Phosphate-buffered saline (PBS), a buffer solution commonly used in many biological research fields, contains monobasic potassium phosphate, sodium chloride, and dibasic sodium phosphate. These solutions are widely used in both in vitro and in vivo studies and in laboratory protocols for dilutions, washing cell suspensions, rinsing culture flasks, and plates; they are also used as an additive to cell culture media.1–9 PBS is commercially available in different formulations, supplemented with calcium (Ca++) and magnesium (+/+ PBS) or without (–/– PBS) (Table 1).
Table 1 - Phosphate-buffered saline formulations*
Calcium functions as a major intracellular second messenger in many signal transduction pathways and triggers a broad range of cellular actions such as muscle contractility,10 bone metabolism,11 hormone secretion,12 and vascular tone adjustment.13 Furthermore, calcium has been shown to influence cellular interactions in sepsis14 and tissue inflammation by triggering chemotaxis directed to sites of infection or tissue injury.15 Since heightened extracellular calcium concentrations have been related to recruitment of immune cells, the authors hypothesized whether the addition of –/– or +/+ PBS might have an influence on human peripheral blood mononuclear cells (PBMCs) under different culture conditions. These alterations in the extracellular ion microenvironment may affect several cellular functions, including secretion of cytokines, chemoattraction, and cell death.16,17 Previous studies have shown that extracellular fluids at sites of dermal wounds or inflammation reportedly contain high concentrations of calcium,18,19 and chronic inflammatory conditions such as atherosclerosis are associated with tissue deposition of calcium.20,21
It has recently been shown that monocyte–macrophage cell lines can detect increasing extracellular calcium concentrations via the G-protein-coupled calcium-sensing receptor (CaR). Triggering of this receptor results in increased cytokine secretion, e.g., transforming growth factor-beta (TGF-β).22,23 Furthermore, a similar response has been described for keratinocytes and PBMCs.24,25 Interleukin-1 receptor antagonist (IL-1ra) is released when cells are exposed to increasing levels of extracellular calcium, further confirming the immunoregulatory function of Ca++.
Olszak et al.15 determined that mononuclear cells up-regulate the expression of the membrane-bound CaR in response to heightened levels of extracellular calcium concentrations. By this response, cells detect gradients of increasing calcium levels at sites of inflammation, which induces cellular chemotaxis and local immune cell accumulation.
The authors hypothesized that this response is also important in in vitro conditions, such as cell cultures, and could be further supported by increased chemokine secretion.26 Therefore, they planned to evaluate whether the addition of +/+ PBS in comparison to calcium-free PBS solutions has an impact on chemokine secretion by human PBMC. They sought to screen for a broad range of CXC chemokines such as interleukin-8 (IL-8); epithelial neutrophil activating peptide-78 (ENA-78); growth-related oncogene-alpha (GRO-α); granulocyte chemoattractant protein-2 (GCP-2); interferon-gammainduced protein-10 (IP-10); stromal cell-derived factor-1 (SDF-1); and CC chemokines, such as monocyte chemoattractant protein-1 (MCP-1), regulated upon activation, normal T-cell expressed, and secreted (RANTES), and CX3C chemokines (fractalkine).
The study was conducted at the research laboratory of the Department of Cardiac and Thoracic Surgery (Medical University Vienna) according to the principles of the Helsinki Declaration and Good Clinical Practice. Informed consent was obtained from all participants in the study. Major inclusion criteria were: body mass index of 18–28 kg/m2, no intake of anti-inflammatory drugs during the last two weeks, no acute infection during the last month, no chronic inflammatory disease, and no physical activity during the last hours prior to testing.
Separation of PBMCs
PBMCs were separated by Ficoll density gradient centrifugation, as previously described. In short, venous blood was drawn from healthy volunteers (n = 7). Anticoagulated blood specimens were processed immediately, diluted 1:2 in Hank’s balanced salt solution (HBSS, Lonza, Basel, Switzerland), and shifted carefully into 50-mL tubes containing Ficoll-Paque solution (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Tubes were centrifuged for 15 min at 800 g at room temperature continuously, and buffy coats with mononuclear cells were obtained. Cells were washed in HBSS and resuspended in fresh GIBCO® RPMI 1640 medium (Invitrogen) supplemented with Glutamax ™ (Invitrogen). Cell concentrations were determined on a Sysmex KX-21N automated cell counter (Sysmex, Mundelein, IL).
PBMCs were seeded on 24-well cell culture plates (PerkinElmer, Waltham, MA) at a concentration of 1 × 106 per milliliter, containing 20% of either PBS (Invitrogen) with (+/+ PBS) or without (–/– PBS) calcium and magnesium. For further investigations, increasing concentrations of +/+ PBS were added. Dilutions of 1.25×, 1.5×, 2×, and 3× +/+ PBS were generated from a stock solution of 10× +/+ PBS. Final molar concentrations for calcium and magnesium in medium supplemented with increasing dilutions of +/+ PBS were as follows: 1× +/+ PBS 0.50 mM Ca++, 0.50 mM Mg++; 1.25× +/+ PBS 0.64 mM Ca++, 0.52 mM Mg++; 1.5× +/+ PBS 0.69 mM Ca++, 0.55 mM Mg++; 2× +/+ PBS 0.78 mM Ca++, 0.60 mM Mg++; and 3× +/+ PBS 0.96 mM Ca++, 0.69 mM Mg++. Autologous EDTA plasma was utilized to block Ca++-dependent effects. In the control group, volumes were adjusted by adding fresh RPMI 1640 medium. Cell-free supernatants were collected by centrifugation after a culture period of 30 min and 1, 2, 3, 4, and 24 hr in an incubator at 37° C and kept frozen at –20° C until further testing.
