Iara de Messias Reason, R. Padre Camargo 280, 80·069–900 Curitiba – PR Brazil. E-mail: firstname.lastname@example.org
Although mannose-binding lectin (MBL) is known to be involved in the primary defense against microorganisms, there are emerging lines of evidence for an active proinflammatory role for MBL in different chronic diseases. In this study we determined the circulating levels of MBL in patients with rheumatic heart disease (RHD). A total of 100 patients (77 women, 23 men; mean age 45·8 ± 11 years, range 19–76 years) with chronic RHD, and a previous diagnosis of rheumatic fever, were studied. Transthoracic echocardiography was performed in all patients to evaluate valvular heart disease. Ninety-nine healthy individuals matched for age, sex and ethnic origin were included as controls. MBL concentration was measured by enzyme-linked immunosorbent assay and C3 and C4 levels by turbidimetry. MBL levels were significantly higher in patients with RHD than in healthy subjects (mean ± SEM: 3036·2 ± 298·9 ng/ml versus 1942·6 ± 185·5 ng/ml, P < 0·003). In addition, MBL deficiency was more prevalent in controls (17·1%) than in patients (9% P < 0·09). Concentrations of C4 were within the normal range (22·7 ± 0·8 mg/dl, normal: 10·0–40·0 mg/dl), while C3 concentrations were found to be elevated (109·2 ± 3·6 mg/dl, normal: 50·0–90·0 mg/dl). No correlation was observed between serum MBL levels and valve area or the type of surgical procedure. The significantly elevated circulating MBL levels in patients with RHD together with the greater prevalence of MBL deficiency in controls suggest that MBL may cause undesirable complement activation contributing to the pathogenesis of RHD.
Rheumatic fever (RF) is the most common cause of acquired valular disease in children and young adults worldwide. Although the incidence of RF has decreased over the last few decades in developed countries, the disease continues to be a serious health problem in developing countries, including Brazil. Mitral stenosis, the classical cardiac manifestation of chronic rheumatic heart disease (RHD), is a late complication of RF which takes at least 2 years to develop after the initial outbreak of RF. Generally, a period of 10–20 years is necessary after an initial episode of carditis for the clinical manifestations of rheumatic mitral stenosis to become evident .
The pathogenic mechanisms responsible for the development of RF/RHD are associated to an abnormal host immune response (both at humoral and cellular level) to crossreactive streptococcal antigens. In fact, RF/RHD is considered to be a streptococcal-induced autoimmune disease, due to antigenic mimicry between streptococcal antigens and relevant host antigens that occurs in genetically susceptible individuals . Although extensively studied, the pathogenesis of the disease is not yet fully understood.
Mannose-binding lectin (MBL) is a calcium dependent lectin shown to play an important role in the first line of host defense against microorganisms. MBL binds to different sugars such as mannose, fucose, glucose and N-acetyl-D-glucosamine present on the surface of bacteria, fungi, protozoa and viruses. Binding of MBL to microbial surface, promotes C1- and antibody-independent activation of complement by the lectin pathway . In addition, MBL mediates the opsonization and phagocytosis of microorganisms through interaction with collectin receptors present on phagocytic cells . The concentration of MBL in plasma is determined genetically, primarily by the genetic polymorphism of the first exon of the structural gene and promoter region. Normal serum levels of MBL range from 800 to 1000 ng/ml in healthy Caucasians, however, wide variations can occur due to point mutations in codons 52, 54 and 57 of exon 1 and/or in the promotor region of the MBL gene . The exon 1 mutations are referred to as D, B and C variants, respectively, in contrast to the wild type A. Heterozygous individuals for these mutations have a substantial decrease in MBL serum concentration, whereas MBL is undetectable in the serum of homozygous individuals. MBL deficiency is considered to be the most common immunodeficiency, with B mutation occurring in 26% of Caucasian populations . While it is well known that MBL deficiency is associated with increased susceptibility to infection during infancy  and adulthood , evidence of an active proinflammatory role for MBL in different chronic diseases is emerging. Recent studies have demonstrated that MBL can bind to the endothelium causing excessive complement activation and subsequent tissue damage . On the other hand, MBL deficiency may be advantageous in some circumstances since MBL may lead to an increased cytokine secretion by macrophages . It has also been shown that MBL is associated to disease severity in both infectious and autoimmune disease [10,11]. There is no data available on the role of MBL in RHD. In the present study we present evidence that significantly elevated levels of MBL may contribute to the pathogenesis of chronic RHD.
