C4d as a marker of complement activation in dermatomyositis muscle tissue
Correspondence Address: Source of Support: None, Conflict of Interest: None DOI: 10.4103/0028-3886.237014
Source of Support: None, Conflict of Interest: None
Keywords: C4d, complement, dermatomyositis, muscle biopsy
The idiopathic inflammatory myopathies (IIMs) are a heterogenous group of acquired muscle diseases which are potentially treatable. The pathologic classification and identification of disease subgroups are extremely important for assessing treatment options and prognosis in individual patients.
Dermatomyositis (DM) differs from other IIMs by the characteristic skin rash and distinct histological and immunopathological features. It is also characterized by its association with interstitial lung disease, connective tissue disease (CTD), or malignancy. Early diagnosis and institution of adequate therapy are essential to alleviate muscle damage, improve muscle weakness and quality of life. DM is considered a microvasculopathy, characterized by injury to the capillary endothelium leading to depletion of capillaries in the muscle; the damage to muscle is manifested as perifascicular atrophy (PFA) and is accompanied by inflammatory cell infiltrates. However, PFA is seen in only 50% of adults with DM when biopsied early in the course of the disease. The microangiopathy in DM is complement mediated. The earliest abnormality is formation and deposition of membrane attack complex (MAC) along with complement components on the endomysial blood vessels before structural changes take place or inflammatory infiltrates occur.,,, Detection of MAC was used as a diagnostic tool in the diagnosis of DM with high sensitivity and specificity., Both classical and alternate complement cascade members along with regulators are highly upregulated in DM patients. Therapies that intercept complement activation/assembly and deposition of MAC have shown beneficial effects in DM., Hence, identification of complement activation helps in diagnosing DM and in differentiating it from other IIMs.
Complement split products have emerged as useful markers of antibody-mediated rejection in solid organ transplants. C4d is now increasingly recognized as a potential biomarker where antibodies can cause tissue damage such as systemic autoimmune diseases. The role of C4d as a core diagnostic tool in the detection of antibody-mediated injury in renal allografts and other transplants has been established. As C4d antibody is widely used for several indications in a routine surgical pathology laboratory, we aim to study C4d expression in the microvasculature of DM muscle biopsies, as a marker of complement activation.
Muscle biopsies from patients diagnosed as definite DM (10), nonspecific myositis (9) including myositis associated with systemic lupus erythematosus (SLE) (2), rheumatoid arthritis (RA) (2), systemic sclerosis (SS) (2), overlap myositis (3), and one each of necrotizing autoimmune myopathy (NAM), inclusion body myositis, and normal muscle, according to ENMC criteria 2004, were included in the study. The demographic data, clinical and laboratory findings were retrieved from medical records. The muscle biopsy was performed from vastus lateralis in all patients. The biopsies were reviewed with hematoxylin and eosin, ATPase preincubated at pH 9.4 and 4.6, succinic dehydrogenase (SDH), nicotinamide adenine tetrazolium reductase, cytochrome c oxidase (COX), COX-SDH, modified Gomori trichrome, and Masson's trichrome.
The biopsies were evaluated for PFA, endomysial/perivascular/perimysial inflammation, rimmed vacuoles, muscle fiber necrosis, degeneration, regeneration, and fibrosis. Immunohistochemistry was done on cryostat sections using avidin-biotin with antibodies for CD34 (1:50 Novocastra) and C4d (prediluted ready to use antibody Biogenex). C4d is a protein A purified rabbit polyclonal antibody diluted in phosphate-buffered saline, pH 7.6 containing 1% BSA and 0.09% sodium azide [Figure 1] and [Figure 2].
The capillary loss on CD34 was assessed in perifascicular and endomysial areas as mild, moderate, and severe. C4d was assessed as present or absent with mild, moderate, or severe intensity in the endomysial capillaries and perimysial arterioles.
Immunohistochemistry for inflammatory cell categorization, MHC-1 staining, and MAC deposition were not done. Election microscopic studies also were not performed.
The demographic, clinical, laboratory, and pathological features are given in [Table 1].
Definite dermatomyositis (n = 10)
There were 4 males and 6 females with their age ranging from 22 to 65 years (mean 47.4 years). All patients presented with rash, proximal muscle weakness, and elevated creatine kinase (CK) [400–2000 IU/L]. Electromyoneurography (EMNG) was available in 8 patients, and it showed a myopathic pattern.
All biopsies showed perifascicular atrophy; necrotic, degenerating, and regenerating fibers were seen in predominantly perifascicular distribution. Perivascular inflammatory infiltrate was seen in all biopsies. There was no endomysial inflammation. Perimysial and endomysial fibrosis was seen in six biopsies. CD34 immunostaining showed moderate degree of capillary loss in the endomysium. It was multifocal involving small groups of muscle fibers, with predominant perifascicular distribution. Immunohistochemistry with C4d showed staining of perimysial arterioles with moderate intensity in all biopsies. Endomysial capillaries, predominantly in the perifascicular area, showed positive staining in 3 biopsies [Figure 1] and [Figure 2].
Nonspecific myositis (n = 9)
There were 9 females with age ranging from 17 to 44 years (mean 33.57 years). These included two patients each of SLE, RA, and SS associated with myositis and overlap myositis in three patients including SS (2) and RA (1). All patients had proximal muscle weakness.
Muscle biopsy showed perivascular inflammation with scattered necrotic and degenerating fibers in all biopsies. There was no PFA; moderate capillary loss in two-third, and C4d positivity in perimysial arterioles in two-third biopsies of patients with overlap myositis were seen. In all other biopsies, there was no capillary loss and no C4d positivity. Necrotic muscle fibers in all the biopsies showed positivity.
