The Second Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan
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Abstract |
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Methods. We compared the lymphocytic response to in-vitro stimulation by group A streptococcal M proteins of apparent virulence factor between IgAN, non-proliferative glomerulonephritis (NPGN), and normal subjects. M proteins were extracted from group A streptococcal strain type 5 and type 12 determined serologically.
Results. M protein-induced proliferation of lymphocytes from IgAN was higher than in NPGN but not in healthy control subjects. Flow cytometric analysis indicated that stimulation by M protein extracts derived from type 5 streptococci (M5) increased surface IgA-positive B cells in IgAN, but did not activate the production of soluble IgA. We also showed that M5 induced significant increases in TGF-ß, in culture supernatants of lymphocytes from patients with IgAN.
Conclusion. Our results suggest that Streptococcal infection may play an important role in the pathogenesis of IgAN by stimulating IgA production through TGF-ß synthesis.
Keywords: glomerulonephritis; IgA nephropathy; lymphocyte proliferation; streptococcal M protein; TGF-ß
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Introduction |
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Transforming growth factor-ß (TGF-ß) induces IgA isotype expression on surface IgA negative (sIgA-) B cell in certain B cell activation systems [813], and inhibits the production of IgM and IgG [10,13,], T cell function [15,16], and NK cell function [17,18]. In the present study, we examined T cell responses and B cell class switching associated with TGF-ß in vitro following stimulation, by group A streptococcal M proteins, of peripheral blood mononuclear cells (PBMC) obtained from patients with IgAN, non-proliferative glomerulonephritis (NPGN), and control subjects.
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Subjects and methods |
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Preparation of streptococcal M proteins
Group A streptococci were kindly provided by Dr Y. Ishii (Toho University School of Medicine, Tokyo, Japan). Group A streptococci were cultured in ToddHewitt broth, and M protein extracts derived from type 5 streptococci (M5) and type 12 streptococci (M12) were prepared using the mild pepsin digestion method, described previously by Beachey et al. [19]. The protein content of the crude extract was determined by Lowry's method and lyophilized preparations were used in all experiments.
Cultures of peripheral blood mononuclear cells
Heparinized venous blood samples were obtained from each subject after consent. PBMC were separated by FicollConray density gradients. PBMC (1x106/ml) were incubated in flat-bottomed 24- or 96-well plastic plates with or without 1 µg/ml of M protein at 37°C in 5% CO2 in air. The culture medium was Cosmedium-001 (Cosmo Bio Co., Tokyo, Japan) supplemented with 100 U/ml of penicillin and 100 mg/ml of streptomycin.
Proliferative assay of cultured cells
PBMC (1x106/ml) were incubated in 96-well plate with or without 1 µg/ml of M protein. After 5 days culture, [3H]thymidine (final concentration, 3.7 kBq/ml) was added to the plate and incubation was extended for another 16 h. Cultured cells were then harvested and the level of incorporated radioactivity was determined by a scintillation counter (Aloka, Tokyo). The stimulation index reflected the level of proliferation of PBMC in response to M protein and was calculated by a/b, where a=c.p.m. in the supernatant of PBMC mixed with M protein, and b=c.p.m. in the supernatant of PBMC mixed with equivalent content of inactive pepsin.
Surface phenotyping of PBMC and cultured cells
PBMC and cultured cells were stained by phycoerythrin-labelled anti-CD20 (Leu16-PE, BectonDickinson, Milan, Italy), a marker for mature B cells, and FITC-labelled anti-human immunoglobulin-F(ab,)2 (TAGO, Inc., Burlingame, CA). Two-colour fluorescent flow cytometric analysis was used to identify the surface immunoglobulin class of CD20 positive cells. The ratio of CD3+/CD20+ and CD4+/CD8+ lymphocytes was evaluated by two-colour fluorescent flow cytometric analysis using phycoerythrin-labelled anti-CD20, anti-CD4 (BectonDickinson) and FITC-labelled anti-CD3, anti-CD8 (BectonDickinson).
Enzyme-linked immunosorbent assay (ELISA)
The concentrations of IgA and IgG in the culture supernatant were measured after 5 days incubation by using ELISA. The concentrations of TGF-ß and interferon- (IFN-
) in culture supernatant were measured by the TGF-ß ELISA system (King Brewing Co., Hyougo, Japan) and IFN-
system (TFB Co., Tokyo) respectively.
