1 Departments of Clinical and Laboratory Medicine and 2 Otorhinolaryngology, Fukui Medical University, Fukui and 3 Department of Medicine (II), Niigata University School of Medicine, Niigata, Japan
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Abstract |
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Methods. We used tonsillar lymphocytes isolated from the palatine tonsils of 15 patients with IgAN and 16 patients with chronic tonsillitis but without renal disease. We examined lymphocyte proliferation and production of IgA, IgG, and IgM antibodies against HP antigens by measuring thymidine uptake and concentrations of these antibodies in culture supernatants after lymphocyte incubation with HP antigens by ELISA.
Results. Lymphocytes from patients with IgAN showed a significantly higher stimulation index (SI) on exposure to HP antigens (thymidine incorporation in tonsillar lymphocytes exposed to HP (c.p.m.)/ thymidine incorporation in unstimulated tonsillar lymphocytes (c.p.m.)) than did controls (P=0.0015). Lymphocytes from patients with IgAN also showed a significantly higher IgA SI (concentrations of IgA against HP antigens in supernatants from HP-stimulated lymphocytes/IgA against HP antigens in supernatants from unstimulated tonsillar lymphocytes) than did controls (P=0.0004). We found positive correlations between concentrations of IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against HP antigens after HP stimulation.
Conclusions. Our results suggest that HP antigens stimulate tonsillar T and B lymphocytes in patients with IgAN and that these patients have polyclonal activation of lymphocytes against HP antigens, with isotype switching of antibody production from IgM to IgA.
Keywords: B cell; Haemophilus parainfluenzae; IgA nephropathy; T cell; tonsil
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Introduction |
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We have shown earlier that mucosal infections such as tonsillitis are often associated with an acute onset of IgAN [11]. IgAN is, therefore, an immune complex disease that is caused by a poor mucosal immune response to chronic environmental antigen exposure. We have previously observed that Haemophilus parainfluenzae (HP) is more commonly isolated from the pharynx of patients with IgAN than from patients with other glomerular diseases [12]. We also have identified glomerular deposition of antigens from the outer membrane of HP (OMHP) and noted increased serum concentrations of IgA against OMHP antigens in patients with IgAN [12]. In addition, we have shown that patients with IgAN have a specific increase in the production of IgA against OMHP antigens secondary to polyclonal lymphocyte activation against OMHP antigens, with switching of antibody production from one isotype to another, e.g. from IgM to IgA [13]. Furthermore, a significant relationship between serum concentrations of IgA antibody against OMHP and degrees of mesangial matrix proliferation exists in patients with IgAN [14]. We describe here our study of the immune response to HP by lymphocytes of the palatine tonsils in patients with IgAN.
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Subjects and methods |
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Cell preparation
Tonsils were cut into small pieces with scissors [15], then pressed through 200-gauge stainless steel mesh. Tonsillar lymphocytes were isolated by density gradient centrifugation using Lymphocyte Separation Medium (Flow Labs, McLean, VA), washed twice with minimal essential medium supplemented with 7.5 mol/l HEPES, and 2% heat-inactivated newborn calf serum (NCS), then resuspended in phosphate-buffered saline (PBS 0.1 mol/l, pH 7.2) supplemented with 2% heat-inactivated NCS. Monocytes (detected as cells bearing CD116) comprised less than 4% of tonsillar mononuclear cells. The respective proportions of T and B cells in tonsils of patients with IgAN and chronic tonsillitis were examined by fluorescence-activated cell sorter (FACS). There were no significant differences between two groups in respective proportions.
HP and Ps antigens
HP antigen was prepared from a strain isolated from the pharynx of a normal individual and subsequently cultured to stationary phase in brainheart infusion broth. Sonicates of HP (SHP) were prepared by 5 min of high-power ultrasound pulses and stored at -80°C in PBS (pH 7.4) at a protein concentration of 3.2 mg/ml [12]. Pseudomonas aeruginosa (Ps), a Gram-negative organism (ATCC 27580) was the gift of Mr Yasuo Nakatomi, Research and Development Division, Denka Seiken Co. Ltd. Sonicated Ps (SPs) was prepared as Ps antigen and stored at -80°C in PBS (pH 7.4) at a protein concentration of 7.4 mg/ml. For comparison, Ps was also examined, because it had been reported that Ps infection was frequently coincident with glomerular IgA deposition in pulmonary diseases [16].
