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. 2012 Aug 7;14(4):R181.
doi: 10.1186/ar4011.

Human monocytes and macrophages differ in their mechanisms of adaptation to hypoxia

Monique Fangradt, Martin Hahne, Timo Gaber, Cindy Strehl, Roman Rauch, Paula Hoff, Max Löhning, Gerd-Rüdiger Burmester, Frank Buttgereit

Human monocytes and macrophages differ in their mechanisms of adaptation to hypoxia

Monique Fangradt et al. Arthritis Res Ther. .

Abstract

Introduction: Inflammatory 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).

Methods: Isolated 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).

Results: We 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.

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

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Figures

Figure 1
Figure 1
Hypoxia-inducible factor alpha (HIF-1α) is stabilized under hypoxia but remains in the cytoplasm. (A) Scheme of sample acquisition for kinetic analyzes of monocytes incubated in a water-jacket chamber sealed with a Clark-type oxygen electrode, which facilitates the constant monitoring of oxygen saturation during the experimental setup (sample acquisition is indicated by arrows). (B) Detection of HIF-1α and, for normalization purposes, β-actin in monocyte whole cell protein samples acquired as shown in (A) by immunoblot. Under hypoxia, monocytes stabilize HIF-1α in a time-dependent manner. (C) Detection of HIF-1α, β-actin and for normalization purposes, the nuclear protein Lamin B, in monocyte nuclear fraction (NF+) and cytosolic (NF-) cell fractions using immunoblot. HIF-1α was exclusively detected in the cytoplasm in unstimulated monocytes incubated under hypoxic conditions (n = 6).
Figure 2
Figure 2
Toll-like receptor (TLR) stimulation does not affect hypoxia-inducible factor alpha (HIF-1α) localization. (A) Table of TLRs and their corresponding ligands used in our experiments within the given concentration range. (B-D) Detection of HIF-1α, Lamin B and β-actin in monocyte nuclear (NF+) and cytosolic (NF-) cell fractions using immunoblot. Monocyte protein lysates were acquired after TLR stimulation and incubation for 5 h under hypoxia using (B) TLR1/2 stimulation by Pam3CSK4 3HCl, (C) TLR4 stimulation by LPS or (D) TLR7/8 stimulation by R-848.
Figure 3
Figure 3
Protein kinase (PKC)-α/β1 is essential for hypoxia-inducible factor alpha (HIF-1α) translocation. (A+B) Detection of HIF-1α, Lamin B and β-actin in nuclear (NF+) and cytosolic (NF-) cell fractions of primary human monocytes using immunoblot. (A) Monocyte protein lysates were acquired after PMA stimulation and incubation for 5 h under hypoxia and under normoxia as indicated. (B) Monocyte protein lysates were acquired after PMA stimulation following PKC-α/β1 inhibition using Gö6976 as indicated and subsequent incubation for 5 h under hypoxia. (A, B) PMA stimulation for 5 h under hypoxia leads to nuclear translocation of HIF-1α, and specific inhibition of PKC-α/β1 by Gö6976 prevents translocation of HIF-1α.
Figure 4
Figure 4
Hypoxia-inducible factor alpha (HIF-1α) translocation after differentiation of monocytes to macrophages and co-culture with endothelial cells. (A, B) Detection of HIF-1α, Lamin B and β-actin in monocyte or hMDM nuclear (NF+) and cytosolic (NF-) cell fractions using immunoblot. (A) Protein lysates were acquired after incubation of human monocytes and hMDM for 5 h under hypoxia. Differentiation of monocytes to macrophages leads to HIF-1α translocation into the nucleus. (B) Monocyte protein lysates were acquired after incubation of human monocytes on human endothelial cells (HMEC-1, human microvascular endothelial cells) coated well plates for 5 h under normoxia and under hypoxia. Co-culture of monocytes with endothelial cells is not sufficient for the translocation of HIF-1α.
Figure 5
Figure 5
Monocytes, in contrast to human monocyte derived macrophages (hMDMs), show hypoxia-induced gene expression. Real-time PCR analysis of mRNA expression levels of the glycolytic enzymes LDHA (A) and PGK1 (B), the hypoxia inducible factor (HIF)-1 target gene CXCR4 (C) and the transcription factor HIF1A (D) in monocytes and hMDM that were incubated under normoxia (white bars) and under hypoxia (black bars), respectively (n = 6). The mRNA expression level of ß-actin (ACTB) was used for normalization. ΔCt = Ct(GOI) - Ct(ACTB). Values are means ± SD. +P < 0.1;* P < 0.05; (Wilcoxon t-test).
Figure 6
Figure 6
Incubation of monocytes under hypoxia leads to translocation of transcription factor, nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB1), into the nucleus. Detection of hypoxia-inducible factor alpha (HIF-1α), Lamin B and β-actin in nuclear (NF+) and cytosolic (NF-) cell fractions of primary human monocytes using immunoblot. (A-C) Monocyte protein lysates were acquired after incubation for 5 h under hypoxia and under normoxia as indicated. The cellular localization of transcription factors NFκB p100/p52, c-Rel, and c-Jun (Figure 6A), NFκBp105/p50 and c-Fos (Figure 6B), and Jun B and NFκB p65 (Figure 6C) were determined by immunoblot. NFκBp105 (the inactive form of NFκB1) remains in the cytoplasm, while the active form NFκBp50 is translocated into the nucleus under hypoxic conditions (Figure 6B). Other factors showed either no change in their localization or, as for c-Jun and NFκBp52, were no longer translocated into the nucleus under hypoxic incubation.
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