Clinical and immunological implications of increase in CD208+ dendritic cells in tonsils of patients with immunoglobulin A nephropathy

Abstract

Background: The therapeutic effect of tonsillectomy for immunoglobulin A nephropathy (IgAN) has been widely recognized, but the mechanism by which tonsillar immunity leads to glomerulonephritis has been unclear. We investigated subtypes and localization of dendritic cells (DCs) in tonsils and looked for relationships between the tonsillar DCs and the clinical features and renal histological changes of patients with IgAN.

Methods: We examined tonsils from 33 IgAN patients, using as control tonsillar specimens from subjects without glomerulonephritis. Five distinct markers of DCs (CD303, CD1c, CD209, CD208 and CD1a) were analyzed by immunohistochemistry and flow cytometry. The mRNA levels of these DC markers were evaluated using real-time polymerase chain reaction. The clinical data and histological results obtained evaluating renal biopsy tissues were statistically compared with immunological data.

Results: Of the five subtypes of DCs, CD208(+) DCs were significantly increased in the tonsils of IgAN patients compared with that of controls. Furthermore, the number of CD208(+) DCs in the tonsils was positively and linearly correlated with the proportion of crescentic glomeruli in renal biopsy tissues and with the urinary protein level. Only few CD208(+) cells, however, were found in the kidney biopsy specimens of IgAN patients.

Conclusions: These observations suggest that increased CD208(+) DCs in tonsils may play a directive role in the pathogenesis of IgAN. The present results support the therapeutic significance of tonsillectomy for IgAN patients.

Keywords: CD208; IgA nephropathy; dendritic cells; glomerular crescent; tonsillectomy.

FIGURE 1:

Histological structure of tonsils. Original magnification; ×10. (A and B) Representative photomicrograph of HE-stained tonsillar tissue. (A) A patient with chronic tonsillitis. (B) A patient with IgA nephropathy (IgAN). Enlarged interfollicular areas and small germinal centers are characteristic in the tonsils of patients with IgAN. (C) Germinal center area and total tonsil area were measured, and proportions of germinal center area to total tonsillar area were calculated. Results are means ± SE for each group. The proportion of germinal center to total tonsillar area in IgAN patients was significantly smaller than that in controls. *P < 0.01 versus controls. Control: n = 9, IgAN: n = 33.

FIGURE 2:

Various subtypes of DCs in tonsils (A–E) and kidneys (F) of patients with IgAN. Original magnification; A–C, E and F: ×200, D: ×100. (A) IF staining for CD303 (under-epithelial area). The arrows show epithelium. (B) IF staining for CD1c (interfollicular area). (C) Immunoperoxidase staining for CD209 (interfollicular area). The arrow shows a follicle. (D) Immunoperoxidase staining for CD208 (interfollicular area). The arrow shows a follicle. CD208⁺ cells are clustered in the interfollicular area. (E) IF staining for CD1a (under-epithelial area). The arrows show epithelium. (F) IF staining for CD208 in the renal tissue of an IgAN patient. CD208⁺ cells are observed in the renal interstitium. Photograph shows CD208⁺ cells around a globally sclerotic glomerulus.

FIGURE 3:

Quantification analysis of different markers of subtypes of DCs in tonsillar tissues from controls and IgAN patients. Results are means ± SE for each group. (A) CD303⁺, (B) CD1c⁺, (C) CD209⁺, (D) CD208⁺ cells. The number of CD208⁺ cells in the tonsils of IgAN patients was significantly greater than that in the tonsils of controls. *P < 0.05 versus controls. (E) CD1a⁺ cells. Control: n = 9, IgAN: n = 33.

FIGURE 4:

Triple IF staining of tonsils of patients with IgAN. (A–D) Triple IF staining for CD208 (Alexa Fluor 594: red), CD3 (Alexa Fluor 350: blue) and HLA-DR (Alexa Fluor 488: green). (A) CD208, (B) CD3, (C) HLA-DR. HLA-DR is known to be expressed on DCs, macrophages, B-cells and activated T-cells. (D) Merged image of A–C. The arrows indicate activated T-cells (CD3⁺ HLA-DR⁺). (E–H) Triple IF staining for CD20 (B-cells; Alexa Fluor 594: red), CD3 (T-cells; Alexa Fluor 350: blue) and IgA (FITC: green). (E) CD20. The arrows indicate follicles. (F) CD3, (G) IgA and (H) merged image of E–G.

FIGURE 5:

Flow cytometry analysis of a patient with IgAN (A–F) and a control subject (G). (A–C) Isolated cells were double-stained with antibodies for HLA-DR and either CD303, CD1c or CD1a. The HLA-DR-positive population was analyzed and is shown in graphs. HLA-DR⁺ and DC-marker⁺ cells in each staining were identified to be DCs. (A) HLA-DR and CD303 staining. (B) HLA-DR and CD1c staining. (C) HLA-DR and CD1a staining. (D–G) Isolated cells were triple-stained with antibodies for HLA-DR, lineage marker and either CD209 or CD208. The lineage marker-negative population (indicated lin⁻ in graph D) was analyzed and is shown in E–G. Lineage marker⁻, HLA-DR⁺ and DC-marker⁺ cells in each staining were identified to be DCs. (E) HLA-DR and CD209 staining in the lineage marker⁻ population in a patient with IgAN. (F) HLA-DR and CD208 staining in the lineage marker⁻ population in a patient with IgAN. (G) HLA-DR and CD208 staining in the lineage marker⁻ population in a control subject. (H–L) Quantification analysis results for the different subtypes of DCs in cells isolated from tonsils of IgAN patients and controls. (H) CD303⁺ DCs, (I) CD1c⁺ DCs, (J) CD209⁺ DCs, (K) CD208⁺ DCs. The percentage of HLA-DR⁺ cells in the lineage marker⁻ population that were CD208⁺ was significantly greater in the tonsils of IgAN patients than that in the tonsils of controls. *P < 0.05 versus control. (L) CD1a⁺ DCs. Results are expressed as means ± SE for each group. Control: n = 3, IgAN: n = 4.

FIGURE 6:

mRNA expression for various markers of DCs. (A) CD303 mRNA, (B) CD1c mRNA, (C) CD209 mRNA, (D) CD208 mRNA and (E) CD1a mRNA. The levels of CD208 and CD1c mRNA in the tonsils of IgAN patients were significantly greater than that in the tonsils of controls. Results are expressed as means ± SE for each group. *P < 0.05 versus controls. Control: n = 8, IgAN: n = 23.

FIGURE 7:

Correlation between the number of tonsillar CD208⁺ DCs and clinical findings. (A) A significant and positive correlation between the number of CD208⁺ cells in the tonsils and the proportion of crescentic glomeruli to total glomeruli in the renal biopsy tissues of patients with IgAN (n = 33). (B) A significant and positive correlation between the number of CD208⁺ cells in the tonsils and the urinary protein level (n = 33). CD303, CD1c, CD209 and CD1a had no correlation with clinical or renal histological data.