PATIENTS AND METHODS
Patients and controls
One-hundred patients with chronic RHD being accompanied by the Cardiology Outpatient Clinic of Hospital de Clínicas of the Federal University of Paraná, Curitiba, Brazil (23 men and 77 women, mean age of 45·8 ± 11 years, range 19–76 years) were studied (Table 1). All subjects had a clinical history compatible with RF and an echocardiogram confirming mitral valve involvement. Twenty-eight of the patients were not submitted to any surgical valve procedure whereas 72 underwent the following surgical intervention: 8 exchanged for a metal valve; 24 exchanged for a biological valve; 14 had balloon valvuloplasty and 26 underwent commissurotomy. Eighty-two patients were of Caucasoid origin, 12 were Mullatos and one was an Indian. Patients with active RF, infection, history of neoplasias, infective endocarditis or other inflammatory diseases were excluded from the study.
Table 1. Demographic, clinical characteristics and MBL, C3 and C4 levels in patients with chronic rheumatic heart disease submitted or not to surgery
Ninety-nine healthy volunteers that matched with the patients for age, sex, ethnic and geographical origin were selected as controls. Both patients and controls signed a free informed consent form, and the study was approved by the Research Ethics Committee of Hospital de Clínicas of the Federal University of Paraná.
Venous blood (10ml) was collected from each patient and control and allowed to clot. Serum was collected after centrifugation, separated and aliquots were stored at − 80°C until used.
MBL, C3 and C4 assays
Serum MBL levels were determined by ELISA using commercial kits (Staten Serum Institute, Denmark). Individuals with MBL values below 50·0 ng/ml were considered to be deficient. C3 and C4 levels were measured in 80 patients by immunoturbidimetry (Dade Behring, Germany), C3 levels between 50·0 and 90·0 mg/dl and C4 levels between 10·0 and 40·0 mg/dl were considered as normal.
Data are given as mean ± SEM. Statistical differences between groups were determined by the two-tailed Student t-test. Pearson and Spearman correlation were used to estimate the strength of association between variables. The chi-square test with Yates correction was used to determine differences between observed and expected values. All statistical analysis was performed using Statistica for Windows version 99 software. Differences were considered to be significant when P < 0·05.
Serum MBL levels
The distribution of circulating MBL concentration among patients and controls is presented in Fig. 1. MBL levels were significantly higher in patients than in controls (mean ± SEM 3036·2 ± 298·9 versus 1942·6 ± 185·5 ng/ml; P < 0·003 Fig. 2). MBL was undetectable in 9/100 (9·0%) patients and in 17/99 (17·1%) controls (P < 0·09 Fig. 1). Circulating MBL concentrations higher than 1000 ng/ml were observed in 71·0% of the patients and 55·5% of the controls (P < 0·035, Fig. 3). No significant difference in MBL levels was observed between the patients who did or did not undergo valve surgery (mean ± SEM 2944·2 ± 185·5 versus 3269·3 ± 298·0 ng/ml; P < 0·66, Table 1) or between female and male patients either in the patient or control group (P = ns). There was no correlation between valve area and age (r = −0·16; P < 0·41) or between valve area and MBL levels in the nonoperated patient group (r = 0·23; P < 0·31).
Regarding the type of surgery, no significant difference was observed in MBL levels between patients submitted to commissurotomy or balloon valvuloplasty (mean ± SEM 3321·5 ± 529·4 ng/ml, n = 38) and patients submitted to valve exchange (mean ± SEM 2451·4 ± 380·0 ng/ml, n = 34; P < 0·29)
Serum C3 and C4 levels
Serum C3 levels were above the normal range in RHD patients with a mean of 109·2 ± 3·6 SEM mg/dl (n = 80) and significantly higher in operated (n = 58) than in nonoperated patients (n = 22) (mean ± SEM, 113·6 ± 4·5 versus 97·4 ± 4·2 mg/dl; P < 0·04 Table 1). C4 concentrations in the RHD patients were within the normal range (mean ± SEM 22·7 ± 0·8 mg/dl). No significant differences in C4 levels was observed between the operated and the nonoperated patients (mean ± SEM 23·5 ± 1·0 versus 20·7 ± 0·85 mg/dl; P < 0·16 Table 1). A positive correlation was observed between C3 and C4 levels (r = 0·27; P < 0·01), but not between C3 and MBL levels (r = −0·17; P < 0·23) or between C4 and MBL levels (r = −0·008; P < 0·16).
