Fig 1.
Treatment schedules of CD14+ monocytes to generate macrophages or immature dendritic cells.
CD14+ monocytes were cultured in the presence of M-CSF (30 ng/ml), CXCL4 (1 and 10 μg/ml) or CXCL4L1 (1 and 10 μg/ml) to differentiate into macrophages. For the differentiation into dendritic cells, CD14+ monocytes were cultured in the presence of GM-CSF (50 ng/ml) and IL-4 (20 ng/ml). On day 4 (iMDDC A) or day 0 (iMDDC B), different concentrations of CXCL4 (1 and 10 μg/ml) or CXCL4L1 (0.1, 1 and 10 μg/ml) were added.
Table 1.
Primer and probe sequences for evaluated genes.
Fig 2.
In contrast to CXCL4L1, CXCL4 induces morphological changes in monocyte-derived macrophages and promotes their survival.
CD14+ monocytes were cultured in the presence of M-CSF (30 ng/ml), natural CXCL4 (1 and 10 μg/ml), recombinant CXCL4L1 (1 and 10 μg/ml) (panel A-C) or a combination of CXCL4 (10 μg/ml) and CXCL4L1 (1 and 10 μg/ml) (panel D). Panel A reports bright field pictures illustrating the morphology of monocyte-derived macrophages after 6 days of culture (20x magnification; scale bar 10μm). Panel B: After 6 days of culture cells were stained with the viability dye Zombie Aqua before flow cytometry analysis. Dot plots (forward scatter versus fluorescence) from one representative experiment out of 8 are shown. The gate circles the living cells. Numbers indicate the % of viable cells in the sample. Panel C and D report quantification of monocyte-derived macrophages after 6 days of culture by the ATP lite assay. The obtained luminescence values were normalized relative to the luminescence value obtained after culture in the presence of M-CSF (dashed line). Results are shown as mean ± SEM from 6–9 independent experiments. ***p<0.001; Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF) - †††p<0.001; Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations)—$p<0.05; Sign test [CXCL4 (10 μg/ml) versus the combination of CXCL4 (10 μg/ml) plus CXCL4L1 (10 μg/ml)].
Fig 3.
Effects of CXCL4 and CXCL4L1 on the matrix metalloproteinase and chemokine receptor gene expression in differentiated monocyte-derived macrophages.
Relative expression of iNOS, HMOX1, MMP-2, MMP-8, MMP-9, CXCR3A, CXCR3B, IL-1RN and MRC was evaluated in monocyte-derived macrophages following 6 day-treatment with M-CSF- (30 ng/ml), CXCL4 (10 μg/ml) or CXCL4L1 (1 and 10 μg/ml). Results are shown as mean (± SEM) percentage expression relative to the expression in M-CSF-treated monocytes. *p<0.05, Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF); †p<0.05, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations); #p<0.05, Mann-Whitney U test (CXCL4 vs CXCL4L1 1 μg/ml).
Fig 4.
The expression of surface markers and chemokine receptors on macrophages after stimulation with M-CSF, CXCL4 or CXCL4L1.
Purified human CD14+ monocytes were cultured for 6 days in the presence of recombinant M-CSF (30 ng/ml), natural CXCL4 (10 μg/ml) or recombinant CXCL4L1 (1 and 10 μg/ml). On day 6 expression of several surface markers (panel A) and chemokine receptors (panel B) was analyzed by flow cytometry. Results from 4 to 8 independent experiments were expressed as mean fluorescence intensity and are shown as mean (± SEM) percentages, normalized to M-CSF-treated monocytes (100%). *p<0.05, **p<0.01,***p<0.001, Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF); †p<0.05,††p<0.01, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations).
Fig 5.
Cytokine and chemokine production by macrophages after stimulation with M-CSF, CXCL4 or CXCL4L1.
CD14+ monocytes were differentiated in the presence of recombinant M-CSF (30 ng/ml), natural or recombinant CXCL4 (1–10 μg/ml) or recombinant CXCL4L1 (1 and 10 μg/ml). After 6 days of culture, cell supernatants were analyzed by ELISA to determine the amount of CCL18 (panel A), IL-10 (panel B), CCL22 (panel C), CCL2 (panel D) and CXCL8 (panel E). The measured cytokine concentrations were corrected for the number of viable cells in the cultures at the time of sampling. The results are shown as mean ± SEM and are pooled from 8 independent experiments. *p<0.05, **p<0.01,***p<0.001, Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF); ††p<0.01, †††p<0.001, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations).
Fig 6.
Chemokine production by macrophages after stimulation with CXCL4L1 is mediated by CXCR3.
