Technical Innovation. Key terms: flow cytometry; brefeldin A; monensin; intracellular cytokines; monocytes; IL-1 ; IL-6; TNF- ; rheumatoid arthritis

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1 Cytometry (Communications in Clinical Cytometry) 46: (2001) Technical Innovation Evaluation of Monensin and Brefeldin A for Flow Cytometric Determination of Interleukin-1 Beta, Interleukin-6, and Tumor Necrosis Factor-Alpha in Monocytes A.J. Schuerwegh, W.J. Stevens,* C.H. Bridts, and L.S. De Clerck Department of Immunology, Allergology and Rheumatology, University of Antwerp, Antwerp, Belgium Flow cytometry has become a powerful technique to measure intracellular cytokine production in lymphocytes and monocytes. Appropriate inhibition of the secretion of the produced cytokines is required for studying intracellular cytokine expression. The aim of this study was to compare the capacity of cytokine secretion inhibitors, monensin and brefeldin A, in order to trap cytokine production (interleukin-1 beta [IL-1 ], IL-6, tumor necrosis factor-alpha [TNF- ]) within peripheral blood monocytes. A two-color flow cytometric technique was used to measure intracellular spontaneous and lipopolysaccharide (LPS)-stimulated IL-1, IL-6, and TNF- production in monocytes (CD14 ) of whole blood cultures. The viability of monensin-treated monocytes was slightly lower than that of brefeldin A-inhibited monocytes, as measured with propidium iodide (PI). The percentage of IL-6 and TNF- producing monocytes after 8hofculture without stimulation revealed significant lower values for monensin-treated than for brefeldin A-treated monocytes. The percentages for stimulated cells did not differ. The spontaneous intracellular production in molecules of equivalent soluble fluorochrome units (MESF) of IL-1, IL-6, and TNF- after8hofculture was higher in brefeldin A than in monensin-inhibited monocytes. The LPS-stimulated intracellular production of IL-1, IL-6, and TNF- was increased in brefeldin A-inhibited monocytes. In conclusion, for flow cytometric determination of intracellular monocytic cytokines (IL-1, IL-6, and TNF- ), brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin. Cytometry (Comm. Clin. Cytometry) 46: , Wiley-Liss, Inc. Key terms: flow cytometry; brefeldin A; monensin; intracellular cytokines; monocytes; IL-1 ; IL-6; TNF- ; rheumatoid arthritis RATIONALE AND DESIGN Among various inflammatory cells, peripheral blood monocytes have the capacity to produce proinflammatory cytokines, such as interleukin-1 beta (IL-1 ), IL-6, and tumour necrosis factor-alpha (TNF- ). Many techniques are used to determine these monocytic cytokines: at the mrna level, polymerase chain reaction (PCR; 1) and in situ hybridization (2) are performed; enzyme-linked immunosorbent assay (ELISA) measures cytokine secretion in supernatants (3); and flow cytometry determines intracellular cytokine production (4 8). In order to accumulate cytokines within monocytes, reagents are added that inhibit protein secretion during stimulation (5 7). The most widely used reagents are the antibiotics monensin and brefeldin A. The aim of this study was to evaluate and compare the efficiency of both monensin and brefeldin A in inhibiting the cytokine secretion of peripheral blood monocytes in rheumatoid arthritis patients and healthy persons. Viability of Monocytes Monensin treatment induced a decrease of viable monocytes after 6hofincubation, compared with nontreated monocytes (P 0.04; Table 1). The viability of monensintreated monocytes was also lower after 8 and 12 h (P 0.04, P 0.04, respectively; Table 1). In contrast, brefeldin A showed no toxic effect after 8hofexposure. After 12 h, brefeldin A-treated monocytes also had good viability (Table 1). Grant sponsor: Fund of Scientific Investigation (FWO), Flanders, Belgium. *Correspondence to: Prof. Dr. W.J. Stevens, UIA University of Antwerp, Department of Immunology, Allergology and Rheumatology, Universiteitsplein 1, 2610 Antwerp, Belgium. wim.stevens@ua.ac.be Received 21 August 2000; Accepted 16 February Wiley-Liss, Inc.

