Methods for intracellular cytokine staining are becoming widely accepted as an analytical tool in the immunology laboratory. This staining technique allows the delineation of distinct cytokine-producing leukocyte subsets within a mixed cell population. The methodology involves the fixation and permeabilization of the target cells for detection of intracellular cytokines by flow cytometry. This method was first proposed by Jung et al. (9
) as a modification of a protocol that was originally designed for analysis using a fluorescence microscope (21
). Further publications on this subject have resulted in reports addressing the kinetic aspects of cytokine expression (11
) and the development of a whole-blood method (23
). Using this methodology, a number of groups have detected imbalances in cytokine production in different disease states (4
A key aspect of intracellular cytokine detection is trapping the cytokine within the cell. Generally, unstimulated cells produce undetectable amounts of cytokine. Therefore, the cells must be stimulated; a popular choice for stimulation is the combination of phorbol myristate acetate (PMA) and ionomycin (ION), which induces rapid induction of many cytokines. Thus, early quantification (i.e., at 2 to 4 h) of the number of cells expressing a cytokine and determination of the relative amount of cytokine per cell can be made. A protein transport inhibitor is added to the cultures to prevent the release of cytokines from the cells. Two commonly used compounds are monensin (MN) and brefeldin A (BFA). MN is derived from Streptomyces cinnamonensis
and is an Na+
ionophore that disrupts intracellular Na+
gradients, exerting its greatest effects on the regions of the Golgi apparatus that are associated with the final stages of secretory vesicle maturation (13
; E. Chu, J. Elia, D. Sehy, D. Ernst, and C. Shih, Hotlines [Pharmingen] 3:
9-10, 1997). BFA is a macrocyclic lactone that is produced by a variety of fungi and is synthesized from palmitate. BFA was originally isolated from Penicillium brefeldianum
as described by Dinter and Berger (7
) and appears to inhibit protein secretion early in a pre-Golgi compartment (between the endoplasmic reticulum and Golgi). The mechanism of this action is complicated and is best explained by Dinter and Berger in their review of Golgi-disturbing agents (7
). In a review of the literature addressing intracellular staining, we found MN to be the most common choice for the protein transport inhibitor. Differences related to the choice of protein transport inhibitor used for intracellular cytokine staining have been previously reported for the measurement of intracellular cytokine production by CD4+
mouse splenocytes after either anti-CD3-anti-CD28 or PMA-ION stimulation (Chu et al., Hotlines [Pharmingen], 1997). Nylander and Kalies (14
) assessed the effects of BFA and MN on viability, intracellular IFN-γ expression, and surface CD69 expression in CD4+
mouse splenocytes and reported BFA both to have lower cytotoxicity than MN and to be more effective in blocking CD69 surface expression. Recently, it was also shown that the expression of specific cytokines in cultures of isolated monocytes could be altered depending on the protein transport inhibitor used (22
). The present report addresses differences observed between MN and BFA treatments when intracellular cytokine (e.g., tumor necrosis factor alpha [TNF-α] and gamma interferon [IFN-γ]) production, lymphocyte surface marker (CD3, -4, and -8) expression, and expression of the activation marker CD69 were analyzed following PMA-ION stimulation in a human whole-blood culture system. The report addresses the suggested basis for these differences.
MATERIALS AND METHODS
Blood samples were obtained from healthy volunteers who gave informed consent. The number of donors totaled 12; 8 were female (average age, 46 years; range, 23 to 57 years), and 4 were male (average age, 50 years; range, 40 to 61 years). Not all specimens were used in all analyses. This study was approved under New York State Department of Health IRB Study 98-1-08.
