Induction of CD36 and Thrombospondin-1 in Macrophages by Hypoxia-Inducible Factor 1 and Its Relevance in the Inflammatory Process

<div><p>Inflammation is part of a complex biological response of vascular tissue to pathogens or damaged cells. First inflammatory cells attempt to remove the injurious stimuli and this is followed by a healing process mediated principally by phagocytosis of senescent cells. Hypoxia and p38-MAPK are associated with inflammation, and hypoxia inducible factor 1 (HIF-1) has been detected in inflamed tissues. We aimed to analyse the role of p38-MAPK and HIF-1 in the transcriptional regulation of CD36, a class B scavenger receptor, and its ligand thrombospondin (TSP-1) in macrophages and to evaluate the involvement of this pathway in phagocytosis of apoptotic neutrophils. We have also assessed HIF-1α, p38-MAPK and CD36 immunostaining in the mucosa of patients with inflammatory bowel disease. Results show that hypoxia increases neutrophil phagocytosis by macrophages and induces the expression of CD36 and TSP-1. Addition of a p38-MAPK inhibitor significantly reduced the increase in CD36 and TSP-1 expression provoked by hypoxia and decreased HIF-1α stabilization in macrophages. Transient transfection of macrophages with a <em>miHIF-1α</em>-targeting vector blocked the increase in mRNA expression of <em>CD36</em> and <em>TSP-1</em> during hypoxia and reduced phagocytosis, thus highlighting a role for the transcriptional activity of HIF-1. CD36 and TSP-1 were necessary for the phagocytosis of neutrophils induced by hypoxic macrophages, since functional blockade of these proteins undermined this process. Immunohistochemical studies revealed CD36, HIF-1α and p38-MAPK expression in the mucosa of patients with inflammatory bowel disease. A positive and significant correlation between HIF-1α and CD36 expression and CD36 and p38-MAPK expression was observed in cells of the lamina propria of the damaged mucosa. Our results demonstrate a HIF-1-dependent up-regulation of CD36 and TSP-1 that mediates the increased phagocytosis of neutrophils by macrophages during hypoxia. Moreover, they suggest that CD36 expression in the damaged mucosa of patients with inflammatory bowel disease depends on p38-MAPK and HIF-1 activity.</p> </div

HIF-1, p38-MAPK and CD36 correlates in the inflamed mucosa of patients with inflammatory bowel disease.

<p>A) Representative microphotographs showing HIF-1α, p38-MAPK and CD36 immunostaining in the damaged and non-damaged mucosa of patients with inflammatory bowel disease. Biopsy specimens of the intestine were excised, formalin-fixed, paraffin-embedded, cut into 5 µm slices, and stained with hematoxylin; B) Graph shows a quantitative analysis of the number of HIF-1α, p38-MAPK or CD36 positive cells in a total area of 0.135 mm² of the mucosa of patients with IBD. Bars in the graph represent mean± SEM (<i>n></i>3). Significant difference from the respective non-damaged mucosa is shown by *<i>P</i><0.05. C) Graphs show a positive and significant correlation between CD36 and HIF-1α (R Spearman = 0.7170, P = 0.0087**, n = 12) and p38-MAPK and CD36 (R Spearman = 0.6525, P = 0.0215*, n = 12) immunostaining at the damaged mucosa of patients with IBD. No correlation was observed between CD36 and HIF-1α (R Spearman = −0.0513, P = 0.95, n = 5), or p38-MAPK and CD36 (R Spearman = 0.5204, P = 0.2311, n = 7) immunostaining at the non-damaged mucosa.</p

Role of CD36 and TSP-1 in phagocytosis mediated by macrophages.

<p>Graphs show the effects of CD36 and TSP-1 functional antibodies or control IgG on phagocytosis of apoptotic neutrophils mediated by U937 cells or THP1 cells. In both cases, blockade of CD36 or TSP-1 significantly reduced hypoxia-induced phagocytosis. Data show the intensity of fluorescence in arbitrary units (quantified by static cytometry). Bars represent mean± SEM (<i>n></i>3). Groups were compared using ANOVA followed by a Newman Keuls test. *P<0.05 shows significant difference with respect to all groups in the same graph.</p

Patient clinical information.

<p>Patient clinical information.</p

Recruitment of HIF-1 to the promoter of <i>TSP-1</i> gene.

<p>A) Results show a representative chromatin immunoprecipitation (ChIP) experiment performed in samples from U937-derived macrophages in normoxia or hypoxia. Chromatin was immunoprecipitated with anti-HIF-1α antibody, or a non-related antibody anti-IgG as a control. An aliquot of the input chromatin is also shown. Primers specific to the promoter region for TSP-1 gene were used to amplify the DNA isolated from the ChIP assay. B) HIF-1α expression in nuclear lysates derived from non-transfected cells and from <i>miHIF1α</i> or mock-transfected U937cells exposed to normoxia or hypoxia. Interactions between HIF-1α and HRE of the TSP-1 promoter gene were examined by EMSA using synthetic oligonucleotides and nuclear lysates derived from transfected or non-transfected cells exposed to normoxia or hypoxia. Specificity was determined with excess unlabelled probed (XS) or mutated probe (n = 3).</p

Hypoxia induces TSP-1 and CD36 expression and HIF-1α stabilization through activation of p38-MAPK.

<p>U937 cells were maintained under normoxia or hypoxia in the presence or absence of SB 202190 (a p38-MAPK inhibitor, 10 µM, 24 h) and levels of proteins were determined by Western blot. Graphs show quantification of HIF-1α, TSP-1 and CD36 by densitometry. In hypoxia, cells treated with SB 202190 exhibited significantly lower protein expression of HIF-1α, TSP-1 and CD36 than cells treated with vehicle. In all cases bars represent mean± SEM (<i>n></i>3). Comparisons between groups were performed using ANOVA followed by a Newman Keuls test. *P<0.05 and ***P<0.001 with respect to all groups in the same graph and ^###P<0.001 vs. bars in normoxia.</p