2 MONENSIN AND BREFELDIN A EFFICACY 173 Table 1 Viability of Nontreated, Monensin-Treated, and Brefeldin A-Treated LPS-Stimulated Monocytes 3h 6h 8h 12h LPS treated 98 (97 98) 98 (97 98) 97 (96 98) 95 (92 98) LPS and monensin treated 96 (95 97) 89 (87 95)* 88 (75 90)* 85 (69 88)* LPS and brefeldin A treated 97 (96 98) 96 (95 97) 97 (95 98) 93 (91 96) Results are expressed as percentage viable monocytes (range). *P 0.04 versus LPS and brefeldin A-treated monocytes. Influence of Culture Time Incubation with lipopolysaccharide (LPS) and monensin or brefeldin A resulted in a rise of intracellular cytokines compared with intracellular cytokines before incubation (Fig. 1A C). Intracellular cytokine production peaked after 8hofincubation for both monensin and brefeldin A. Thereafter, a decrease of intracellular cytokine accumulation was observed for both secretion inhibitors. A significantly higher production of intracellular cytokines (expressed in molecules of equivalent soluble fluorochrome units [MESF]) was observed after 8hof culture for the measured cytokines (IL-1, IL-6, TNF- ) in LPS-stimulated monocytes (Fig. 1A C). Percentage of IL-1, IL-6, and TNF- Positive Monocytes The percentage of IL-6 and TNF- producing monocytes after 8hofculture without stimulation revealed lower values for monensin-inhibited compared with brefeldin A-inhibited monocytes (P 0.05 and P 0.03, respectively; Table 2). The percentage of LPS-stimulated monensin-treated monocytes did not differ from the percentage of LPS-stimulated brefeldin A-treated cells for the three cytokines (Table 2). Semiquantitative Measurement of IL-1, IL-6, and TNF- Production Expressed in MESF units in Monocytes Representative histograms of the spontaneous and stimulated intracellular IL-1, IL-6, and TNF- production in monensin and brefeldin A-treated monocytes are shown in Figure 2A C. The spontaneous intracellular production of IL-1, IL-6, and TNF- after 8hofculture was higher in brefeldin A-treated than in monensin-treated monocytes (P 0.005, P 0.01, P 0.01, respectively; Fig. 3). For the LPS-stimulated production of IL-1, IL-6, and TNF-, there was a stronger increase in cytokine production in brefeldin A-inhibited monocytes compared with monensin-inhibited monocytes (P 0.005, P 0.005, P 0.005; Fig. 4). To our knowledge, only one study compared the efficacy of these two inhibitors on the expression of the surface activation marker CD69 and interferon-gamma (IFN- ) production by mouse T lymphocytes. The conclusion was that brefeldin A, not monensin, completely inhibited CD69 expression (9). These investigators reported that the percentage of IFN- positive T lymphocytes was independent from the applied inhibitor. However, the quantity of blocked IFN- in the T lymphocytes, reflected by the mean fluorescence, was lower when monensin was FIG. 1. A representative example of the intracellular cytokine production in monocytes after 0, 1, 3, 6, 8, 12 h of stimulation with 1 g/ml LPS and 2 mol/l monensin (open square) or 1.4 mol/l brefeldin A (closed square). Results are expressed as mean MESF units. A: Intracellular IL-1 production in monocytes. B: Intracellular IL-6 production in monocytes. C: Intracellular TNF- production in monocytes.