The antibodies used for surface and intracellular staining were obtained either from BD Pharmingen (San Diego, Calif.) as fluorescein isothiocyanate (FITC), phycoerythrin (PE), or allophycocyanin (APC) conjugates or from BD Immunocytometry Systems (San Jose, Calif.) as a peridinin chlorophyll protein (PerCP) conjugate. The FACSLyse (commercial fixative solution) used to fix cells after surface staining was obtained from BD Immunocytometry Systems. The lysing buffer used for viability assessment (PharmLyse) and the reagents used in the intracellular staining protocol for cell fixation (Cytofix/Cytoperm) and permeabilization (PermWash) were obtained from BD Pharmingen. Sodium heparin Vacutainers used for blood collection were obtained from Becton Dickinson (Franklin, N.J.). MN, BFA, PMA, ION, and propidium iodide (PI) were obtained from Sigma (St. Louis, Mo.). Medium (RPMI 1640) was obtained from BioWhittaker. The OptEIA enzyme-linked immunosorbent assay (ELISA) kits used to determine cytokine concentrations were obtained from BD Pharmingen.
A whole-blood culture system was used as previously described (23
). Briefly, whole, heparinized (sodium heparin) blood was diluted 1:2 with RPMI 1640 (1 part blood to 2 parts medium), and aliquots (2 ml) were aseptically pipetted into 13-ml tubes. A total of six tubes were required per individual assayed; three tubes were left unstimulated, and the remaining three tubes were stimulated with PMA (50 ng/ml) and ION (1 μM). Of the three stimulated tubes, one was treated with BFA (10 μg/ml, or approximately 35.7 μM), one was treated with MN (2 μM), and one was left untreated. Surface CD69 expression, cytokine amounts in the culture supernatants, and viability were assessed after 2, 4, and 18 h of culture. The intracellular expression of CD69, IFN-γ, and TNF-α and the surface expression of lymphocyte markers (CD3, -4, and -8) were assessed following 4 h of culture. All cell cultures were incubated for the prescribed periods at 37°C, 5% CO2
, with and without protein transport inhibitors.
The viability of lymphocytes was assessed by PI incorporation (n = 5). From each culture tube, 300 μl of blood-medium was transferred to a Falcon tube (12 by 75 mm; BD Falcon, Bedford, Mass.). Two milliliters of a 1× PharmLyse solution was added to each tube, and the red blood cells were lysed for 10 min. The cells were centrifuged for 10 min at 400 × g, washed with phosphate-buffered saline (PBS), and resuspended in 0.5 ml of PBS; 10 μl of a 0.5-mg/ml solution of PI was added to the cells. The cells were incubated for 5 min at room temperature and then kept on ice. Specimens were analyzed using the flow cytometer within 20 min of staining.
Intracellular cytokine staining.
Following 4 h of PMA-ION stimulation, cell aliquots (n = 7) were surface stained with a combination of FITC-conjugated CD3, PerCP-conjugated CD4, and APC-conjugated CD8, fixed with 2 ml of a 1× FACSLyse solution, and stored overnight in fixative at 4°C. The following day, the fixed cells were centrifuged at 500 × g for 10 min, washed with 1 ml of staining buffer (3% heat-inactivated FBS in PBS with 0.1% sodium azide), and resuspended in 600 μl of staining buffer; 200-μl aliquots were then dispensed in triplicate into a 96-well round-bottom plate. The plates were centrifuged as described earlier, and intracellular staining for cytokines was performed via a slightly modified version of the protocol supplied by BD Pharmingen (supplied in their intracellular cytokine staining kit). Briefly, cells were treated with Cytofix/Cytoperm Buffer (for permeabilization) and washed with a 1× PermWash solution following each step in the procedure. For intracellular cytokine staining, one of the following antibodies was added per well for each set of triplicate wells: mouse immunoglobulin G1 (IgG1) isotype control (clone MOPC-21), mouse anti-IFN-γ (clone 4S.B3), or mouse anti-TNF-α (clone MAb11).
