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Arthritis Research and Therapy

, 14:R181

First Online: 07 August 2012Received: 29 February 2012Revised: 30 June 2012Accepted: 07 August 2012


IntroductionInflammatory arthritis is a progressive disease with chronic inflammation of joints, which is mainly characterized by the infiltration of immune cells and synovial hyperproliferation. Monocytes migrate towards inflamed areas and differentiate into macrophages. In inflamed tissues, much lower oxygen levels hypoxia are present in comparison to the peripheral blood. Hence, a metabolic adaptation process must take place. Other studies suggest that Hypoxia Inducible Factor 1-alpha HIF-1α may regulate this process, but the mechanism involved for human monocytes is not yet clear. To address this issue, we analyzed the expression and function of HIF-1α in monocytes and macrophages, but also considered alternative pathways involving nuclear factor of kappa light polypeptide gene enhancer in B-cells NFκB.

MethodsIsolated human CD14 monocytes were incubated under normoxia and hypoxia conditions with or without phorbol 12-myristate 13-acetate PMA stimulation, respectively. Nuclear and cytosolic fractions were prepared in order to detect HIF-1α and NFκB by immunoblot. For the experiments with macrophages, primary human monocytes were differentiated into human monocyte derived macrophages hMDM using human macrophage colony-stimulating factor hM-CSF. The effects of normoxia and hypoxia on gene expression were compared between monocytes and hMDMs using quantitative PCR quantitative polymerase chain reaction.

ResultsWe demonstrate, using primary human monocytes and hMDM, that the localization of transcription factor HIF-1α during the differentiation process is shifted from the cytosol in monocytes into the nucleus in macrophages, apparently as an adaptation to a low oxygen environment. For this localization change, protein kinase C alpha-beta 1 PKC-α-β1 plays an important role. In monocytes, it is NFκB1, and not HIF-1α, which is of central importance for the expression of hypoxia-adjusted genes.

ConclusionsThese data demonstrate that during differentiation of monocytes into macrophages, crucial cellular adaptation mechanisms are decisively changed.


ATPadenosine triphosphate

c-AMPcyclic adenosine monophosphate

CDcluster of differentiation

cDNcomplementary deoxyribonucleic acid

CO2carbon dioxide

ctthreshold cycle

CXCRC-X-C motif chemokine receptor

DCdendritic cell

DRKDeutsches Rotes Kreuz German Red Cross

EGFendothelial growth factor

FCSfetal calf serum

GÖ 697612-2-Cyanoethyl-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo2,3-apyrrolo3,4-ccarbazole

HeLaHenrietta Lacks cell line

HIFhypoxia-inducible factor

HL-60human promyelocytic leukemia cells

hM-CSFhuman macrophage colony-stimulating factor

hMDMhuman monocyte derived macrophages

HMEC-1human microvascular endothelial cell line

HRPhorseradish peroxidase

IgGimmunoglobulin G

LDHAlactate dehydrogenase A


MACSmagnetic activated cell sorting


MHCmajor histocompatibility complex

mmHgmillimeter of mercury

N2molecular nitrogen

NFnuclear fraction

NFκBnuclear factor of kappa light polypeptide gene enhancer in B-cells

O2molecular oxygen

OxPhosoxidative phosphorylation


PCRpolymerase chain reaction

PGK1phosphoglycerate kinase 1


PKCprotein kinase C

PMAphorbol 12-myristate 13-acetate

pO2oxygen partial pressure

qPCRquantitative polymerase chain reaction


RArheumatoid arthritis

RNAribonucleic acid

RPMI 1640Roswell Park Memorial Institute media 1640

SDstandard deviation

SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresis

THP-1human acute monocytic leukemia cell line

TLRtoll like receptor

TNFtumor necrosis factor

U-937human histiocytic leukemia cell line

v-vvolume-volume percent.

Electronic supplementary materialThe online version of this article doi:10.1186-ar4011 contains supplementary material, which is available to authorized users.

Monique Fangradt, Martin Hahne contributed equally to this work.

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Autor: Monique Fangradt - Martin Hahne - Timo Gaber - Cindy Strehl - Roman Rauch - Paula Hoff - Max Löhning - Gerd-Rüdiger Burm


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