Figure 1.
GLP-1(9-36) inhibits chemokine-induced migration of isolated human CD4-positive lymphocytes.
A. and B.: Cells were pretreated with GLP-1(9-36) for 30 minutes at concentrations indicated before migration experiments using SDF-1 (100 ng/mL) (A) or RANTES (100 ng/mL) (B) were performed for three hours in the modified Boyden chamber. The data show that GLP-1(9-36) inhibits both SDF- and RANTES- induced migration of human CD4-positive lymphocytes. C: Scrambled GLP-1(9-36) does not reduce SDF-1-induced T-cell migration. Cells were pretreated with 1 nM GLP-1(9-36) and 1 nM scrambled GLP-1(9-36) for 30 minutes before migration experiments using SDF-1 (100 ng/mL) were performed for three hours in the modified Boyden chamber. Data are expressed as fold induction of unstimulated cells. Bars represent mean±SEM (n = 7); ***p<0.001, **p<0.01, *p<0.05 compared with SDF-stimulated cells.
Table 1.
GLP-1 does not decrease the expression of the chemokine receptor CXCR4 and does not influence cell viability; n = 4; the data is displayed as mean ± SEM.
Figure 2.
The PI3-kinase signaling cascade is involved in the inhibitory effect of GLP-1(9-36) on human lymphocyte migration.
A: GLP-1(9-36) reduces SDF-1-induced PI-3 kinase activity in isolated human CD4-positive lymphocytes. Cells were pretreated with GLP-1(9-36) for 30 minutes at concentrations indicated before stimulation with SDF-1 (100 ng/mL) for 5 min. An arrow indicates PIP3. Three independent experiments showed similar results. B+C: GLP-1(9-36) inhibits SDF-1-induced phosphorylation of cofilin (B) and MLC (C) in isolated human CD4-positive lymphocytes. Cells were pretreated with GLP-1(9-36) for 30 min at concentrations indicated before stimulation with SDF-1 (100 ng/mL). Western blot analysis for p-cofilin and p-MLC was performed after 5 min. ß-actin protein expression served as a loading control. Representative results of 8 (p-cofilin) and 6 (p-MLC) independent experiments are shown.
Figure 3.
GLP-1(9-36) reduces SDF-1-induced ICAM3 translocation in isolated human CD4-positive lymphocytes.
A.: Cells were pretreated with GLP-1(9-36) for 30 min at concentrations indicated before stimulation with SDF-1 for 30 min. ICAM3 translocation was determined using immunofluorescence staining. T-cells were considered to be resting if ICAM-3 was located evenly. If a clear clustering of ICAM3 at the uropod region was visible, ICAM3 translocation was considered to be present. B: Statistical analysis of cells positive for ICAM3 translocation as fraction of polarized cells; bars represent mean±SEM; n = 6; **p<0.01; ***p<0.001 compared with SDF-1-stimulated cells.
Figure 4.
GLP-1 reduces f-actin formation but does not change intracellular cAMP-concentrations in isolated human CD4-positive lymphocytes.
A: GLP-1(9-36) reduces SDF-1 induced f-actin formation in isolated human CD4-positive lymphocytes. Cells were pretreated with GLP-1 for 30 min (10 nmol/L) before stimulation with SDF-1. Actin polymerisation was assayed by flow cytometry at time points indicated. Dots represent mean. *p<0.05 for comparison between SDF-1-treated cells and GLP-1-pretreated cells before stimulation with SDF-1 at times indicated. # p<0.001 for comparison between SDF-treated cells and untreated cells; n = 12. B: GLP-1(9-36) does not change cAMP-concentration in CD4-positive lymphocytes. CD4-positive lymphocytes were treated with 10 nM GLP-1(9-36), SDF-1 (100 ng/mL) or pretreated with 10 nM GLP-1(9-36) for 30 minutes before stimulation with SDF-1 (100 ng/mL) for times indicated. Bars represent mean±SEM; n = 12; ***p<0.001 compared with untreated cells.
Figure 5.
Transfection of isolated human CD4-positive lymphocytes with GLP-1 receptor siRNA reduces both GLP-1 receptor mRNA and protein levels.
A. and B.: Transfection with 200 nM GLP-1R siRNA effectively reduces the amount of GLP-1 receptor mRNA (A.) and protein (B.) levels in isolated human CD4-positive lymphocytes. Gene expression (A.) and a representative Western blot are depicted together with quantitative analysis using densitometry (B.). Quantitative analysis for both GLP-1 receptor mRNA (A.) and protein levels (B.) are displayed as mean±SEM; n = 4 for (A.) and n = 5 for (B.); **p<0.01, ***p<0.001 compared to controls.
Figure 6.
GLP-1(9-36) reduces SDF-1-induced ICAM3 translocation in GLP-1R siRNA transfected human CD4-positive lymphocytes.
A: MOCK and siRNA transfected CD4-positive lymphocytes were pretreated with 10 nM GLP-1(9-36) for 30 min before stimulation with SDF-1 for 30 min. ICAM3 translocation was determined using immunofluorescence staining as described in figure 4. B: Statistical analysis of cells positive for ICAM3 translocation are presented as fraction of polarized cells; bars represent mean±SEM; n = 5; ***p<0.001 compared with SDF-1-stimulated cells.