Effect of anti-TGF-ß on DNA synthesis
In the neutralization test, PBMC (2x105/well) of control subjects were mixed with M protein (0.2 µg/well), IgG fraction of rabbit antisera to TGF-ß (5 mg/well) (King Brewing), and serially diluted recombinant human TGF-ß (1, 2, and 5 ng/well) (King Brewing). The mixture was incubated in 96-well plates for 5 days. The proliferation assay was then performed using the method described above. Phytohaemagglutinin (PHA) was used instead of M protein as an example of T cell mitogen in other control experiments.
In-situ hybridization of cultured cells
After 5-days culture, cells were washed with PBS and resuspended in RPMI 1640 with L-glutamine 2 mM, penicillin 100 U/ml, and streptomycin 100 µg/ml. Cell suspension (100 µl), containing 25x104 cells, was added to a cytospin cuvette (Shandon Inc., Pittsburgh, PA) and adhered to glass slides coated with 3-aminopropyl-triethoxy-silane (APS; A-3648; Sigma Chemical Co., St Louis, MO). Cytospin preparations were allowed to air-dry for 30 min and were then fixed with 4% paraformaldehyde (PFA) in PBS. In-situ hybridization was performed according to the technique developed in our laboratory [20]. Briefly, cells were fixed with 4% PFA in PBS and washed in PBS and then treated with HCl and digested with proteinase K (Sigma P4914) at 37°C in a solution containing 4x standard saline citrate (SSC), 5x Denhardt's solution, 0.2 mg/ml salmon testis DNA (Sigma D-7656), 0.2 mg/ml yeast tRNA (Sigma R-8508), and 50 mM sodium phosphate, pH 8.0. Cells were drained and hybridized overnight with digoxigenin (DIG)-labelled oligonucleotide probe (human TGF-ß cDNA) in the prehybridization buffer at 37°C. Cells were then washed twice with 2x and 1x SSC at room temperature. Visualization of hybridized DIG-labelled probe was performed by immunohistochemical staining, as described previously [20].
Statistical analysis
Data were expressed as mean±SD. Differences between groups were examined for significance using the paired and unpaired t-tests. A P value <0.05 denoted statistical significance.
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Results |
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DNA synthesis in cultured cells
In-vitro stimulation of cultured PBMC with M protein resulted in proliferation of these cells. Flow cytometric analysis showed a dominant increase in CD3+ cells. Generally, the ratio of CD3+/CD20+ cells increased from approximately 4.74 to approximately 10.75 after incubation with M5 (in five patients with IgAN and control subjects). This result suggested that the majority of the proliferative cells were T cells. Furthermore, the ratio of CD4+/CD8+ cells decreased from approximately 2.3 to approximately 1.8 under the conditions described above (data not shown). Proliferation of PBMC from IgAN in response to M protein was of moderate to high level compared to those from NPGN. There was no significant difference between the response to M5 and M12 proteins. In control subjects, the level of M protein-stimulated proliferation of PBMC was similar to that in IgAN (Figure 1).
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Immunoglobulin class of cultured B cells
Following confirmation of the population of cultured cells as described above, we performed two-colour flow cytometric analysis to characterize the class switching on cultured B cells using anti-CD20 antibody and anti-human immunoglobulin antibodies (see Subjects and methods). After incubation, M5 induced a significant increase in surface IgA-positive (sIgA+) B cells, but not surface IgG-positive (sIgG+) B cells in IgAN. All other tested conditions failed to switch the surface immunoglobulin class of B cells (Table 1).
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Soluble immunoglobulin in culture supernatants
We also measured the concentrations of soluble IgA and IgG in the culture supernatant containing M proteins. The concentration of soluble IgA in supernatant of IgAN was significantly higher than in control samples under all conditions. However, M proteins did not specifically increase the concentration of soluble IgA in the culture supernatant. There was no difference in the concentration of soluble IgG between IgAN and control (data not shown).
TGF-ß and IFN- in culture supernatants
We also measured the concentrations of two cytokines in the culture supernatant. These included TGF-ß of IgA class switching factor, and IFN- of IgG2a class switching factor. M5 induced a significant increase in TGF-ß in supernatant of IgAN, but not in control samples (Table 2
). On the other hand, there was no difference in the concentration of IFN-
between IgAN and control (data not shown).
