Published Online First: 10 July 2006.
doi:10.1136/ard.2005.051086
Annals of the Rheumatic Diseases
2006;65:1565-1571
? 2006 by BMJ Publishing Group Ltd & European League
Against Rheumatism
EXTENDED REPORT |
1 Rheumatology Unit, Department of Medicine,
Karolinska University Hospital, Karolinska Institutet, Solna, Sweden
2
Department of Laboratory Medicine, Division of Pathology, Karolinska University
Hospital, Huddinge, Sweden
Correspondence to:
C Dorph
Rheumatology Clinic, D2:01, Karolinska University Hospital, Solna, 171
76 Stockholm, Sweden; Christina.Dorph@karolinska.se
Accepted 26 June 2006
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ABSTRACT |
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(IL1) and major
histocompatibility complex (MHC) class I and II antigens on
muscle fibres.
Methods: Biopsy specimens from
clinically symptomatic (proximal
muscles) and asymptomatic (all distal but two proximal) muscles
in eight patients with polymyositis, three patients with dermatomyositis
and six healthy controls were analysed by immunohistochemistry for
the presence of T cells and macrophages, and expression of IL1 and of MHC class I and II antigens. Results
were evaluated by conventional light microscopy and by computerised
image analysis.
Results: Inflammatory infiltrates
with T cells and macrophages
were observed to an equal degree in both symptomatic and asymptomatic
muscle. The numbers of capillaries with IL1 expression were significantly
higher (p<0.05) in the symptomatic and asymptomatic muscles of
patients than in controls. The total IL1
expression per tissue section assessed by computerised image analysis
was significantly higher in symptomatic muscles but not in
asymptomatic muscles compared with that in controls. Neither the
number of IL1-positive capillaries nor the total IL1 expression differed significantly
between symptomatic and asymptomatic muscles.
Expression of MHC class I and II antigens on muscle fibres was
detected in both symptomatic and asymptomatic muscles but rarely in
healthy controls.
Conclusions: Presence of
inflammatory infiltrates, T cells and
macrophages, and expression of MHC class I and II antigens and
of IL1 on muscle fibres were independent of clinical
symptoms, and were present to an equal degree in both proximal and
distal muscles. Thus, other factors seem to determine the development
of clinical symptoms. One such factor could be variations in physical
demands.
Abbreviations: EMG, electromyography; HLA, human leucocyte antigen; MHC, major histocompatibility complex; MRI, magnetic resonance imaging
Polymyositis and dermatomyositis are chronic inflammatory muscle diseases that are clinically characterised by proximal muscle weakness, and dermatomyositis by skin abnormalities. Typical histopathological findings in the muscle tissue are inflammatory infiltrates, mainly composed of T lymphocytes and macrophages.
Inflammation may induce muscle weakness by several mechanisms.
Muscle cell necrosis induced by infiltrating inflammatory cells
is often proposed. However, there is a lack of correlation between
the clinical symptoms and the degree of inflammatory infiltrates
in patients with myositis.1?4 Other phenotypic changes
that have been related to muscle weakness include immune function-related
molecules produced locally in muscle tissue, such as major histocompatibility
complex (MHC) class I and II antigens and interleukin (IL)1
expressed on muscle fibres and microvessels. MHC class I antigen,
which normally is not expressed on muscle fibres, is expressed
on muscle fibres independent of inflammatory cell infiltrates
in patients with myositis and muscle weakness. This was observed
in patients in both early and late chronic phases of the disease.2,5?11
A role of MHC class I antigens in causing muscle weakness is
supported by the observation that transgenic mice with specific up
regulation of MHC class I antigens on muscle fibres develop muscle
weakness before infiltration of inflammatory cells.12
The most consistently expressed cytokines in muscle tissue of
patients with polymyositis and dermatomyositis are the proinflammatory
cytokines IL1 and IL1? which have been found in both
the early and the chronic phases, during and after immunosuppressive
treatment.2,6,13 Notably, in muscle tissue from patients with
muscle weakness but without detectable inflammatory infiltrates,
increased IL1 expression was observed in the endothelium of
capillaries, suggesting that microvessels with IL1
expression could cause muscle symptoms in patients with polymyositis
or dermatomyositis.2,5 Involvement of microvessels in the
disease mechanisms of dermatomyositis was already proposed by the
observation of a decreased number of capillaries in patients with
dermatomyositis, even in patients with early disease without
inflammatory infiltrates.14?17
To further determine the role of the observed phenotypes of
microvessels with IL1 expression and that of muscle fibres with
MHC class I expression in causing clinical symptoms, we compared the
expression of these molecules in two different muscles from the same
patient, one from a clinically symptomatic (ie, with subjective
muscle weakness) and the other from a clinically asymptomatic muscle.