CD14+ monocytes were treated with control medium or AMG487 (100 nM) and differentiated in the presence of recombinant M-CSF (30 ng/ml), natural CXCL4 (1–10 μg/ml) or recombinant CXCL4L1 (10 μg/ml). After 6 days of culture, cell supernatants were analyzed by ELISA to determine the amount of CXCL8. The measured cytokine concentrations were corrected for the number of viable cells in the cultures at the time of sampling. The results are shown as mean ± SEM and are pooled from 8–10 independent experiments. *p<0.05, **p<0.01,***p<0.001, Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF); †p<0.05, Sign test (inhibition by AMG487 of CXCL8 release).
Fig 7.
Modulation of the surface marker and chemokine receptor expression on immature MDDC by CXCL4 and CXCL4L1.
Purified human CD14+ monocytes were cultured in the presence of 50 ng/ml GM-CSF and 20 ng/ml IL-4. On day 4, different concentrations of natural CXCL4 (10 μg/ml) or recombinant CXCL4L1 (1 and 10 μg/ml) were added to the cultures. On day 6 (i.e. 2 days after addition of chemokines), iMDDCs A were collected and the expression of surface markers (panel A) and chemokine receptors (panel B) was analyzed by flow cytometry. Results from 3 to 7 independent experiments were expressed as mean fluorescence intensity and are shown as mean (± SEM) percentages, normalized to iMDDC cultured in the absence (100%) of CXCL4 or CXCL4L1. **p<0.01, Mann-Whitney U test (CXCL4 versus untreated); †p<0.05, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations).
Fig 8.
Effects of CXCL4 and CXCL4L1 on gene expression in differentiated immature MDDC.
Relative expression of CCL2, iNOS, MMP-8, MMP-9, MMP-12 and VEGF was evaluated in iMDDC A following treatment with GM-CSF (50 ng/ml) and IL-4 (20 ng/ml) in the absence (Co) or presence of CXCL4- (10 μg/ml) or CXCL4L1 (1 and 10 μg/ml). The results are shown as mean (± SEM) percentage expression relative to the expression on untreated iMDDC. *p<0.05, Mann-Whitney U test (CXCL4 or CXCL4L1 versus CO); †p<0.05, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations).
Fig 9.
Cytokine and chemokine production by iMDDC A after stimulation with CXCL4 or CXCL4L1.
Purified human CD14+ monocytes were cultured in the presence of 50 ng/ml GM-CSF and 20 ng/ml IL-4. On day 4, different concentrations of natural CXCL4 (10 μg/ml) or recombinant CXCL4L1 (0.1, 1 and 10 μg/ml) were added to the cultures. Cell supernatants were collected after 6 days of culture. The amount of CCL18 (panel A), IL-10 (panel B), CCL22 (panel C), CCL2 (panel D) and CXCL8 (panel E) produced was measured by sandwich ELISAs. The results (mean ± SEM) are derived from 5–7 independent experiments.
Fig 10.
Chemokine production by iMDDC B after a 6 day-treatment with CXCL4 or CXCL4L1.
Purified human CD14+ monocytes were cultured for 6 days in the presence of 50 ng/ml GM-CSF and 20 ng/ml IL-4. From the start of the culture different concentrations of natural CXCL4 (10 μg/ml) or recombinant CXCL4L1 (0.1, 1 and 10 μg/ml) were added (iMDDC B). Cell supernatants were collected after 6 days of culture. The amount of CCL22 (panel A) and CXCL8 (panel B) produced was measured by sandwich ELISAs. The results (mean ± SEM) are derived from 6 independent experiments. *p<0.05, **p<0.01, Mann-Whitney U test (CXCL4 or CXCL4L1 versus M-CSF); †p<0.05, Mann-Whitney U test (CXCL4 versus CXCL4L1 at identical concentrations).
Fig 11.
CXCL4- or CXCL4L1-stimulated phagocytosis of S. aureus by dendritic cells.
CD14+ monocytes were cultured in the presence of 50 ng/ml GM-CSF and 20 ng/ml IL-4. On day 4 (panel A; iMDDC A) or day 0 (panel B;iMDDC B), different concentrations of natural CXCL4 (10 μg/ml) or recombinant CXCL4L1 (0.1, 1 and 10 μg/ml) were added. After 6 days of culture, DC were exposed to pHrodo-labeled S. aureus as described in Methods. The phagocytic capacity of CXCL4- and CXCL4L1-stimulated iMDDC was assessed by flow cytometry and is expressed relative to the net fluorescence of control iMDDC, differentiated with GM-CSF plus IL-4 without addition of chemokine (Co). Results of 3–4 independent experiments, each performed in duplicate are shown. *p<0.05; Mann-Whitney U test (CXCL4 versus M-CSF) - †p<0.05; Mann-Whitney U test (CXCL4 versus CXCL4L1).