Table 1.
Antibodies used in the study.
Figure 1.
Time-dependent dependent mast cell releasate-induced changes in macrophage scavenger receptor mRNA expression.
A) Monocyte-derived macrophages (MDM) from 3 donors were cultured in human primary mast cell releasate for 72 h, and LOX-1 mRNA expression was analyzed and compared to non-treated cells at the beginning of the incubation. B) MDMs from 6 donors were cultured in LAD2 mast cell releasate for 4 h and 24 h, and LOX-1, CD36 and MSR1 mRNA expression was measured and compared to non-treated cells at each time point. C) MDMs from 3 different donors were incubated in complete releasate, soluble releasate, or granules derived from LAD2 mast cells for 4 h and 24 h. The results are presented as means ± SEM. *p<0.05, **p<0.01 vs. non-treated cells.
Figure 2.
Effect of individual mast cell releasate components on macrophage LOX-1 expression.
Monocyte-derived macrophages were incubated with A) histamine, B) TNF-α, and C) TGF-β1 for 4 h and 24 h, and analyzed for LOX-1 mRNA expression (n = 6 donors). D) The combined effect of the components was studied by incubating macrophages with histamine (10 µM) and/or TNF-α (1 ng/ml) and/or TGF-β1 (0.1 ng/ml) for 4 h and 24 h (n = 4 donors). The results are presented as means ± SEM. *p<0.05, **p<0.01 vs. non-treated cells.
Figure 3.
Time-dependent expression of LOX-1 protein in histamine-treated human macrophages.
A) Monocyte-derived macrophages were cultured in the presence of 10 µM histamine for 6, 8, and 16 h. The cells were then lysed and LOX-1 protein expression was analyzed by Western blot technique. B) LOX-1 protein levels were quantified, normalized to b-actin, and compared to the levels at the beginning of the incubation (controls). The results are presented as means ± SEM (n = 4 donors). *p<0.05 vs. control cells.
Figure 4.
Effect of histamine H1 and H2 receptor antagonists on histamine-induced macrophage LOX-1 mRNA expression.
Monocyte-derived macrophages were incubated in the presence of 10 µM histamine and the indicated concentrations of the histamine H1 and H2 receptor antagonists (pyrilamine and ranitidine, respectively) for 4 h and analyzed for LOX-1 mRNA expression. The results are presented as means ± SEM (n = 3 donors). All statistical comparisons were non-significant.
Figure 5.
Time-dependent dependent mast cell releasate-induced effects on oxLDL-stimulated macrophage scavenger receptor mRNA expression.
Monocyte-derived macrophages were incubated with 50 µg/ml of DiI-oxLDL and/or in LAD2 (A, C, E) or primary mast cell (B, D, F) releasate for 4 h and 24 h, and analyzed for LOX-1, MSR1 and CD36 mRNA expression. The results are presented as means ± SEM (n = 4 donors). *p<0.05; **p<0.01.
Figure 6.
Effect of LAD2 and primary mast cell releasates on macrophage oxLDL uptake.
Monocyte-derived macrophages were incubated for 16 h in the presence of 50 µg/ml of DiI-oxLDL and/or with LAD2 (A) or primary mast cell (B) releasate, and/or with specific blocking antibodies for CD36 and MSR1 (2 µg/ml), or with isotype control antibodies IgG1 and IgA (2 µg/ml), or with 20-fold excess of unlabeled oxLDL to determine the magnitude of the high-affinity uptake of Dil-oxLDL. DiI fluorescence was measured from cell lysates. The results are presented as means ± SEM (n = 4 donors). *p<0.05, **p<0.01, ***p<0.001.