3 174 SCHUERWEGH ET AL. Table 2 Percentage of Cytokine (IL-1, IL-6, TNF- )-Positive Monocytes After Unstimulated Cultures and LPS-Stimulated Cultures of Monensin and Brefeldin A-Treated Monocytes Unstimulated IL-1 IL-6 TNF- Monensin Brefeldin A Monensin Brefeldin A Monensin Brefeldin A 16.9 ( ) 21.7 ( ) 10.4 ( ) 12.5* ( ) 10.4 ( ) 13.2** ( ) LPS stimulated IL-1 IL-6 TNF ( ) 99.3 ( ) 97.4 ( ) 96.1 ( ) 95.4 ( ) 97.3 ( ) Results are expressed as median percentage (range). *P 0.05 versus % IL-6 positive monensin-treated unstimulated monocytes. **P 0.03 versus % TNF- positive monensin-treated unstimulated monocytes. used instead of brefeldin A. In accordance with these data, the percentage of cytokine (IL-1, IL-6, TNF- ) producing monocytes in our study did not differ between monensin FIG. 2. Histograms representing irrelevant staining (a) and intracellular cytokine staining in unstimulated (b) and stimulated (c) monocytes (CD14 gated events). A: Intracellular IL-1 production in monocytes treated with monensin (left) or brefeldin A (right). B: Intracellular IL-6 production in monocytes treated with monensin (left) or brefeldin A (right). C: Intracellular TNF- production in monocytes treated with monensin (left) or brefeldin A (right). and brefeldin A-treated LPS-stimulated monocytes. The mechanism of the capacity to inhibit intracellularly produced proteins is well known for both inhibitors. Monensin is a monovalent ion-selective ionophore that disrupts the trans-golgi transport of proteins by interacting with the Golgi membrane Na /H transport (10). Brefeldin A redistributes the intracellularly produced proteins from the cis/medial Golgi complex to the endoplasmatic reticulum, avoiding further transport of these proteins to the trans-golgi site of the Golgi complex (11). Lower MESF values were found for LPS-stimulated cytokine production in monocytes after inhibiting secretion with monensin. An explanation may be that in monensintreated monocytes, the intracellularly produced cytokines bypass the Golgi complex using an unknown alternative secretory pathway (9,10). For IgM, this mechanism has been reported in a human B-lymphoblastoid cell line (12): although monensin was added to this cell line, transport of incomplete glycosylated IgM could be demonstrated, indicating that the interference of monensin with intracellular processing of IgM did not influence the membrane expression of IgM. Although the appropriate concentrations for brefeldin A and monensin were applied in this study (9), the viability of the monensin-treated monocytes was lower in our study compared with untreated cells. The more toxic effect of monensin on the viability of monocytes during culture, compared with brefeldin A, could be explained by the fact that membrane traffic between the endoplasmic reticulum and the Golgi complex continues during exposure to brefeldin A. Although proteins are rapidly returned to the endoplasmic reticulum via the retrograde pathway, proteins are still transported within the cell but are not secreted (11). In contrast, treatment with monensin can lead to full blockage of the Golgi complex, which might be more stressful for the cells as hypothesized by Nylander and Kalies (9) and lead to lower production of cytokines and more necrosis of the monocytes as observed in our study. Thus, leakage of produced cytokines by an alternative secretory pathway combined with the toxicity of monensin might explain the lower amounts of intracellular cytokines in LPS-stimulated monocytes. Indeed, we found that the supernatants of monensin-treated

4 MONENSIN AND BREFELDIN A EFFICACY 175 FIG. 3. Intracellular IL-1, IL-6, and TNF- spontaneous production in monocytes of RA patients (closed circles) and controls (open circles) after8hofculture with 2 mol/l monensin (mon) or 1.4 mol/l brefeldin A (bref). Results are expressed as MESF units. monocytes contained high levels of IL-6 as detected by an ELISA (data not shown). In conclusion, brefeldin A is a more potent, effective, and less toxic inhibitor of cytokine secretion than monensin for flow cytometric determination of intracellular monocytic cytokines (IL-1, IL-6, TNF- ) in whole blood cultures. METHODOLOGY Study Population Five patients with a median age 49 years (range 35 61) fulfilled the diagnostic criteria for rheumatoid arthritis (RA) as defined by the American College of Rheumatology. Five healthy volunteers, age and sex matched with the RA patients, were also evaluated. Controls had no history of cancer or of immunological or allergic disorders. They did not have an active infection nor had they undergone a surgical or other invasive procedure during the 6 weeks preceding the study. All patients and controls gave informed consent before entering the study. Cell Culture Heparinized blood (Vacuette, Greiner, Labortechnik, Kremsmüster, Austria) was obtained by venipunction. FIG. 4. Intracellular IL-1, IL-6, and TNF- production in monocytes of RA patients (closed circles) and sex and agematched controls (open circles) after 8 h of stimulation with 1 mol/l LPS and 2 mol/l monensin (mon) or 1.4 mol/l brefeldin A (bref). Results are expressed as MESF units.