For assessing surface CD69 expression (n = 5), cells were stained with CD3-PerCP and either FITC-conjugated mouse IgG1 (isotype control) or CD69-FITC and then fixed and stored overnight as previously described. Specimens not used to assess intracellular CD69 (from the 2- and 18-h time points) were washed once and resuspended in staining buffer prior to analysis. Specimens that were evaluated for intracellular CD69 (4-h time point; n = 5) were permeabilized, split into two aliquots, and stained intracellularly with either PE-conjugated mouse IgG1 (isotype control) or CD69-PE. All staining was performed as described earlier for the intracellular cytokines; following staining, the cells were resuspended in 0.5 ml of staining buffer and analyzed.
Flow cytometric analysis.
The specimens were analyzed on a BD FACSCalibur using CellQuest software. Intracellular and surface CD69 was analyzed by gating on the CD3+ lymphocytes in a CD3-versus-side scatter (SSC) dot plot. One-dimensional histograms of surface and intracellular CD69 expression were based on the CD3+ gate. Histogram markers were set based on the isotype controls in conjunction with the unstimulated controls.
Surface marker expression was analyzed by first drawing a gate around the lymphocytes in a forward scatter-versus-SSC dot plot; a dot plot was drawn of CD3 versus SSC based on the lymph gate. A second gate was drawn around the CD3+ lymphocytes in the CD3-versus-SSC plot. To analyze CD3 expression, a histogram was created gated on the lymph gate; to analyze CD4 and CD8 expression, histograms were gated on the CD3+ gate. Markers used to assess a shift in fluorescence of the surface markers were set based on the unstimulated (control) cultures.
To analyze intracellular cytokine expression by CD4+ and CD8+ lymphocytes, a gate was first drawn around the lymphocytes in a dot plot of forward scatter versus SSC. A dot plot of CD3 versus SSC was drawn based on the lymph gate, and a second gate was drawn around the CD3+ cells. Dot plots based on the CD3+ gate were created to analyze either CD4 or CD8 versus the PE-conjugated antibody (either the isotype control, IFN-γ, or TNF-α). Quadrant markers were set according to both the unstimulated control cells and the isotype control. The percent of cytokine expressed by the lymphocyte subsets was computed using the following formula based on the percentage of gated values given for the double positive cells: (stimulated − stimulated isotype control) − (unstimulated − unstimulated isotype control).
Quantification of cytokines by ELISA.
Culture supernatants (n = 5) from all treatment groups were saved following 2, 4, and 18 h of incubation, frozen at −80°C until analysis, and then analyzed by ELISA. The culture supernatants were analyzed for IFN-γ and TNF-α according to kit instructions. Standard curves for the cytokines employed 300, 150, 75, 37, 18, 9, and 5 pg/ml for IFN-γ and 500, 250, 125, 63, 31, 15, and 7 pg/ml for TNF-α. The plates were analyzed using a BIO-TEK series UV900C plate reader, and data reduction was performed with KC jr. software (BIO-TEK).
Statistical analysis of results.
The results from the flow cytometric data were analyzed for statistically significant differences using SigmaStat software (version 2.0). Surface expression of CD3, CD4, CD8, and CD69 among the treatment groups (unstimulated [UNS]; stimulated, [STM]; stimulated, brefeldin A-treated [STM-BFA]; and stimulated, monensin-treated [STM-MN]) were analyzed using one-way analysis of variance. Statistically significant results were identified by a P value of <0.05. The expression of intracellular cytokines was analyzed using Student’s t test; BFA- and MN-treated groups were compared to ascertain whether the observed differences in cytokine expression were significant.
In this report, two protein transport inhibitors commonly used in intracellular cytokine staining (BFA and MN) were compared with regard to expression of CD69 (an activation marker), CD3, CD4, and CD8 (lymphocyte surface markers), and intracellular expression of cytokines (IFN-γ and TNF-α). Unstimulated cells and cells stimulated with PMA and ION with or without treatment with either BFA or MN were assayed by flow cytometry. Significant differences among groups were obtained with the different protein transport inhibitors.