Whereas MBL deficiency has been associated with different infectious and auto-immune diseases, including rheumatic disorders, high MBL levels associated to disease has only been recently reported . Low levels of the protein have been related to a poor prognosis in rheumatoid arthritis perhaps due to the modulatory action that MBL exerts on the secretion of tumour necrosis factor α, a central molecule in the pathogenesis of rheumatoid arthritis . Low MBL levels have also been associated with adult dermatomyositis and are probably related to a reduced clearance of apoptotic keratinocytes . Genotypes related to a lower production of MBL have also been linked to the development of systemic lupus erythematosus [15,16] and increased susceptibility to infection in this disease .
In the present study, serum MBL levels were found to be significantly elevated in RHD, but were not correlated with the severity of heart disease as determined by a transthoracic echocardiogram. In addition, a significantly larger number of individuals with MBL levels above 1000 ng/ml were observed in the patient group. On the other hand, MBL deficiency was more prevalent among healthy controls. These results suggest that MBL deficiency may represent an advantage against the development of rheumatic mitral stenosis and that increased MBL levels may be related to the development of the disease. Although the MBL genotype cannot be predicted with precision based on serum concentration, it is known that individuals homozygous for the A allele (A/A) typically show MBL levels above 1500 ng/ml, levels 6–8 times higher than those observed for heterozygous individuals (A/O, where O can be B, C or D) and that the O/O combination is related to undetectable MBL .
In this study, C3 levels were increased in the patients with RHD and significantly higher in the operated patients when compared to the nonoperated patients. These findings suggest the presence of ongoing inflammation in these patients with probable complement activation due to the presence of more severe mitral stenosis or of an artifical valve. However, MBL levels did not differ between the latter two groups. C3 is an acute-phase protein whose levels significantly increase in response to infections or to an inflammatory reaction, while MBL levels only show a moderate increase in these circumstances . In addition, there was no correlation between C3 and MBL levels, indicating that elevated MBL concentration might be an independent event.
Involvement of complement system in RF/RHD has been shown in different studies [17–19]. Antibodies found in the serum of patients with RF that show molecular mimicry between streptococcal and human antigens were able to cause human fibroblasts lyses in vitro in the presence of complement . Serum levels of C3d and immune complexes were shown to be increased in both the patients with acute RF and chronic RHD when compared to normal controls . Also, complement deposition in heart tissue was observed in autopsy samples of patients with RHD . In addition, the high frequency of the rare C4A*6 allele in patients with RHD, suggests an immunogenetic role for complement in the disease . The most severe sequela of acute RF is chronic RHD. In adults mitral stenosis almost always results from postrheumatic inflammation and degenerative disease . Evidence of a continuous inflammatory reaction in chronic RHD has been demonstrated by several authors. Increased serum levels of adhesion molecules (E-selectin, ECAM-1 and VCAM-1) , apolipoprotein B , high sensitivity C-reactive protein  and advanced oxidation protein products (unpublished data) have been observed in patients with chronic RHD when compared to normal controls.
Elevated serum levels of MBL have been implicated in the pathogenesis of renal manifestations of Henoch-Schönlein purpura , in IgA nephropathy , in other forms of human glomerulonephritis , and in vascular complications of diabetes mellitus type 1 . Although MBL is an acute-phase protein produced by the liver , its levels only show a moderate increase in inflammatory diseases and are determined genetically. The elevated MBL levels observed in patients with chronic RHD might corroborate the chronic inflammatory activity present in these individuals and contribute to valve injury through complement activation. In addition, MBL may act as an immunomodulatory molecule, inducing a higher secretion of cytokines by macrophages .
Under normal conditions, MBL does not bind to the organism's own tissues, but in situations of cellular hypoxia, glycosylation of cell surfaces may occur, leading to the deposition of MBL followed by complement activation . In an experimental study, the blockade of MBL with monoclonal antibodies reduced neutrophil infiltration and the amplitude of the inflammatory response in myocardial tissue reperfused after a period of hypoxia . The significantly elevated levels of MBL observed in chronic RHD suggest that MBL may represent a pathogenic factor in the complex physiopathology of the disease, whereas MBL deficient individuals might be less susceptible to develop chronic RHD.