We also investigated histopathological changes, cellular infiltration
of T cells and macrophages in the same biopsy specimens.
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PATIENTS AND METHODS |
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). Five patients had definite polymyositis and three
had probable polymyositis (all had symmetrical proximal muscle
weakness, raised serum creatine kinase levels and muscle biopsy
abnormalities typical of polymyositis). One patient had definite
dermatomyositis, and two patients had probable dermatomyositis with
muscle symptoms and a raised creatine kinase level in addition to the
classic rash. No patient fulfilled the diagnosis of inclusion-body
myositis according to Griggs?s criteria.20 The mean age
at the time of the biopsies was 58 (range 38?76) years.
We aimed to take the biopsy specimens before starting
immunosuppressive
treatment. Concomitantly, however, treatment had to be started in two
patients for clinical reasons before one of the biopsies was carried
out (patients 2 and 9 in table 1
). Patient 1 had been prescribed a low dose of prednisolone
2 weeks before biopsy sampling and diagnosis by a general
practitioner.
Muscle weakness was confirmed by a reduced functional index, performed on all but two patients.21 The median (range) values for the right and left side of the body were 40 (10?63) and 41 (10?63) points, respectively; 64 points indicated maximum capacity.
Controls
Muscle biopsy specimens from five healthy
people and one from a patient with myalgia without clinical or
histopathological signs of muscle disease were included as controls
(4 women and 2 men). The mean age was 51 (range 47?56) years. Muscle
biopsy sites were the musculus vastus lateralis in the five controls
and the musculus tibialis anterior in the patient with
myalgia.
Muscle biopsies
On the basis of where the patients experienced subjective symptoms,
primarily muscle weakness, biopsy was carried out on two muscles, one
symptomatic and the other asymptomatic. From each site, at least two
biopsy specimens were taken, one for diagnosis and the other for
research. The biopsy specimens were obtained under local anaesthesia
using the semi-open biopsy technique, and frozen in isopenthane on
dry ice and stored at ?80?C.22,23 The coded biopsy specimens were
analysed for histopathological signs of myositis (inflammation,
regenerating and degenerating muscle fibres, muscle fibre atrophy,
and central nuclei) by an experienced neuropathologist (IN). Results
were assessed on a five-point scale for inflammation or abnormal
fibres: 0, absent/absent; +, scattered inflammatory cells/close to
normal; ++; mild inflammation/few; +++, moderate/moderate; ++++,
pronounced/many. Degenerating muscle fibres were defined as necrotic
fibres invaded by inflammatory cells. Atrophic fibres were defined as
muscle fibres with prominent size reduction.
The local ethics committee at Karolinska University Hospital, Solna, Sweden, approved the study, and all patients gave informed consent for participation.
Immunohistochemistry
Table 2 lists the antibodies used for immunohistochemistry.
Staining for CD3 (T cells), CD163 (macrophages), human leucocyte
antigen (HLA)-A/B/C (MHC class I) and HLA-DR (MHC class II)
expression was performed with a standard immunohistochemistry
protocol.24 Staining with anti-IL1 antibody was performed according to
a protocol described earlier,13
with modifications.2 For all stains, an isotype-matched
irrelevant antibody was included as a negative control. The first and
last sections were also stained with haematoxylin?eosin to evaluate
the histopathology and to confirm that the histopathological changes
remained unchanged throughout the biopsy specimen.
Evaluation of immunohistochemistry
All
immunohistochemical stains for patients and controls were analysed on
coded slides on whole-tissue sections by conventional microscopic
assessment (Polyvar microscope; Reichert-Jung, Nussloch, Germany) by
two independent observers (CD and IEL). Results are given as the mean
of the two assessments. The whole-tissue section was analysed (median
area 4.2 mm2, range 0.1?12.1 mm2). CD3 and
CD163 expression is presented as the number of CD3-positive and
CD163-positive cells/mm2. Expression of MHC class I
(HLA-A/B/C) and class II (HLA DR) antigens was assessed as follows:
?, negative fibres, positively stained endothelial cells only; +,
1?10% positively stained fibres; ++, 11?25% positively stained
fibres; +++, 26?50% positively stained
fibres; ++++, 51?75% positively stained fibres; and +++++, 76?100%
positively stained fibres.