Both protein transport inhibitors aided in the retention of cytokines within the cell; however, there were differences in the amount of cytokine retained between groups treated with either BFA or MN. Previous publications have reported differences in CD69 and cytokine expression between cultures treated with BFA or MN [14
; Chu et al., Hotlines (Pharmingen), 1997], but these results were obtained using cultures of isolated CD4+
mouse splenocytes or human monocytes. Herein, we investigated whether these observations applied to whole-blood cultures containing multiple human lymphocyte subsets as well as whether there were differential effects on the expression of surface markers and cytokines by CD4+
The effect of protein transport inhibitors on CD69 expression.
Although MN and BFA differentially blocked the expression of surface CD69 after cell activation, neither BFA nor MN inhibited protein synthesis based on intracellular CD69 expression. However, it is possible there could be differential effects on the expression of other proteins. Dual staining of lymphocytes with CD69 labeled with two fluors (FITC and PE) demonstrated BFA produced a more effective blockage of surface CD69 expression than MN; there was a greater amount of CD69 intracellularly in the BFA-treated cells than on the surface of the cells, while the opposite was true for the STM-MN cell group (Fig. 2
Analysis of CD69 kinetics indicated that while the percentages of surface CD69+
cells for the STM and STM-MN cells were equivalent, the amounts of surface CD69 expressed on the STM-MN-treated cells were significantly lower than those on the STM cells. Two conclusions can be drawn from these results: either MN-treated cells have a lesser amount of surface CD69 because of an overall inhibitory effect on the transport of proteins to the surface, or the CD69 is being surface expressed in an altered nonimmunoreactive form. Because CD69 is a type II transmembrane glycoprotein, evidence for these possibilities can be drawn from other experiments using MN to block protein secretion. Kubo and Pigeon (10
) demonstrated that in the presence of MN there were alterations in the intracellular processing of proteins in Daudi cells. Specifically, the terminal glycosylation of surface IgM was affected. However, this alteration did not affect the surface expression of the altered form of IgM. Another study (15
) analyzed the effect of MN on the glycosylation of α1
-protease inhibitor in cultured rat hepatocytes. The researchers found that the conversion of α1
-protease inhibitor to its final form was blocked in the presence of MN and that there was evidence for the existence of an intermediately processed form of the protein.
The results of the kinetic experiment showed that the STM-BFA cells expressed greater percentages of surface CD69 with increasing incubation time. The experimental results corroborated previously published work demonstrating that BFA induces a delay in the expression of glycosylated proteins. Using cultured rat hepatocytes to examine the glycosylation of α1
-protease inhibitor, Misumi et al. (12
) reported the glycosylation process to be delayed by BFA; however, unlike the altered form of α1
-protease inhibitor produced in the presence of MN, a fully glycosylated, unaltered protein was eventually expressed in the presence of BFA.
Our data, in conjunction with previous data, suggest the observed difference between STM and STM-MN surface CD69 expression after activation may be due to the expression of two structurally different forms of the same protein. MN may alter the glycosylation of the CD69 protein; however, unlike the effects of fixation, the glycosylation differences did not substantially inhibit recognition by the antibody. The expression of surface CD69 differed in the STM-BFA cells because of a delay in the processing of the glycosylated protein, a suggestion supported by the data shown in Fig. 2
. For the STM-BFA cell group, the amount of intracellular CD69 was threefold greater than the amount on the surface of the cells, while the opposite was true for the STM cultures. Adding together the intracellular and surface amounts of CD69 for these two cell groups, the total amount (GM) of CD69 in the two treatment groups was equivalent (STM-BFA, 253 ± 60; STM, 194 ± 35) differing only in the distribution of the protein.
The effect of protein transport inhibitors on lymphocyte surface marker expression.
