Table 1.
Patient characteristics.
Fig 1.
DP delays constitutive neutrophil apoptosis in normal subjects.
(A) Neutrophils were isolated from the peripheral blood of normal subjects (n = 59) and then incubated for 24 h in the absence (Con) and presence of DP (10 μg/mL) and DF (10 μg/mL). (B) Neutrophils (n = 4) were incubated for 24 h in the absence (Con) and presence of DP in the indicated concentration. (C) Neutrophils (n = 3) were incubated for 24 h in the absence (Con) and presence of DP, DP-LPS [LPS-removed DP using Endo Trap Red (Lonza, MD)], or LPS. (D) Normal neutrophils (n = 3) were pre-treated for 1 h with and without polymyxin B in the indicated concentration, after which the cells were incubated for 24 h in the absence and presence of DP (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.
Fig 2.
DP induces the inhibition of neutrophil apoptosis via activation of TLR4, Lyn, PI3K, Akt, ERK and NF-κB, and suppression of the caspase9/3 pathway.
(A-B) Normal neutrophils (3<n<7) were pre-treated for 1 h with and without TLR4i in the indicated concentration (A) or 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, 10 μM PD98059 (PD) and 10 μM BAY-11-7085 (BAY) (B), after which the cells were incubated for 24 h in the absence and presence of DP (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are presented relative to the control, which was set at 100% of the means ± SD. **p < 0.01 indicates a significant difference between the control and DP-treated groups, and ##p < 0.01 represents a significant difference between the DP-treated group and the inhibitor-treated group. (C) Normal blood neutrophils were incubated with DP (10 μg/ml) for the indicated time. Phosphorylation of Lyn, Akt and ERK in the lysates was detected by Western blotting. (D) Normal blood neutrophils were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY) and 10 μM AKTi, and then incubated with DP (10 μg/ml) for 30 min. Phosphorylation of ERK in the lysates was detected by Western blotting. (E) Normal neutrophils were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, and 10 μM PD98059 (PD) and then incubated with DP (10 μg/ml) for 8 h. The nuclear fraction was extracted, and the NF-κB DNA binding activity was assessed using an transcription factor kit. **p < 0.01 indicates a significant difference between the control and DP-treated groups, and ##p < 0.01 represents a significant difference between the DP-treated group and the inhibitor-treated groups. (F) Normal blood neutrophils were incubated with DP (10 μg/ml) for the indicated time. Procaspase 9, procaspase 3 and Mcl-1 proteins were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control.
Fig 3.
Both S100A8 and S100A9 are released after DP treatment and suppress neutrophil apoptosis.
(A) Neutrophils were incubated with and without 10 μg/ml of DP for 24 h. The supernatant (Sup) was collected and added to the fresh neutrophils obtained from the peripheral blood of normal individuals (n = 9). Media were incubated with DP for 24 h in a 5% CO2 incubator at 37°C. The media were collected and added to the fresh neutrophils obtained from the peripheral blood of normal individuals (n = 5). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. **p < 0.01 indicates a significant difference between the control and the DP-treated group, and *p < 0.05 represents a significant difference between the control group and the Sup with DP treatment-treated group. (B) Neutrophils were incubated with and without 10 μg/ml of DP for 24 h. The supernatant (Sup) was collected and analyzed by 2DE and MALDI-TOF/TOF. (C) Neutrophils (n = 3) were incubated with 10 μg/ml of DP for the indicated time. The cells were fixed and permeabilized with 0.37% paraformaldehyde solution and 0.2% Triton X-100 solution, respectively, then incubated with anti-S100A8 or anti-S100A9 antibodies and analyzed on a FACSort cytofluorimeter. The mean intensity of untreated cells was considered 100%. Alteration of intracellular S100A8 and S100A9 expression after DP treatment was evaluated as the mean intensity of DP-treated cells/the mean intensity of untreated cells × 100. (D) Neutrophils (3<n<6) were incubated for 24 h in the absence (Con) and presence of S100A8 and S100A9 (10 μg/mL) in the indicated concentration. (E) Normal neutrophils (n = 4) were pre-treated for 1 h with and without 50 μg/ml polymyxin B after which the cells were incubated for 24 h in the absence and presence of S100A8 or S100A9DP (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups.
Fig 4.