Evaluation of cell culture supernatants
Supernatant levels of chemokines were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits for the quantification of IL-8, ENA-78, GRO- α, GCP-2, MCP-1, RANTES, fractalkine, IP-10, and SDF-1 (Duoset®, R&D Systems , Minneapolis, MN) according to the manufacturer’s protocol. In short, 96-well multiple-well plates were coated overnight at room temperature with the appropriate capture antibody. After blocking of plates, samples of plasma, supernatants, and standard protein were added to the wells. After a washing step, a biotin-labeled antibody was added to each well and incubated for 2 hr. Plates were washed and streptavidin-horseradish peroxidase was added. Color reaction was achieved using tetramethylbenzidine (TMB) (Sigma-Aldrich, St. Louis, MO) and was stopped by a sulfuric acid stop solution (Merck, Darmstadt, Germany). Optical density values were measured at 450 nm on a Victor3 multilabel ELISA plate reader (PerkinElmer). The intraassay coefficient of variation (CV) for IL-8 was 4.6%, ENA-78: 5.4%, GRO-α: 4.5%, GCP-2: 5.0%, MCP-1: 4.2%, RANTES: 3.6%, fractalkine: 10.0%, IP-10: 2.9%, and SDF-1: 3.9%. Minimum detectable doses for IL-8 were 3.5 pg/mL, ENA-78: 15.0 pg/mL, GRO-α: 10.0 pg/mL, GCP-2: 1.6 pg/ mL, MCP-1: 5.0 pg/mL, RANTES: 2.0 pg/ mL, fractalkine: 200 pg/mL, IP-10: 1.67 pg/ mL, and SDF-1: 18.0 pg/mL.
Statistical analyses were performed using Graph Prism 5 software (GraphPad Software, La Jolla, CA). Data are given as mean ± standard error of the mean (SEM). Two-sided Student’s t-tests for paired comparisons were used to calculate significances. Bonferroni–Holm correction was used to adjust obtained p-values for multiple testing. A p-value of <0.05 was considered statistically significant.
Figure 1 - Chemokine levels in supernatants from PBMC collected after 24 hr. The addition of calcium and magnesium containing PBS to cell cultures increases values of IL-8, whereas the addition of EDTA–plasma leads to a strong reduction of chemokine release (a). This response was also detected for MCP-1, although to a lesser extent (b). *p <0.05 vs control.
Cell culture supernatants acquired from PBMCs after 24 hr evidenced IL-8 levels of 576.9 ± 194 pg/mL in the control group (medium) and 607.4 ± 165 pg/mL in wells supplemented with PBS without calcium and magnesium. When adding PBS containing both calcium and magnesium (20%) to cell cultures, the value increased to 1464.1 ± 671 pg/mL. This effect could be totally abrogated by adding autologous EDTA containing plasma, as evidenced by values of 40.2 ± 13 pg/mL (Figure 1a). A slight increment could also be detected for ENA-78.
Since the addition of +/+ PBS induced an increment of IL-8 levels, the authors also tested for other chemokines (MCP-1, RANTES, and fractalkine). In addition, MCP-1 and RANTES showed a moderate increase of chemokine release in the +/+ PBS group. No changes were found for GRO-α, GCP-2, fractalkine, IP-10, or SDF-1 (Figure 1b).
Figure 2 - Dose-dependent effect of supplementing PBMC cell cultures with increasing concentrations of calcium and magnesium containing PBS, resulting in heightened levels of IL-8. *p <0.05 vs control.
Figure 3 - Short-term responses of IL-8 secretion by human PBMC after incubation times of 30 min to 24 hr. Heightened extracellular ion concentrations in medium containing increasing levels of +/+ PBS seem to have an influence on cellular IL-8, even within short incubation intervals (e.g., within 2 or 3 hr). *p <0.05, ** p <0.01 vs control.
To further prove the data, the same set of experiments was performed, and increasing doses of calcium and magnesium containing PBS were added to the PBMC cultures. A significant dose-dependent increment of IL-8 was found in supernatant levels (levels of 1464.1 ± 671 pg/mL at 1× +/+ PBS, 1198.1 ± 363 pg/mL at 1.25× +/+ PBS, 1887.9 ± 487.6 pg/mL at 1.5× PBS, 2781.2 ± 1008.8 pg/mL at 2× +/+ PBS, and 3896.5 ± 242.5 pg/mL at 3× +/+ PBS). MCP-1 demonstrated a significant dose-dependent response, although not as prominent as for IL-8. No dose dependency was observed for PBMC cell culture supernatants analyzed for ENA-78, GRO-α, GCP-2, RANTES, IP-10, SDF-1, and fractalkine (Figure 2a and b).
During most laboratory applications (rinsing of plates or flasks, intermittent culture intervals), immune cells or cell lines are exposed to PBS solutions for short incubation intervals such as minutes or a few hours. Thus, the authors investigated whether chemokine secretion could be induced within 30 min to 4 hr. For the chemokine IL-8, significant differences after incubation periods between 2 and 4 hr were apparent, suggesting that even short-term exposure to heightened extracellular ion levels during cell culture is sufficient to influence secretion of chemokines, predominantly IL-8 (Figure 3).