IL1 expression was estimated separately in different structures.
The numbers of IL1-positive infiltrating mononuclear cells,
capillaries, larger blood vessels and muscle fibres were calculated
per square millimetre. Total IL1
expression was also analysed using a previously described
computerised image analysis system5 under 680x magnification. The
images were analysed with a Quantimet 600 image analyser (Leica,
Cambridge, UK). The image processor was directed by a personal
computer, and a special program was written in a high-level
programming language, QUIPS, for this
application. Computerised image analysis measures the total
positively stained area for IL1 as well as the total area per
tissue section. The image analysis results are presented as the
percentage positively stained IL1 area in each tissue section.
Statistical analyses
Data were analysed using the software program Statistica V.6.0 from
Stat Soft (Tulsa, Oklahoma, USA) Owing to the small number of
patients and controls and the non-normality of the dataset,
non-parametric tests were used to test for significance. We used the
Mann?Whitney U test and Wilcoxon?s matched
pairs test; p<0.05 was considered significant; p>0.05 was
considered not significant.
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RESULTS |
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, fig 1). These changes included both presence of inflammatory
cells and regenerating and degenerating fibres. Muscle fibre atrophy
was similarly absent, close to normal or with a few atrophic fibres
in both the symptomatic and asymptomatic muscle, with the exception
of one patient with polymyositis who had moderate muscle fibre
atrophy in the symptomatic muscle and no muscle fibre atrophy in the
asymptomatic muscle. We also compared the two samples taken from each
biopsy site, in both the symptomatic and asymptomatic muscles (table
3). Regeneration muscle fibres were observed more often in
the biopsy specimen taken for diagnosis rather than in the specimen
taken for research. We found no difference in histopathological
findings between the first and last sections in the consecutive
series of sections. We aimed to take the biopsy specimens before
starting immunosuppressive treatment, but this was not always
feasible for ethical and practical reasons (table 1). In two patients (patients 2 and 9), there was a time
lapse of 4 and 2 months respectively between the time points at which
the biopsies on the symptomatic and asymptomatic muscle were
performed. The difference in time and treatment, however, did not
affect the histopathological changes or MHC and IL1 expression. Even so, the patient who was
receiving low-dose prednisolone still had inflammation, and MHC and
IL1
were expressed in similar amounts as in the other patients. The
biopsy samples that were taken after starting prednisolone treatment
still showed histopathological changes, presence of T cells and
macrophages, expression of MHC class I and II antigens on muscle
fibres, and IL1 expression on capillaries in both
symptomatic and asymptomatic muscles in comparably large amounts.
Specimens from controls showed no histopathological changes, with the
exception of one, which had a few scattered mononuclear inflammatory
cells surrounding one fibre and a few atrophic fibres.
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IL1
expression
Using conventional microscopy, we recorded a
significantly increased number of IL1-positive capillaries (median, range) in
symptomatic (13.3/mm2, 0.6?76.5/mm2) and
asymptomatic (12.4/mm2, 2.3?35.7/mm2)
muscles compared with controls (3.4/mm2, 1.4?5.3/mm2;
fig 2A). We found no significant difference between the numbers of
IL1-positive capillaries in symptomatic versus asymptomatic
muscles. Some of the IL1-expressing capillaries were morphologically
changed, with thick, high endothelium venule (HEV)-like or swollen
endothelial cells in both the symptomatic and asymptomatic muscle
biopsy specimens (fig 3).
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expression was significantly increased in symptomatic muscles (4.9/mm2,
0.1?25/mm2) compared with healthy control muscles. We
found no significant difference between symptomatic and asymptomatic
muscles (4.1/mm2, 0.9?8.2/mm2) or between asymptomatic
and control muscles (1.9/mm2, range: 0.6?6.0/mm2). IL1
was not expressed on muscle fibres in any of the three groups.
