The Interleukin-1 Receptor Antagonist Gene and the Inhibitor of Kappa B-Like Protein Gene Polymorphisms Are Not Associated with Myocardial Infarction in Slovene Population with Type 2 Diabetes

In this study we investigated the association of the interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism and of the inhibitor of kappa B-like protein (IKBL) gene polymorphism with myocardial infarction (MI) in a group of patients with type 2 diabetes. The IL1RN VNTR and the IKBL+738T>C gene polymorphisms were tested in 374 Caucasians: 151 cases with MI and 223 subjects with no history of coronary artery disease. The IL1RN VNTR polymorphism was not a risk factor for MI in Caucasians with type 2 diabetes (genotype 22 vs. the rest: odds ratio (OR) 1.6; 95% confidence interval (CI) = 0.8–3.5; p=0.2). We also failed to demonstrate that IKBL+ 738T>C gene polymorphism was associated with MI in patients with type 2 diabetes (OR =0.9; 95% CI = 0.3–2.6; p=0.9). We provide evidence that the IL1RN VNTR and the IKBL+738T>C gene polymorphisms are not risk factors for MI in Caucasians with type 2 diabetes

FPR1 cell surface expression on human neutrophils, monocytes, and macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1. A) Neutrophils. B) Monocytes. C) 9 day old macrophages. Autofluorescence of the cells is shown in light grey, the isotype control in grey and cells labeled with FPR1 Ab in black. D) Quantitative representation of the FPR1 median fluorescence intensity (antibody MFI minus isotype MFI) NG: neutrophils (n = 3). MO: monocytes (n = 5). MA: 9 day old macrophages (n = 9).</p

fLMF chemotaxis assays with human macrophages.

<p>Control (Ctrl) and IL-4 (10 ng/ml), IL-13 (10 ng/ml), IFNγ (50 ng/ml), LPS (100 ng/ml), and 3M-002 (3 µM) stimulated macrophages were allowed to migrate towards the chemotactic factor fLMF (+). Medium without chemotactic factor (−) was used as control for basal migration in each experiment. Graphs show the mean migration index compared to each individual control (n = 3) and error bars display the standard deviation (±SD). *p<0.05; **p<0.01.</p

Regulation of FPR1 mRNA expression in human macrophages.

<p>A) Relative mRNA expression of FPR1 after stimulation of human macrophages with different stimuli for 24 hours. IL-3 (20 ng/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml), IL-1β (5 ng/ml), IL-6 (10 ng/ml), INFγ (50 ng/ml), TNFα (1 ng/ml), CpG (100 ng/ml), LPS (100 ng/ml), Poly I:C (1 ng/ml), 3M-001 (3 µM), 3M-002 (3 µM). B) Time-course experiment of FPR1 mRNA expression in macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. The values are normalized for GAPDH mRNA expression and are presented relative to non-stimulated control macrophages (black). Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Regulation of FPR1 cell surface expression in human macrophages.

<p>FACS analysis was performed to investigate cell surface expression of FPR1 upon treatment with the indicated stimuli. A) FPR1 protein expression after stimulation of human macrophages for 48 hours with stimuli which were shown to regulate FPR1 mRNA expression (IL-4 (10 ng/ml), IL-13 (10 ng/ml), INFγ (50 ng/ml), LPS (100 ng/ml), 3M-002 (3 µM)). B) Time-course experiment of FPR1 protein expression in controls macrophages (black), or macrophages stimulated with IL-4 (10 ng/ml) (dotted) and IL-13 (10 ng/ml) (white) for 24, 48 and 72 hours. Values are presented relative to unstimulated macrophages. Bars display the mean and the standard deviation (±SD) of three independent experiments. *p<0.05, **p<0.01.</p

Characterization of the promoter and the transcriptional regulation of the Lipoxin A4 receptor (FPR2/ALX) gene in human monocytes and macrophages

The lipoxin A4 receptor FPR2/ALX plays an important part in host defense and inflammation. The receptor binds structurally diverse agonistic ligands, which mainly regulate chemotaxis and activation of leukocytes. However, little is known about the promoter region of the FPR2/ALX gene and its transcriptional regulation in leukocytes. We identified two TATA-less promoter regions, separated by 224 bp, that drive the expression of FPR2/ALX in macrophages. Both promoter regions increased transcription in a reporter assay, and the basal transcription factors OCT1 and SP1 were shown to bind the first and the second promoter, respectively, and to transactivate transcription. Although monocytes expressed high levels of FPR2/ALX mRNA from the second promoter region, differentiation into macrophages abrogated FPR2/ALX expression. Stimulation of macrophages with a set of cytokines revealed that only IFN-γ and LPS increased FPR2/ALX expression from the first promoter to levels similar to those detected in monocytes. The upregulation by IFN-γ is in part mediated by the interaction of IFN regulatory factor 1 with an IFN-responsive sequence element transcription factor binding site located in the first promoter region of the FPR2/ALX gene. However, this upregulation on the mRNA level did not translate into FPR2/ALX protein expression in macrophages owing to reduced translation of the longer mRNA from the first promoter. In contrast, FPR2/ALX mRNA transcribed from the second promoter was translated into surface expression of FPR2/ALX in monocytes. These data support a model in which FPR2/ALX plays a role in chemotaxis and activation of monocytes; however, they also suggest that its function in resident tissue macrophages is limited

Regulation of the formyl peptide receptor 1 (FPR1) gene in primary human macrophages

The formyl peptide receptor 1 (FPR1) is mainly expressed by mammalian phagocytic leukocytes and plays a role in chemotaxis, killing of microorganisms through phagocytosis, and the generation of reactive oxygen species. A large number of ligands have been identified triggering FPR1 including formylated and non-formylated peptides of microbial and endogenous origin. While the expression of FPR1 in neutrophils has been investigated intensively, knowledge on the regulation of FPR1 expression in polarized macrophages is lacking. In this study we show that primary human neutrophils, monocytes and resting macrophages do express the receptor on their cell surface. Polarization of macrophages with IFNγ, LPS and with the TLR8 ligand 3M-002 further increases FPR1 mRNA levels but does not consistently increase protein expression or chemotaxis towards the FPR1 ligand fMLF. In contrast, polarization of primary human macrophages with IL-4 and IL-13 leading to the alternative activated macrophages, reduces FPR1 cell surface expression and abolishes chemotaxis towards fMLF. These results show that M2 macrophages will not react to triggering of FPR1, limiting the role for FPR1 to chemotaxis and superoxide production of resting and pro-inflammatory M1 macrophages