Table 1.
Correlation between mouse ELF GSH and Alveolar Macrophage GSH.*
Figure 1.
Extracellular GSH alters intracellular levels.
J774 (A), or primary alveolar macrophages (B) were grown in media supplemented with or without 500 µM GSH and intracellular GSH levels were measured. Data represented as mean ± SEM, with ***p<0.001.
Figure 2.
GSH supplementation alters inflammatory cytokine release.
The J774 (A) and primary AM (B) were grown in media with or without 500 µM GSH and treated with LPS for 2 hours. TNFα release was analyzed in the media by ELISA. J774 nuclear fractions were examined for the nuclear translocation of the p65 subunit of NF-κB (C), HDAC used as a protein loading control with the ratio of p65 to HDAC area density listed below. Data represented as mean ± SEM, with ***p<0.001 compared between PBS and GSH.
Figure 3.
Uptake of GSH is through macropinocytosis.
J774 cells were grown in media supplemented with 500 µM GSH either containing buthionine sulfoxime (BSO), acivicin, or methionine to inhibit synthesis, breakdown or transport of GSH, respectively (A). Cytochalasin D was used as an inhibitor of endocytosis and amiloride was used as more specific inhibitor of macropinocytosis. Data represented as mean ± SEM, with *p<0.05, ***p<0.001 compared to control.
Figure 4.
Anti-inflammatory effects of GSH are dependent on macropinocytosis.
Brighfield images of control (A), fluorescent labeled GSH (Fl-GSH) (B) or Fl-GSH with cytochalasin D (C) provide further evidence for the direct uptake of extracellular GSH. Furthermore, LPS induced TNFα release was examined after supplementation with GSH and/or cytochalasin D to block that uptake (D). Data represented as mean ± SEM, with ***p<0.001 compared to either LPS only.
Figure 5.
In vivo uptake of fluorescent labeled GSH.
Fluorescent labeled GSH (Fl-GSH) was administered intratracheally and examined after a period of 2 h. Representative image of BAL cells recovered from control (A) and Fl-GSH (B) administered mice. Quantization of resulting Fl-GSH concentrations 2 h after administration (C), control samples contain no detectable fluorescence.
Figure 6.
Different smoke exposure paradigms result in different GSH levels.
Mice were exposed to acute 2 h cigarette smoke (CS) exposure with little rest before sampling of the ELF and BAL cell GSH which resulted in depleted GSH levels. However, after a 24 h exposure which includes a rest period there is a full adaptive response with elevated ELF and BAL cell GSH levels. Data represented as mean ± SEM, with *p<0.05, ***p<0.001 compared to either ELF or BAL cell control.
Figure 7.
Altered GSH status in the airways results in different cytokine response to LPS.
Alveolar macrophages were obtained from mice sampled immediately after a 2 h (closed bars) cigarette smoke (CS) exposure or 24 h (open bars) recovery from CS exposure and treated with LPS for 2 h. TNFα release was measured by ELISA. Data represented as mean ± SEM, with ***p<0.001 compared between 2 h CS and 1 d CS.