5 176 SCHUERWEGH ET AL. One milliliter of whole blood was stimulated during 3, 6, 8, or 12 h with 1 g/ml LPS Escherichia coli (Sigma, St. Louis, MO) alone or together with 2 mol/l monensin (Sigma) or 1.4 mol/l brefeldin A (Sigma). Some cells were incubated with 2 mol/l monensin or 1.4 mol/l brefeldin A without LPS stimulation to evaluate spontaneous cytokine production during culture. Incubation was performed in sterile 15-mL polypropylene tubes (Sarstedt, Nümbrecht, Germany ) in order to prevent adherence of cells. Viability of Cells After culturing, red blood cells were lysed with lysis buffer (155 mmol/l NH 4 Cl, 1 mmol/l NaHCO 3, 0.1 mmol/l Na 2 EDTA) for 20 min at room temperature and washed with phosphate- buffered saline (PBS; Gibco, Life Technologies, Paisley, Scotland). After, 50 g/ml propidium iodide (PI; Molecular Probes, Eugene, OR) in PBS (Gibco) was added to the cell pellet. After 5 min of incubation, cells were measured on a FACScan flow cytometer (Becton Dickinson [BD], San Jose, CA) and analyzed with WinMDI 2.8 software (provided by J. Trotter through Monocytes were gated on forward scatter (FSC) and side scatter (SSC) plots. Within this window, PI fluorescence was analyzed on a histogram. Flow Cytometric Detection of Cytokines in Monocytes After incubation, 100 L of cultured whole blood was incubated with CD14-FITC for 15 min at 4 C to identify the monocytes. Subsequently, red blood cells were lysed and remaining white blood cells were fixed at room temperature with Facslysis (BD). Cell membranes were made permeable with a 0.3% saponin (Sigma) solution in PBS, followed by incubation with phycoerythrin (PE)-labeled anti-cytokine antibodies against IL-1, IL-6, and TNF- (BD) during 30 min at room temperature. Cells were washed with 0.3% saponin in PBS and centrifuged for 10 min at 1,000 g and the cell pellet was resuspended in 0.3 ml PBS and measured. Acquisition and Analysis List mode data were acquired on a FACScan flow cytometer (BD) and analyzed using WinMDI 2.8 software. Analysis gates were set on CD14-positive cells according to FITC emission and SSC. In this gate, cytokine production (PE emission) of 2,000 monocytes was assessed. Measurements were standardized using microspheres (Fluoro- Spheres, Code No. K 0110; DAKO, Glostrup, Denmark), according to the manufacturer s instructions. Results were expressed in two different ways: as MESF units, reflecting a semiquantitative value for the amount of intracellular cytokine (14), and as the percentage of cytokine-producing monocytes, using markers on the 95th percentile of isotype-matched antibodies (mouse IgG1 R-PE, Serotec, UK; 4). Histograms showed cytokine staining (PE emission) in the gated window (Figs. 2A C). Statistics The Friedman and Wilcoxon signed ranks tests were used to calculate differences among untreated, monensin, and brefeldin A-treated monocytes. 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