Tonsillar lymphocyte proliferation
Isolated tonsillar lymphocytes were resuspended in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Gibco), L-glutamine (2 mmol/l), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 2x106 cells/ml. Cells were incubated for 72 h in microculture plates at 37°C in a humidified atmosphere with 5% CO2 with or without SHP (0.1 µg/ml), SPs (0.1 µg/ml), and phytohaemagglutinin (PHA); (Sigma, St Louis, MO) (20 µg/ml). Preliminary experiments established that these concentrations of SHP, SPs, and PHA induced maximal lymphocyte proliferation. Lymphocyte proliferation was measured by thymidine uptake. After 72 h of incubation, 0.25 µCi of [3H]thymidine was added to each well of the microculture plates (Costar, Cambridge, MA), and the cultures were terminated after 16 h. The incorporated radioactivity, expressed as counts per minute (c.p.m.), was determined.
Production of antibody against HP and Ps antigens
Isolated tonsillar lymphocytes were resuspended in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, L-glutamine (2 mmol/l), penicillin (100 U/ml), and streptomycin (100 µg/ml) at 1x106 cells/ml. Cells were incubated for 72 h at 37°C in humidified atmosphere with 5% CO2, with or without SHP (0.1 µg/ml) or SPs (0.1 µg/ml). Preliminary experiments established that these concentrations of SHP or SPs induced maximal production of antibody against HP or Ps antigens, respectively. Supernatant samples were stored at -80°C until used.
Enzyme-linked immunosorbent assay (ELISA) for antibodies against HP and Ps antigens
ELISA was carried out according to the modified method of Borradori et al. [17]. Briefly, a 96-well polystyrene microtitre plate was coated with 100 µl SHP at a final protein concentration of 1.07 µg/ml in 0.05 mol/l carbonate buffer (pH 9.5). After overnight incubation at 4°C, wells were washed three times with PBSTween and shaken dry. Unoccupied absorption sites in the wells were blocked by overnight incubation at 4°C with PBSTween containing 0.5% (w/v) bovine serum albumin. Supernatant (100 µl) diluted 1:10 with PBSTween was added to wells and incubated for 60 min at 37°C. Plates were then washed three times with PBSTween, and 100 µl of peroxidase-conjugated rabbit antibody against human IgG, IgA, or IgM (Dako Japan, Kyoto, Japan), diluted 1 : 6000, 1 : 4000, or 1 : 1000 with PBSTween, was added to each well and incubated for 60 min at 37°C. Wells were washed, and 100 µl of o-phenylenediamine at a concentration of 33 mg/ml and 0.018% (w/v) H2O2 in 0.1 mol/l phosphatecitrate buffer (pH 4.9) were added to each well. After incubation at room temperature for 30 min in the dark, the reaction was stopped by addition of 100 µl 0.75 mol/l H2SO4. Absorbance was then read at 492 nm.
Statistical analysis
Numerical data are expressed as means±SD. Differences between groups were tested for significance by the MannWhitney U test. Simple linear correlation and Spearman's test were used to evaluate correlations between variables. A P value <0.05 was accepted as statistically significant.
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Results |
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Tonsillar lymphocyte proliferation |
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Correlations between IgA, IgG, and IgM antibody concentrations for HP and Ps-stimulated lymphocytes
Lymphocytes from patients with IgAN showed positive correlations between concentrations of IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against HP or Ps antigen after HP or Ps stimulation (Table 1). Controls showed a positive correlation between concentrations of IgG and IgM antibodies against HP or Ps antigens after HP or Ps stimulation.
