Figure 1.
CXCL8 levels are elevated in brain lysates of HIV-1 infected patients.
Human brain mRNA and protein lysates collected from frontal cortex of HIV-1 infected individuals and age matched controls were analyzed for CXCL8 levels. A) CXCL8 mRNA expression analyzed by real-time PCR normalized to GAPDH. B) CXCL8 protein levels assayed by ELISA shown as ng/µg total protein. Results are expressed as mean ± SEM of indicated number of donors. Statistical analyses were performed using unpaired student’s t-test.
Figure 2.
Increased CXCL8 expression in astrocytes activated by IL-1β and TNF-α.
Cultured human astrocytes were treated with IL-1β (20 ng/ml) or TNF-α (20 ng/ml). A) CXCL8 mRNA expression 8 h post-activation normalized to GAPDH determined by real-time PCR. B) CXCL8 protein levels 24 h post-activation normalized to MTT determined by ELISA. Results are representative of three independent experiments performed in triplicates. Results are expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons.
Figure 3.
MAPK and SHP2 regulate IL-1β-mediated increased CXCL8 expression in astrocytes.
Cultured human astrocytes were pretreated with MAPK and SHP2 inhibitors for 2 h, followed by activation with IL-1β (20 ng/ml). Untreated controls were maintained in parallel. RNA was isolated 8 h post-activation and supernatants were collected 24 h post-activation. CXCL8 mRNA was measured using real-time PCR, while protein expression was measured by ELISA. A) CXCL8 mRNA levels normalized to GAPDH after pretreatment with inhibitors specific to p38, SB202190 and ERK, U0126. B) CXCL8 protein levels normalized to MTT after pretreatment with inhibitors specific to p38, SB202190 and ERK, U0126. DMSO was used as a solvent control. C) CXCL8 mRNA levels normalized to GAPDH after pretreatment with inhibitors specific to SHP2, PHPS1 and PTP, Na3VO4. D) CXCL8 protein levels normalized to MTT after pretreatment with inhibitors specific to SHP2 (PHPS1) or PTP (Na3VO4). Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons.
Figure 4.
CXCL8 upregulation by SHP2 and p38 overexpression in astrocytes.
Primary human astrocytes were transfected with SHP2WT and p38 overexpression plasmids with corresponding dominant negative mutants SHP2CS and p38agf. Twenty-four h post-transfection supernatants were analyzed for CXCL8 protein levels by ELISA. 24 h post-transfection astrocytes were treated with IL-1β (20 ng/ml) for 24 h, protein lysates and supernatants were collected and analyzed for SHP2 and p38 enzyme activity. A) CXCL8 protein levels assayed by ELISA normalized to MTT. B) SHP2 in vitro phosphatase assay showing phosphate released as a measure of SHP2 phosphatase activity. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons. C) In vitro p38 kinase assay based on immunoprecipitation of p38, followed by incubation with ATF-2 as substrate. P-ATF-2 band at 40 kD, which is a function of higher p38 kinase activity, is shown with densitometry. Results are representative of three independent experiments.
Figure 5.
Effect of SHP2 overexpression and p38 inhibition on CXCL8 levels.
SHP2WT- and SHP2CS-transfected astrocytes were treated with PTP inhibitor, Na3VO4; p38 inhibitor, SB203580, or negative control inhibitor, SB202474. CXCL8 levels measured by ELISA in cellular supernatants collected 24 h post-treatment with A) Na3VO4 B) SB203580 C) SB202474. ND denotes not detectable. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by one-way ANOVA and Newman-Keuls post-test for multiple comparisons or student’s t-test.
Figure 6.
Effect of p38 overexpression and SHP2 inhibition on CXCL8 levels.
p38- and p38agf-transfected astrocytes were treated with PTP inhibitor, Na3VO4; p38 inhibitor, SB203580, or negative control inhibitor, SB202474. CXCL8 levels measured by ELISA in cellular supernatants collected 24 h post-treatment with A) Na3VO4 B) SB203580 C) SB202474. ND denotes not detectable. Results are representative of three independent experiments performed in triplicate and expressed as mean ± SEM, analyzed by student’s t-test.
Figure 7.
SHP2, p38 and ERK phosphorylation following IL-1β stimulation of SHP2WT-, SHP2CS-, p38- and p38agf-transfected astrocytes.
Astrocytes were transfected with SHP2WT, SHP2CS, p38 or p38agf plasmids and stimulated with or without IL-1β for 5, 15 and 25 min. Whole cell protein lysates were collected and equivalent amounts were resolved by SDS-PAGE, transferred and immunoblotted for P-SHP2, SHP2, P-p38, p38, P-ERK and ERK. β-actin was used as loading control. A) Immunoblot of SHP2WT- and SHP2CS-transfected astrocytes following stimulation with IL-1β. B) Immunoblot of p38- and p38agf-transfected astrocytes following stimulation with IL-1β.
Figure 8.
Signaling pathways involved in increased CXCL8 production in activated human astrocytes.
Proinflammatory cytokines, such as interleukin (IL)-1β and tumor necrosis factor (TNF)-α, stimulate astrocytes in turn activating protein tyrosine phosphatase SHP2. Specific inhibitors are indicated adjacent to target. SHP2 is upstream of p38, which directly or indirectly modulates extracellular signal regulated kinase (ERK) mitogen-activated protein kinases activity, leading to increased expression of CXCL8 protein.