High Glucose Upregulates C-Reactive Protein Synthesis in Macrophages

To the Editor:

C-reactive protein (CRP)1 released from hepatocytes during the acute-phase response is a diagnostically sensitive systemic marker for inflammation; CRP also demonstrates substantial proinflammatory effects (1). Although CRP might have an important role in the pathogenesis and prediction of coronary heart diseases(2), the factors influencing its concentration are not yet well understood. Recent histologic investigations have demonstrated that CRP is present in the human arterial intima of atherosclerotic lesions(3) and is located in macrophages of the arterial plaque. In addition, macrophages have been shown to produce CRP mRNA(4).

Diabetes mellitus is associated with premature and accelerated atherosclerosis (5), and hyperglycemia accelerates atherosclerosis by inducing vascular dysfunction and an increased inflammatory burden. Our goal was to analyze the possible modulation of CRP production in macrophages after their exposure to high glucose concentrations.

J-774A.1 macrophage-like cells (ATCC) were plated in DMEM with 100 000 U/L penicillin, 100 mg/L streptomycin, and 2 mmol/L glutamine (P/S/G), and used within 7 days. THP-1 human monocyte cells were maintained in RPMI-1640 medium with P/S/G, and macrophage induction was achieved by incubation with phorbol myristate acetate. Cells were incubated with either 5–40 mmol/L glucose for 18 h or with 22 nmol/L mannitol (an osmotic control that did not appreciably affect CRP cellular production).

We extracted cellular RNA with MasterPure™ RNA Purification Kit (Epicentre Biotechnologies) and prepared cDNA with the Verso™ cDNA Kit (Thermo Scientific) according the manufacturers’ instructions. CRP production was measured by quantitative real-time PCR by means of Rotor-Gene 6000 (Corbett Life Science/Qiagen) amplification with ABsolute Blue QPCR ROX Mix (Thermo Scientific) and primers and probes for the CRP2 (C-reactive protein, pentraxin-related) and ACTB (actin, beta) genes (PrimerDesign) (6).

CRP in sections on slides was stained with antihuman CRP antibody (Sigma–Aldrich) and subsequently counterstained with secondary antibody (antimouse fluorescein isothiocyanate–conjugated IgG; Sigma-Aldrich) (7). All control sections were processed without primary antibody. The slides were photographed with the aid of a fluorescence digital microscope camera [Zeiss Axioskop 2 plus microscope, with image-processing software; Image-Pro Plus 6.0 (Media Cybernetics)]. Light intensity and contrast were calibrated with an appropriate control section. Image-Pro Plus 6.0 software (Media Cybernetics) was used to quantify CRP on the slides, and CRP was measured by ELISA (Mouse CRP 96-well ELISA; Life Diagnostics) as recommended by the manufacturer. Lactate dehydrogenase was measured to assess cell viability, and incubation with 5–40 mmol/L glucose did not affect cell viability. Analysis of variance was used for statistical analyses. Results are expressed as the mean (SD).

THP-1 macrophages incubated with 30 or 40 mmol/L glucose exhibited increased production of CRP mRNA [61% and 109%, respectively; mean ratio of CRP mRNA to β-actin mRNA, 1.144 (0.08) and 1.487 (0.09), respectively], compared with cells incubated with only 5 mmol/L glucose [ratio of CRP mRNA to β-actin mRNA, 0.71 (0.03)]. J-774A.1 mouse macrophages incubated with 40 mmol/L glucose increased CRP mRNA production by 313% [ratio of CRP mRNA to β-actin mRNA, 1.28 (0.05)], compared with cells incubated with only 5 mmol/L glucose [ratio of CRP mRNA to β-actin mRNA, 0.31 (0.01)].

When macrophages were incubated with increasing glucose concentrations (5–40 mmol/L) for 18 h and analyzed by immunohistochemistry for CRP protein content, the intensity of fluorescein isothiocyanate staining of the cells was proportional to the detected CRP concentration. Cells incubated with 20, 30, or 40 mmol/L glucose exhibited increased cellular CRP concentrations (19%, 48%, and 103%, respectively), compared with control cells incubated with 5 mmol/L glucose (Fig. 1⇓ ). Measured CRP concentrations in media harvested from incubated macrophages treated with 40 mmol/L glucose were significantly higher (by 2.5-fold, 8.8 μg/L) than the concentrations in media harvested from incubated macrophages treated with 5 mmol/L glucose (3.5 μg/L).

We hypothesize that CRP secreted by arterial macrophages could be up-regulated under atherogenic conditions such as diabetes and could therefore actively accelerate inflammatory processes in atherosclerotic lesions. Macrophages exposed to high glucose concentrations modulate the regulation of cellular CRP expression and CRP protein biosynthesis and secretion. Therefore, proinflammatory effects mediated by CRP in the arterial wall could be triggered by CRP secreted locally by macrophages, in addition to the effect of circulating, hepatic-derived CRP. Use of pharmacologic therapy to modulate CRP synthesis at the site of the atherosclerotic lesion possibly could reduce the inflammatory cascade markedly during atherosclerosis development and hence help prevent coronary heart disease.