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Discussion
The characteristic feature of IgAN is diffuse mesangial deposition of immunoglobulins, with IgA predominating. The deposited IgA is likely to be directed against environmental factors such as viral, bacterial, or dietary antigens, although the pathogenesis of IgAN is not known. It is aetiologically significant that clinical manifestations of IgAN occasionally appear after upper respiratory tract infections such as tonsillitis or pharyngitis. IgAN is thought to be related to focal infection of the tonsils because of the improvement in urinary findings and renal function after tonsillectomy [10,18]. Regions of lymphoepithelial symbiosis (LES) are characteristic of the palatine tonsil and are the sites through which exogenous antigens enter tonsillar tissue. Several investigators have shown that LES regions consist of B cells and T cells [19,20]. With regard to abnormalities of peripheral T cells in IgAN, several investigators have reported an increase in IgA-specific helper T cells, an increase in activated T cells, and a decrease in IgA-specific suppressor T cells in IgAN [2124]. However, there are few studies of T-cell function in tonsils from patients with IgAN. We have previously reported glomerular deposition of HP antigens and the presence of IgA against HP antigens in patients with IgAN, suggesting that HP has a role in the aetiology of IgAN [12]. In this study we demonstrated that tonsillar lymphocytes from IgAN patients have significantly stronger responses to HP antigens than do tonsillar lymphocytes from controls. These data suggest that an immune response to HP antigens plays an important role in the pathogenesis of IgAN. Furthermore, this study suggests that HP antigens may be inhibiting proliferation by the control tonsillar lymphocytes because it showed that the controls have stimulation indices below 1.
It has been reported that IgA-positive plasma cells are increased [25], IgA-positive cells are abundant in the subepithelial sinusoids [26], and the IgA1 subclass is found in follicular dendritic cells (FDC) in the palatine tonsils of patients with IgAN. These findings are not present in non-IgAN controls [27]. Serum IgA concentrations are significantly decreased after tonsillectomy in patients with IgAN [28,29]. Human tonsillar lymphocytes include numerous antigen-primed B cells [30]. These findings suggest a relationship between the tonsillar immune response and the pathogenesis of IgAN. Continuous stimulation of the mucosal immune system by HP antigens may be necessary for the pathogenesis of IgAN. In addition, it has been reported that IgA produced by tonsillar B lymphocytes binds to the glomerular mesangium in IgAN [31]. In this study we showed that patients with IgAN had significantly higher production of specific IgA by tonsillar lymphocytes cultured with HP antigens than did controls. This study also suggests that HP antigens are efficient in triggering specific IgM and IgG responses in vitro from tonsillar lymphocytes.
We have previously observed that the outer membrane protein (OMP) P6 precursor, OMP P5, and OMP P2 (porin) of OMHP are deposited in the glomeruli of patients with IgAN, and that IgA, IgG, and IgM antibodies against these OMHPs are present in the sera of patients with IgAN [12,13]. The present study demonstrated that tonsillar lymphocytes from patients with IgAN produce IgA, IgG, and IgM antibody against HP antigens. These results suggest that IgA, IgG, and IgM antibody against OMHP produced by tonsillar B lymphocytes circulating in the bloodstream may explain the presence of IgG and IgM accompanying the predominant IgA deposits in the glomeruli of patients with IgAN.
It has been reported that the production of IgA, IgG, and IgM by unstimulated cultures of peripheral blood mononuclear cells is increased in patients with IgAN [32]. We have shown previously that patients with IgAN have significantly higher concentrations of IgA and IgG antibody against OMHP and that patients with IgAN show positive correlations between serum concentrations of IgA and IgG antibodies, between IgA and IgM antibodies, and between those of IgG and IgM antibodies against OMHP [13]. This study demonstrated that after in vitro OMHP stimulation of tonsillar lymphocytes from IgAN patients, positive correlations existed between concentrations of IgA and IgG antibodies, between IgA and IgM antibodies, and between IgG and IgM antibodies against OMHP. These data suggest that the immune response of tonsillar lymphocytes to HP antigens plays an important role in the pathogenesis of IgAN.
Additional studies will be necessary to determine the antigenic epitope of the predominant anti-HP antibody, to isolate T-cell clones reactive to HP, and to clarify the mechanism of glomerular IgA deposition induced by tonsillar lymphocytes. Nevertheless, our demonstration of immunoreactivity of tonsillar lymphocytes to HP indicates that HP might be the aetiologic agent of IgAN.
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Acknowledgments |
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Notes |
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References |
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