Total IL1 expression per tissue section analysed by
computerised image analysis was significantly higher in biopsy
specimens of symptomatic muscle than in those of controls, but when
comparing specimens of asymptomatic muscle and normal controls, the
difference did not reach significance (p = 0.05). We found no
significant difference in total IL1
expression in the symptomatic muscles compared with asymptomatic
muscles (fig 2B).
Expression of MHC class I and II antigens in muscle
fibres
MHC class I (HLA-A/B/C) expression was similarly strongly
(75?100% positively stained area per muscle tissue section) expressed
in muscle fibres in eight specimens each from symptomatic and
asymptomatic muscles, and one patient did not express any MHC class I
antigen on muscle fibres in either biopsy specimen. Although this
patient did not have any inflammatory cell infiltrates in the biopsy
specimens, they nonetheless had muscle and skin symptoms,
electromyography (EMG) and creatine kinase results typical of
dermatomyositis. There was strong agreement of expression of MHC
class I antigens between symptomatic and asymptomatic muscles, with a
few exceptions; one patient expressed it strongly only in the
asymptomatic muscle (table 4) and another patient moderately in the symptomatic
muscle biopsy and strongly in the asymptomatic muscle biopsy
specimen. Six patients had a weak to moderate expression of MHC class
II (HLA-DR) in muscle fibres both in the symptomatic and asymptomatic
muscles. One patient had stronger expression in the symptomatic than
in the asymptomatic muscle. No MHC class II antigens were expressed
on muscle fibres in four patients. Neither MHC class I nor class II
antigens were expressed on muscle fibres in controls, so we did not
think it necessary to carry out statistical analyses comparing
patients and controls (table 4).
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DISCUSSION |
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-expressing microvessels, which was
increased in both symptomatic and asymptomatic muscles compared with
that in control specimens.
To our knowledge, this is the first study to investigate two different muscles from the same patient with myositis, one from a clinically symptomatic (all proximal) and the other from an asymptomatic (mostly distal) muscle. As the histopathological changes and the immunohistochemistry data including T cells and macrophages were similar in the two biopsies from each patient, we believe that the data are valid and may be generalised despite the limited number of patients; it would be unethical to include more patients in the study. To further strengthen our results, we analysed two biopsy samples from each site. Thus, four samples were analysed from each patient, all with similar histopathological findings, two from symptomatic and two from asymptomatic muscles, with the exception of the numbers of regenerating fibres, which are often scattered and unevenly distributed. We found that the presence and degree of muscle fibre atrophy, with one exception, was similar in both the symptomatic and symptomatic muscle, and thus could not explain the clinical symptoms. The choice of biopsy location in each patient was, in our study, based on where the patients experienced subjective clinical symptoms such as weakness, tenderness and pain. Magnetic resonance imaging (MRI) was not included in the protocol, and was performed on only five patients for clinical purposes and did not correspond to muscle biopsy sites. Two patients nonetheless had MRI results typical of myositis. EMG was performed on all patients (seven had EMG results typical of myositis), but even so, muscles analysed did not correspond to muscle biopsy sites. It is therefore not possible to draw any conclusion between MRI and EMG results and findings in the muscle biopsy specimens. To confirm impaired muscle function, we used the functional index test. A limitation of this test is that the tasks in the functional index do not correspond exactly to the specific muscles that we have investigated. All specimens from symptomatic muscle were taken from proximal muscles and those from asymptomatic muscles were taken from distal muscles. This corresponds well to the classical distribution according to textbooks on muscle weakness in patients with myositis.25 Our results, however, suggest that both proximal and distal muscles are affected by infiltration of inflammatory cells in polymyositis and dermatomyositis, and, thus, as previously reported, the degree of inflammatory infiltrates does not correspond to the localisation of the patient?s subjective muscle symptoms. One of the controls had a few mononuclear inflammatory cells surrounding one muscle fibre, but T cells and macrophages were present in equal proportions as in other normal controls. This control patient had no clinical muscle symptoms, thus we believe that this finding could be within normal limits.
