Ubiquitin receptor binding and signaling in primary human leukocytes

Abstract

Utilizing the human monocyte/macrophage cell line THP1, we recently identified extracellular ubiquitin as an endogenous agonist of the G protein-coupled receptor CXC chemokine receptor (CXCR) 4. Because receptor binding and signaling properties of extracellular ubiquitin have not been evaluated in primary human leukocytes, we analyzed its binding characteristics and subsequent Ca²⁺ signaling in freshly isolated human B-cells, T-cells and monocytes. Ubiquitin binding shows typical receptor binding characteristics and promotes intracellular Ca²⁺ flux within seconds in all three cell populations. The K_d for the ubiquitin receptor interaction in freshly isolated human monocytes is consistent with the affinity of the ubiquitin CXCR4 interaction that we reported for THP1 cells. As detected in THP1 cells previously, the ubiquitin induced Ca²⁺ flux can be attenuated with a phospholipase C inhibitor in all primary leukocyte cultures. Our observations further support the finding that ubiquitin is a CXCR4 agonist and demonstrate that extracellular ubiquitin induces physiological relevant signaling events in primary human leukocytes. Although the exact mechanism of the ubiquitin CXCR4 interaction, its receptor selectivity and subsequent signaling events remain to be determined, our findings identify a novel and unexpected biological role of extracellular ubiquitin as an endogenous immune modulator.

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Ubiquitin is a post-translational protein modifier in all eukaryotic cells.1 Besides its intracellular localization and function, ubiquitin has also been detected in various extracellular fluids, such as plasma, cerebrospinal fluid, bronchoalveolar lavage fluid, seminal plasma, amniotic fluid or urine and increased levels of extracellular ubiquitin have been described in a variety of diseases.2 Multiple lines of evidence suggest that extracellular ubiquitin has pleiotropic functions in the innate immune system and that administration of exogenous ubiquitin attenuates inflammation and reduces organ injury in several disease models.2 However, its mechanism of action remained unknown. Utilizing the human acute monocytic leukemia cell line THP1, we recently identified ubiquitin as an agonist of the G protein-coupled receptor (GPCR) CXC chemokine receptor (CXCR) 4.3 Although we demonstrated by flow cytometry that N-terminal fluorescein labeled ubiquitin (FITC-ubiquitin) binds to various human and murine monocyte/macrophage cell lines and freshly isolated human monocytes at 4°C,3 the receptor binding and signaling properties of ubiquitin have not been evaluated in primary human leukocytes. To confirm that ubiquitin binding to the cell surface of primary human monocytes shows also typical receptor binding characteristics, we isolated monocytes from freshly prepared buffy coats by density gradient centrifugation, followed by plastic adherence.3 Buffy coats from healthy blood donors were obtained from Lifesource, Chicagoland's blood center. Ubiquitin receptor binding was tested after incubation of the cells with FITC-ubiquitin (Boston Biochem) for 1 min at 4°C, as described.3 Reactions were performed in the presence of 1% bovine serum albumin to prevent nonspecific binding. As shown in Figure 1A, FITC-ubiquitin binding to monocytes was saturable and could be prevented by an excess of unlabeled ubiquitin. Based on the specific FITC-ubiquitin binding curve from experiments with monocytes obtained from seven blood donors, the K_d was 130 ± 60 nM in saturation binding experiments. This affinity of the ubiquitin receptor interaction is comparable with its receptor affinity that we determined previously in THP1 cells.3 As CXCR4 is abundantly expressed on lymphocytes,4 we also performed initial experiments with B and T cells. Pan B cells, T cells and monocytes were isolated from PBMCs via negative selection using an indirect magnetic labeling system (MACS LS, Miltenyi Biotech Inc., CA). Figure 1B shows typical specific receptor binding curves with leukocytes from a single donor. Consistent with the cell surface expression of CXCR4, ubiquitin receptor binding was detectable in all three cell types. In agreement with a higher expression of CXCR4 on the cell surface of B-cells,5 the B_max value that we determined in B-cells was higher than the B_max values determined for T-cells and monocytes (RFU; B-cells: 406 ± 30; T-cells: 317 ± 26; monocytes: 326 ± 16; p = 0.042). As CXCR4 activation promotes intracellular Ca²⁺ flux, we further confirmed that ubiquitin possess biological activity in primary human leukocytes using the Fluo-4NW calcium assay (Molecular Probes).3,6 Figure 1C shows the results of the Ca²⁺ flux measurements with the leukocyte preparations that we used in Figure 1B. As expected for a CXCR4 agonist, addition of ubiquitin to the cell cultures induced intracellular Ca²⁺ flux within seconds. Consistent with our observations in THP1 cells, the phospholipase C (PLC) inhibitor U73122 was able to inhibit ubiquitin induced Ca²⁺ flux in all cell cultures, as compared with the weak PLC inhibitor U73343 (both from EMD Biosciences). These additional data further support our finding that ubiquitin is a CXCR4 agonist and demonstrate that it induces physiological relevant signaling events in primary human leukocytes.

