Cell culture oxygen and signaling: Supraphysiologic in vitro oxygen signals through the HIF-CXCR4 pathway in human B cells

Abstract

Abstract Current understanding of B lymphocyte function relies heavily upon in vitro assays. However, standard incubators maintain cells at supraphysiologic oxygen (16–19% oxygen). Physiologic oxygen is far lower (blood 5–10%, secondary lymphoid tissues 1–5%, regions of inflammation, tumor, and bone marrow 0–1%). Previously, we showed that transcripts for hypoxia-induced factor 1 alpha (HIF-1a), an oxygen-sensing transcription factor, are upregulated in peripheral B cells after vaccination1. HIF-1a is also a therapeutic target in multiple myeloma2. Downstream of HIF-1a are genes tied to metabolism, proliferation, and the unfolded protein response. One gene downstream of HIF1a that is critical for B cell migration and function is CXCR4. We set out to determine if room air incubation might affect B cells in vitro, our null hypothesis being that physiologic oxygen would not affect B cell HIF-1a levels or CXCR4 function. We used C-chamber incubator sub-chambers and Pro-Ox 110 gas controllers to control the incubation oxygen and carbon dioxide levels. Setting the reference frame to a range relevant to secondary lymphoid organs (1–5% oxygen), we set the chambers to 5% CO2 and 1% (hypoxic), 4% (physioxic), or 19% (supraphysioxic) oxygen. In primary human B cells and myeloma cell lines, HIF-1a protein levels stabilized at 24 hrs of culture at 1% and 4% oxygen, but not at 19% oxygen. More importantly, B cell migration in response to the CXCR4 ligand, CXCL12, was significantly decreased at low oxygen levels, without affecting cell proliferation or viability, disproving our null hypothesis. This has widespread implications for assessments of B cell function in that incubator oxygen drives HIF-1a degradation and CXCR4 responsiveness in vitro.