Inclusion body myositis (n = 1)
The patient was a 55-year old male who presented with both proximal and distal muscle weakness. There was no rash. CK was 1120 IU/L. EMNG showed a myopathic picture.
Muscle biopsy showed endomysial inflammation around nonnecrotic fibers, and there were rimmed vacuoles. There were scattered necrotic and degenerating fibers. There were few COX-negative fibers. CD34 immunostaining showed no capillary loss. Occasional perimysial arteriole showed weak staining with C4d.
Necrotizing autoimmune myopathy (n = 1)
This was a 42-year old female. She presented with proximal muscle weakness. The CK was 2040 IU/L. She was diagnosed to have carcinoma ovary with elevated CA 125 (123.4 U/L).
Muscle biopsy showed numerous necrotic fibers with very sparse inflammation. There was no PFA. There were randomly scattered atrophic and degenerating fibers. CD34 staining showed focal endomysial capillary loss, and C4d showed positivity in perimysial arterioles and endomysial capillaries. Necrotic fibers showed positivity with C4d.
Normal (n = 1)
A 23-year old male presented with myalgias. There was no rash, no muscle weakness, no associated CTD. Muscle biopsy showed an occasional degenerating fiber. There was no PFA and no inflammation. CD34 staining showed no capillary loss. C4d was negative in both endomysial and perimysial vessels.
C4d is an inactive split product formed from C4 during activation of complement by the classical/lectin-mediated pathway. Following activation and degradation of the C4 molecule, thioester groups are exposed which allow transient, covalent binding of the degradation product C4d to endothelial cell surfaces, and extracellular matrix components of vascular basement membranes to accumulate near the sites of C4 activation. C4d forms a tight covalent bond with target cells and remains anchored to the tissue at the site of complement activation even when the antibodies dissociate over time. It is considered as a footprint for antibody-mediated tissue injury. Its role in autoimmune diseases is under investigation. Magro and Dyrsen studied C3d and C4d in skin biopsies of inflammatory skin diseases and observed a prominent mural positivity in the blood vessels of DM. In this study, we investigated C4d immunostaining in muscle biopsies of patients with DM, myositis associated with CTD, and other IIMs.
DM is characterized immunopathologically by a complement-mediated endomysial microangiopathy. Capillaries are an early and specific target of the disease process in DM., The terminal C5b-9 cytolytic MAC is the primary mediator of vessel injury in DM and was observed in the intramuscular microvasculature of a large percentage of patients with juvenile DM and a smaller percentage of adult DM. Pytel studied muscle biopsies of DM patients with both MAC and C4d and reported a high and statistically significant correlation in the pattern of staining with both antibodies. C4d immunostaining showed positivity in all the biopsies of definite DM in the perimysial intermediate-sized blood vessels in this study. This is in agreement with a similar pattern of complement deposits of MAC reported in larger vessels in the perimysial connective tissue in both childhood and adult DM cases., Intermediate-sized vessels are an important focus of immune and inflammatory pathologic processes in DM.
Pytel reported endomysial capillary staining with both MAC and C4d in a subset of DM. We did not perform MAC staining in this study; however, C4d staining was observed in the endomysial vessels in 3 out of 10 adult DM biopsies. The involvement of intermediate-sized perimysial vessels in DM, and the tendency of mononuclear cell inflammation to surround them has been observed in DM., C4d staining in these vessels, as observed in this study, may indicate the site of complement activation in DM. Following activation and degradation of the C4 molecule, thioester groups are exposed which allow transient covalent binding of the degradation product C4d to endothelial cell surfaces and extracellular matrix components of vascular basement membranes near the sites of C4 activations. All biopsies of definite DM showed PFA in the study and C4d positivity in all of them indicates that C4d may remain anchored to the site of complement activation even in late stages of the disease process. This is similar to MAC staining reported by Emslie-Smith and Engel in both early and late stages of DM. Regional variation with MAC staining was reported earlier; however, all biopsies showed a positive staining with C4d with variable distribution between perimysial and endomysial vessels.
Activation of the complement system is also involved in the pathogenesis of systemic autoimmune diseases. SLE is a prototype systemic autoimmune disease characterized by multiorgan involvement and complement components mediate autoantibody-initiated tissue damage. Complement activation is known to occur in RA and SS also.,, None of the muscle biopsies of SLE, RA, and SS showed C4d immunostaining in this study. Two of three biopsies from overlap myositis showed C4d immunostaining.
Despite similarities in clinical presentation, laboratory and electrophysiologic findings, and response to treatment, the pathogenesis of DM (both childhood and adult) is different from that of the other IIMs. Except NAM, the other IIMs and normal muscle biopsy included in the study did not show C4d positivity. Due to the immune mechanism, DM is treated with various immunosuppressive agents. A longer disease duration before the start of immune suppressive treatment is prognostically unfavorable., An early diagnosis and fast institution of adequate therapy are fundamental to achieve good results and quality of life, especially in children.
C5b-9 (MAC) is a marker of necrotic or damaged structures in DM.,, C4d stained the necrotic and degenerated fibers irrespective of the muscle disease in this study. Similar observations were made with MAC and C4d staining in the earlier studies., C4d staining is reported to be relatively inexpensive and easy to perform in the basic laboratory with ease of interpretation and few false positives. Pytel observed that C4d can be used as an alternative to MAC staining because of similar pattern of staining by both antibodies. Our study confirms these observations.
C4d immunostaining can be used as a marker of complement activation in DM muscle biopsies.
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Conflicts of interest
There are no conflicts of interest.
[Figure 1], [Figure 2]