Figure 4.

NGAL^−/− chimeric mice show similar renal myeloid cell, but stronger T_H17 cell influx and increased renal CCL20 expression. MPO^−/− mice immunized with murine MPO were irradiated and transplanted with either WT or NGAL^−/− BM cells as described in Figure 2A. All chimeric mice were analyzed 7–8 weeks after transplantation. (A) Representative FACS plot with gating strategy and corresponding quantification of renal CD45⁺CD11b⁺Ly6G⁺ neutrophils (neutro) and CD45⁺CD11b⁺Ly6C^hi classic monocytes (CM). (B) Representative FACS plots showing IL-17A and IFNγ expression by renal CD4⁺ T cells after PMA/ionomycin stimulation ex vivo. Quantification reveals increased renal total CD4⁺, CD4⁺ T_H1 (IL-17A⁻IFNγ⁺) and CD4⁺ T_H17 (IL-17A⁺IFNγ⁻) T cells in NGAL^−/− compared with WT chimeric mice. (C) Immunohistochemistry of kidney sections stained for the T cell marker CD3 shows that most of the CD3⁺ T cells were detected within and around affected glomeruli (bold arrow), but not in nonaffected glomeruli (dotted arrow). (D) Quantification of renal CCL20 and IL1β mRNA by quantitative PCR with HPRT used as reference gene and renal CCL20 and IL1β protein by ELISA. Both renal CCL20 mRNA and protein were increased in mice transplanted with NGAL^−/− BM. (E) Splenic cells from WT and NGAL^−/− chimeric mice were stimulated with PMA/ionomycin ex vivo and IFNγ and IL-17A was determined in the supernatants by ELISA. Cells isolated from mice transplanted with NGAL^−/− BM produced more IFNγ and IL-17A compared with WT BM-transplanted mice. *P<0.05; **P<0.01.