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Effect of anti-TGF-ß on DNA synthesis
Table 2 demonstrates that M5 induced a significant increase in TGF-ß in the supernatant of IgAN. Therefore, in the next step, we examined the role of TGF-ß on cell growth, since TGF-ß is also known to act as an inhibitory cytokine [10,1318]. Anti-TGF-ß serum enhanced [3H]thymidine uptake in cells cultured with M5, whereas additional recombinant human TGF-ß resulted in a dose-dependent inhibition of [3H]thymidine uptake. These results suggest that TGF-ß acted as an inhibitor in the present experiment. This phenomenon was not observed in cells cultured with PHA (Figure 2
).
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TGF-ß mRNA expression in cultured cells
In-situ hybridization of cultured cells using digoxigenin-labelled oligonucleotide probe (human TGF-ß cDNA) was performed to identify TGF-ß producing cells. A weak expression of TGF-ß mRNA was detected on lymphocytes and a moderate expression was detected on monocytes. The number of positive cells was higher in samples from IgAN containing M5 (Figure 3d) than in controls (Figure 3a
and 3c
) and IgAN without M5 (Figure 3b
).
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Discussion |
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Our results showed that the majority of proliferative cells following stimulation by M-protein were T cells. Accordingly, we investigated immunoglobulin class switching of cultured B cells and the production of soluble IgA to characterize the maturation of B cells. Flow cytometric analysis showed that stimulation by M5 significantly increased sIgA+ B cells in IgAN (Table 1), but did not stimulate the production of soluble IgA (data not shown). These results suggest that the T cell responses induced by M5 include affinity maturation of B cells and induction of IgA isotype expression, but does not involve increased production of IgA in vitro. However, we believe that similar T cell responses may induce IgA production in vivo by inducing other immunological factors.
Previous studies have shown that TGF-ß induces IgA isotype expression on sIgA- B cell in certain B cell activation systems [813], and inhibits the production of IgM and IgG [10,13,14], T cell function [15,16], and NK cell function [17,18]. Accordingly, we postulated that TGF-ß was involved in M-protein-induced enhancement of proliferative lymphocyte response in our experiment, and that certain IgA class switching factors were involved in glomerular IgA deposition in IgAN in vivo. The results shown in Table 2 indicate that M5 significantly increased TGF-ß in the culture supernatant of IgAN. Furthermore, we also used the neutralization test to examine the inhibitory effect of TGF-ß on cell growth. Figure 2
showed that anti-TGF-ß serum enhanced [3H]-thymidine uptake in cells cultured with M5, indicating that neutralization of TGF-ß counterbalanced the inhibitory effect of TGF-ß on cell growth. On the other hand, anti-TGF-ß serum failed to influence cell growth in media containing PHA, since the latter T cell mitogen is known not to involve a specific production of TGF-ß. Figure 3
indicated that the expression of TGF-ß mRNA was clearly observed in monocytes and lymphocytes at a single cell level, and the number of cells expressing TGF-ß mRNA was higher in IgAN cells cultured with M5 than other cell types. However, our results could not confirm that M protein directly stimulated the production of TGF-ß. It is possible that other factor-mediated-interactions produce TGF-ß. Our results suggest that the produced TGF-ß inhibited cell growth in autocrine or paracrine systems. We believe that lymphocytes of IgAN are part of a population of high responders to M protein considering the inhibitory mechanism of TGF-ß on cell growth. In other words, the high response of lymphocytes to streptococcal M protein enhances the development of IgAN, while polymorphism of immunological response must relate to the major histocompatibility complex (MHC; human HLA) and control disease susceptibility [22].
Several investigators have failed to identify a specific pathogen in IgA deposits in the glomerular mesangium of IgAN. IgAN is a syndrome that is derived from many types of pathogens, and it is impossible to divide IgAN into some groups with the same pathogen. If certain pathogens exist in IgA-containing immune complexes within IgA deposits, detectable epitope of the pathogen may disappear by binding of immunoglobulin paratope or endocytosis of mesangial cells. Furthermore, failure to determine the presence of certain immunoglobulin binding antigens may be due to activation of IgA-producing B cells by carbohydrates present in the bacterial cell wall. Future studies should examine changes in glomerular structure and explore the possible presence of pathogens in IgAN.
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Acknowledgments |
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Notes |
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References |
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