Chemokine receptors and their ligands are highly promiscuous, being able to bind multiple receptors/ligands.6,7 While stromal-cell derived factor (SDF)-1α ((CX-C motif) ligand (CXCL) 12) has been shown to bind to CXCR4 and CXCR7, known endogenous CXCR4 ligands are SDF-1α, macrophage migration inhibitory factor (MIF),6–9 and now ubiquitin.3 Because of the promiscuity of chemokine receptors, it appears possible that ubiquitin binds to multiple receptors of this family. Depending on the specific agonist, a single GPCR can signal through different pathways with different efficacies, which is known as biased agonism or functional selectivity.10–12 Biased agonism has been suggested for CXCR4 by findings which imply the presence of alternative agonist-binding sites.13 Although previously reported in vivo and in vitro effects of ubiquitin and SDF-1α are consistent with CXCR4 as their common recep tor,14–26 it cannot be excluded that ubiquitin and SDF-1α activation of CXCR4 evoke distinct biological effects. While the precise mechanism of CXCR4 activation by ubiquitin as well as the subsequent intracellular signaling events remain to be determined, our findings identify a novel biological role of ubiquitin when it is released into the extracellular space. Because ubiquitin is one of the most highly conserved proteins in all eukaryotes,27 its anti-inflammatory actions could constitute a primordial anti-inflammatory mechanism that is conserved throughout the phylogenetic system.

Figures and Tables

Figure 1 (A) FITC-ubiquitin binding to human monocytes (1 min, 4°C). Note that cells were centrifuged for 5 min to remove free FITC-ubiquitin in the cell culture supernatant. Data are mean ± Sem of duplicate measurements with monocytes from seven healthy blood donors. ●, FITC-ubiquitin binding; ■, non-specific binding, as assessed by binding of FITC-ubiquitin in the presence of 300 µm native ubiquitin; dashed line, specific binding curve (= total FITC-ubiquitin binding - non-specific binding; r²: 0.93). (B) Specific FITC-ubiquitin binding curves in monocytes (), B () and t cells () from a single blood donor, determined as in (A). (C) ubiquitin (3 µm) induced Ca²⁺ flux in monocytes (

), B-(

) and t-cells (

) from (B). Data are mean ± SEM from 10 cell cultures per cell type. Arrows indicate the time point when ubiquitin or vehicle (○) was added. (D) Inhibition of the ubiquitin induced Ca²⁺ signal by the PLC inhibitor U73122 (10 µM, open symbols). Data are mean ± SEM from three cell cultures per cell type. The weak PLC inhibitor U73343 (10 µM; grey symbols) was used as a negative control for U73122. Same cells as in B and C. Arrows indicate the time point when ubiquitin (3 µM) was added. RFU: relative fluorescence units.

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