Figure 7
Figure 7. Impaired activation of peritoneal NKT cells partially contributed to the enhanced peritoneal inflammation and delayed resolution in mice with X-CGD. (A) Total peritoneal cell counts in X-CGD and WT mice were determined at day 0, 8 hours, and days 3, 4, and 6 after intraperitoneal (IP) injection. (B, left) The number of neutrophils (GR1high) in the peritoneal cavities of WT or X-CGD mice at baseline and 8 hours after IP injection of sodium periodate was determined by flow cytometry. (Right) Absolute cell numbers of macrophages (F4/80+), granulocytes (GR1high), T cells (CD3+), and B cells (B220+) in the peritoneal cavities of WT or X-CGD mice at baseline and 8 hours after IP injection of sodium periodate was determined by flow cytometry. (C) The numbers of MPO+ (E) or IL-4+ (F) PEMs were determined by ICS in X-CGD and WT mice at day 0 and day 3. At least 3 mice were used for each genotype at each time point. Data represent at least 3 experiments. (D) NKT (CD1d tetramer+ TCRβ +) or non-NKT (CD1d tetramer- TCRβ +) populations from day 4 WT or X-CGD peritoneum were identified as described in Figure 3. Each population was assessed for intracellular IL-4 and IFN-γ. (E) Day 0 or day 4 WT and X-CGD CD4 T cells were ex vivo–stimulated with anti-CD3 and anti-CD28 for 24 hours; IL-4, IL-13, and IFN-γ in supernatants were measured by ELISA. (F) Day 0 and day 4 WT or X-CGD PEM were assessed for surface expression of CD1d by flow cytometry, and mean fluorescence intensity is shown. (G) Activation of N38-2C12 NKT cells by day 4 WT or X-CGD PEMs was determined by measuring IL-2 production by the NKT cells 24 hours after co-culture with the macrophages. (H) Adoptive transfer of WT splenic NKT cells into either WT or X-CGD mice after induction of peritonitis was performed as described in Figure 3. Day 4 peritoneal cell counts were determined. (I) CD4 T cells from recipient mice were ex vivo–stimulated as described in Figure 3, and IL-4 and IFN-γ production were measured by ELISA. (J) WT or X-CGD splenic NKT cells were co-cultured with mCD1d-expressing LMTK cells ex vivo for 48 hours, and IL-4 and IFN-γ production were measured by ELISA. All data represent 2 to 3 independent experiments. PMN, polymorphonuclear leukocytes.

Impaired activation of peritoneal NKT cells partially contributed to the enhanced peritoneal inflammation and delayed resolution in mice with X-CGD. (A) Total peritoneal cell counts in X-CGD and WT mice were determined at day 0, 8 hours, and days 3, 4, and 6 after intraperitoneal (IP) injection. (B, left) The number of neutrophils (GR1high) in the peritoneal cavities of WT or X-CGD mice at baseline and 8 hours after IP injection of sodium periodate was determined by flow cytometry. (Right) Absolute cell numbers of macrophages (F4/80+), granulocytes (GR1high), T cells (CD3+), and B cells (B220+) in the peritoneal cavities of WT or X-CGD mice at baseline and 8 hours after IP injection of sodium periodate was determined by flow cytometry. (C) The numbers of MPO+ (E) or IL-4+ (F) PEMs were determined by ICS in X-CGD and WT mice at day 0 and day 3. At least 3 mice were used for each genotype at each time point. Data represent at least 3 experiments. (D) NKT (CD1d tetramer+ TCRβ +) or non-NKT (CD1d tetramer- TCRβ +) populations from day 4 WT or X-CGD peritoneum were identified as described in Figure 3. Each population was assessed for intracellular IL-4 and IFN-γ. (E) Day 0 or day 4 WT and X-CGD CD4 T cells were ex vivo–stimulated with anti-CD3 and anti-CD28 for 24 hours; IL-4, IL-13, and IFN-γ in supernatants were measured by ELISA. (F) Day 0 and day 4 WT or X-CGD PEM were assessed for surface expression of CD1d by flow cytometry, and mean fluorescence intensity is shown. (G) Activation of N38-2C12 NKT cells by day 4 WT or X-CGD PEMs was determined by measuring IL-2 production by the NKT cells 24 hours after co-culture with the macrophages. (H) Adoptive transfer of WT splenic NKT cells into either WT or X-CGD mice after induction of peritonitis was performed as described in Figure 3. Day 4 peritoneal cell counts were determined. (I) CD4 T cells from recipient mice were ex vivo–stimulated as described in Figure 3, and IL-4 and IFN-γ production were measured by ELISA. (J) WT or X-CGD splenic NKT cells were co-cultured with mCD1d-expressing LMTK cells ex vivo for 48 hours, and IL-4 and IFN-γ production were measured by ELISA. All data represent 2 to 3 independent experiments. PMN, polymorphonuclear leukocytes.

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