S100A8 and S100A9 trigger the suppression of neutrophil apoptosis through activation of TLR4, Lyn, PI3K, Akt, ERK and NF-κB, and via suppression of the caspase9/3 pathway.
(A-B) Normal neutrophils (4<n<6) were pre-treated for 1 h with and without TLR4i in the indicated concentration (A) or 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, 10 μM PD98059 (PD) and 10 μM BAY-11-7085 (BAY) (B), after which the cells were incubated for 24 h in the absence and presence of S100A8 and S100A9 (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. **p < 0.01 indicates a significant difference between the control and DP-treated groups, and #p < 0.05 and ##p < 0.01 represent a significant difference between the DP-treated group and the inhibitor-treated group. (C) Normal blood neutrophils were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY) and 10 μM AKTi, and then incubated with S100A8 and S100A9 (10 μg/ml) for 30 min. Phosphorylation of ERK in the lysates was detected by Western blotting. (D) Normal neutrophils were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, and 10 μM PD98059 (PD) and then incubated with S100A8 and S100A9 (10 μg/ml) for 8 h. The nuclear fraction was extracted, after which the NF-κB DNA binding activity was assessed using an transcription factor kit. **p < 0.01 indicates a significant difference between the control and DP-treated groups, and ##p < 0.01 represents a significant difference between the DP-treated group and the inhibitor-treated group. (E) Normal blood neutrophils were incubated with S100A8 and S100A9 (10 μg/ml) for the indicated time. Procaspase 9 and procaspase 3 were detected by Western blotting. The membrane was stripped and reprobed with anti-ERK2 antibodies as an internal control.
Fig 5.
DP, S100A8, and S100A9 have anti-apoptotic effects in asthmatic neutrophils.
(A-E) (A) Neutrophils were isolated from the peripheral blood of asthmatic subjects (n = 54). The cells were incubated for 24 h in the absence (Con) and presence of DP (10 μg/mL) (B) Normal and asthmatic neutrophils were incubated with and without 10 μg/ml of DP for 24 h. The supernatant (Sup) was collected and added to the fresh neutrophils isolated from the peripheral blood of normal and asthmatic subjects (n = 9). (C) Asthmatic neutrophils (n = 3) were incubated for 24 h in the absence (Con) and presence of S100A8 and S100A9 in the indicated dose. (D) Asthmatic neutrophils (n = 3) were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, 10 μM PD98059 (PD) and 10 μM BAY-11-7085 (BAY), after which the cells were incubated for 24 h in the absence and presence of S100A8 and S100A9 (10 μg/ml). Apoptosis was analyzed by measuring the binding of annexin V-FITC and PI. Data are expressed as the means ± SD and are presented relative to the control, which was set at 100%. (E) Asthmatic neutrophils were pre-treated for 1 h with and without 1 μM TLR4i, 10 μM PP2, 10 μM Ly294002 (LY), 10 μM AKTi, and 10 μM PD98059 (PD) and then incubated with S100A8 and S100A9 (10 μg/ml) for 8 h. The nuclear fraction was extracted, and the NF-κB DNA binding activity was assessed using an transcription factor kit. *p < 0.05 and **p < 0.01 indicate a significant difference between the control and stimulator-treated groups, and #p < 0.05 and ##p < 0.01 represent a significant difference between the stimulator-treated and inhibitor-treated groups.
Fig 6.
Elevated S100A8 level in BALF is associated with an increase of BALF neutrophils and a decrease of physiological function.
(A) The concentration of S100A8 and S100A9 in BALF and serum of normal (6<n<26) and asthmatic subjects (61<n<94) was measured by ELISA as described in the Materials and Method section. **p < 0.01 indicates a significant difference between the normal and asthmatic groups. (B) Linear regression represents the correlation between S100A8 or S100A9 and the number of neutrophils (%) in asthmatic subjects. (C) Linear regression represents the correlation between S100A8 or S100A9 in BALF and FEV1 (%) or FEV1/FVC (%).
Fig 7.
The proposed anti-apoptotic signaling pathway induced by DP in normal and asthmatic neutrophils.
Anti-apoptotic signaling due to DP, S!00A8, and S100A9 involves TLR4, Lyn, PI3K, Akt, ERK and NF-κB, and is associated with suppression of procaspase 3 and procaspase 9 cleavage.