695–9.[Abstract/Free Full Text]
  41. Daar AS, Fuggle SV, Fabre JW, Ting A, Morris PJ. The detailed distribution of MHC class II antigens in normal human organs. Transplantation1984;38:293–8.[ISI][Medline]
  42. Bürrig K-F. The endothelium of advanced arteriosclerotic plaques in humans. Art Thromb1991;11:1678–89.[Abstract/Free Full Text]
Submitted 18 January 2001; Accepted 17 May 2001


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Ann Rheum DisHome page
C Turesson, L T H Jacobsson, G Sturfelt, E L Matteson, L Mathsson, and J Ronnelid
Rheumatoid factor and antibodies to cyclic citrullinated peptides are associated with severe extra-articular manifestations in rheumatoid arthritis
Ann Rheum Dis, January 1, 2007; 66(1): 59 - 64.
[Abstract] [Full Text] [PDF]

Home page
 Rheumatology (Oxford)Home page
H. Lindehammar and B. Lindvall
Muscle involvement in juvenile idiopathic arthritis
Rheumatology, December 1, 2004; 43(12): 1546 - 1554.
[Abstract] [Full Text] [PDF]

Home page
 Ann Rheum DisHome page
C Turesson, W M O'Fallon, C S Crowson, S E Gabriel, and E L Matteson
Extra-articular disease manifestations in rheumatoid arthritis: incidence trends and risk factors over 46 years
Ann Rheum Dis, August 1, 2003; 62(8): 722 - 727.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (16)
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Turesson, C.
Right arrow Articles by Lundberg, I. E.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Turesson, C.
Right arrow Articles by Lundberg, I. E.
Related Collections
Right arrow Rheumatoid Arthritis
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?