The generalised histopathological muscle involvement of both proximal and distal muscle groups was also supported by the extensive expression of MHC class I and II antigens on muscle fibres in both symptomatic and asymptomatic muscles, but controls were all negative. In one patient, the symptomatic muscle specimen did not express MHC class I antigens on muscle fibres, but the asymptomatic muscle expressed it strongly. This could be due to sampling error, as the symptomatic muscle biopsy specimen was small; this strengthens the need for adequate size of the biopsy specimen. One patient with typical dermatomyositis did not express any MHC class I antigen on muscle fibres in any biopsy sample. Although expression of MHC class I antigens on muscle fibres is a common finding in patients with polymyositis or dermatomyositis, it was not present in 38% of patients with polymyositis or dermatomyositis at the time of diagnosis in a recent study.26 Expression of MHC class II antigens on muscle fibres was less often seen in our study, but still without difference between symptomatic and asymptomatic muscles. The persisting expression of MHC class I antigens on muscle fibres, despite immunosuppressive treatment given to a few of our patients before carrying out biopsy, is consistent with a previous finding in patients with polymyositis and dermatomyositis.2,6,26
The number of capillaries expressing IL1 was also increased to a similar
extent compared with the control biopsy specimens in both the
symptomatic and asymptomatic muscles. We have previously reported an
increased number of IL1-expressing microvessels independent
of closely localised inflammatory infiltrates in both the early and
the late chronic phases of polymyositis and
dermatomyositis.2,5,6 Those specimens were obtained mainly from
symptomatic thigh muscles. To those previous observations, we can now
add an increased number of IL1-positive capillaries in distal and asymptomatic muscle. We
believe that the increased number of IL-expressing capillaries in both symptomatic and
asymptomatic muscles indicates a general involvement of the
microvessels in muscles of patients with polymyositis or dermatomyositis.
The larger and thick, high endothelium venule (HEV)-like IL1-positive
capillaries indicate that the endothelial cells of the capillaries
are activated. This is a prerequisite to allow extravasation of
inflammatory cells into the muscle tissue, and is found to an equal
degree in both symptomatic and asymptomatic muscles. Other earlier
findings to support the hypothesis that the endothelial cells might
have an important role in the pathogenesis of myositis are that
intercellular adhesion molecule 1 and vascular cell adhesion molecule
1 have been detected on endothelial cells in all three subtypes of
the disease (polymyositis, dermatomyositis and inclusion-body
myositis).27,28
We could not detect any difference in IL1 expression, the presence of
inflammatory cell infiltrates with T cells and macrophages, or
expression of MHC class I or II antigens, that could explain the
muscle symptoms. Another possibility is that the clinical
symptoms, which are predominantly localised to proximal muscles in
patients with myositis, are caused by different physical demands on
proximal and distal muscles or by different characteristics of muscle
fibres in different muscle groups. Similar mechanisms could also
account for the distribution of muscle weakness in other myopathies
such as muscular dystrophies.29 The physical demands of,
for example, endurance and strength and the physiological demand of
oxygen supply are higher in thigh muscles than in calf muscles. Thus,
a local tissue hypoxia, which could be a consequence of inflammation,
could make the thigh muscles more
prone to clinical symptoms than the distal muscles.30?32
Interestingly, IL1
and as other molecules previously reported to be up regulated in
myositis, such as transforming growth factor ? intercellular adhesion
molecule 1 and vascular cell adhesion molecule 1, could all be up
regulated by hypoxia.13,27,33 In addition, the previously reported
reduced levels of adenosine
triphosphate in muscle of patients with myositis support the
hypotheses of local muscle tissue hypoxia.30 Thus, an increased
physical demand in a muscle tissue with hypoxia could lead to a
further reduction in oxygen tension, which could possibly
explain the reduced functional capacity and pain that the patients
experience.34
In conclusion, we have shown that, independent of clinical
symptoms,
inflammatory cells, expression of MHC class I and II antigens
on muscle fibres and IL1-expressing microvessels are present
in both proximal and distal muscles. These findings indicate that the
inflammatory changes constitute a general phenotype in skeletal
muscle tissue of patients with polymyositis or dermatomyositis, and
suggest that other factors are more important in causing the clinical
symptoms.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Funding: This study was supported by grants from the Swedish Rheumatism Association, King Gustaf Vth 80-year Foundation, the Swedish Research Council 2002-74X-14045-02A, Professor Nanna Svartz Foundation, Magnus Bergvalls Foundation, B?je Dahlin Foundation, Stiftelsen Clas Groschinskys Minnesfond, Karolinska Institutet Foundation and the Vardal Foundation.
Competing interests: None.
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REFERENCES |
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