The activation of lymphocytes by phorbol esters has been shown to downregulate the expression of CD3, CD4, and CD8 on the cell surface (2
). Our data show similar results, with CD4 being the most severely downregulated marker (Fig. 3
). The downregulation of surface CD4 can be initiated by PMA, which activates protein kinase C (PKC); this activation involves the translocation of PKC from the cytoplasm to the plasma membrane and leads to the phosphorylation of serine residues in the cytoplasmic tail of the CD4 molecule; the dissociation of the CD4-tyrosine kinase p56 lck
complex; and an increase in the association of CD4 with clathrin-coated pits, which results in the downregulation of CD4 via endocytosis (17
). Therefore, while the PMA-ION combination is a popular choice for inducing intracellular cytokine expression, analyzing whether the CD4 or the CD8 T-cell subset produces a particular cytokine is difficult due to the loss of the CD4 marker. In our study, we observed that the inclusion of a protein transport inhibitor during PMA-ION stimulation lessened the downregulation of CD4 to different degrees, dependent on the inhibitor used.
The results in Fig. 3
show the order of the degree of CD4 downregulation to be (from most to least) STM, STM-MN, and STM-BFA. The difference in CD4 expression between the BFA and MN groups, while not significant, affected the visual data: the CD4+
groups were distinguishable from each other in the STM-BFA plots, but not in the STM-MN plots (Fig. 4
). Reasons for the differential effect of the inhibitors on CD4 expression likely relate to the mechanisms by which CD4 is downregulated. In the case of MN, Takeuchi et al. (25
) have reported that MN accelerates the proteolytic degradation of PKC in HL60 cells treated with tetradecanoyl phorbol acetate (PMA), an inducer of PKC translocation leading to downregulation; the downregulation of PKC refers to the proteolytic degradation of PKC following its translocation from the cytosol to the plasma membrane. Takeuchi et al. (25
) presented evidence showing the addition of MN to cells stimulated with PMA expedited PKC degradation. This increased rate of PKC degradation could result in the incomplete phosphorylation of the serine residues in the cytoplasmic tail, a necessary step for the downregulation of CD4. In our results, less phosphorylation in the STM-MN cells compared to that in the STM cells could account for the lesser degree of CD4 downregulation in the STM-MN cells compared to the STM cells.
Following the dissociation of the CD4-p56 lck
complex, CD4 associates with clathrin-coated pits prior to endocytosis (17
). Studying the association of GLUT-4 with endocytic clathrin-coated vesicles, Chakrabarti et al. (5
) stated that BFA caused complete disassembly of clathrin lattices at the trans
-Golgi network in 3T3-L1 adipocytes and led to a decrease in the amount of clathrin-coated vesicles that could be purified from the treated cells. Chakrabarti et al. (5
) also found BFA treatment did not affect the number of clathrin-coated vesicles in the plasma membrane. The disruption of clathrin lattices in the trans
-Golgi network could explain the decrease in CD4 downregulation observed in the STM-BFA cells compared to the STM cells; some downregulation of CD4 is posited to occur because of clathrin-coated vesicles available in the plasma membrane, but the degree of endocytosis normally seen in PMA-ION-treated lymphocytes may have been impaired by treatment with BFA because the formation of additional clathrin-coated vesicles is impeded.
The effect of protein transport inhibitors on intracellular cytokine staining.
Significant differences in the intracellular expression of TNF-α were detected between the STM-BFA and STM-MN groups after 4 h of stimulation. After assessing the amount of secreted cytokine in the supernatants from the various treatment groups by ELISA, detectable amounts of both IFN-γ and TNF-α were present in supernatants from the STM and STM-MN (but not the UNS) groups. IFN-γ could be detected in some supernatants from the STM-BFA cells, but only after 18 h of incubation. Thus, the lower levels of intracellular cytokine in the STM-MN cells may be due, at least in part, to increased cytokine secretion compared to the STM-BFA cells.
The reason behind the differences in secretion rates may relate to the intrinsic nature of these cytokines. Human TNF-α is a nonglycosylated type II transmembrane protein of 233 amino acids (aa). After transport to the plasma membrane, the transmembrane form is proteolytically cleaved at the plasma membrane by the action of TNF-α-converting enzyme (TACE), a glycosylated membrane-bound metalloproteinase that cleaves the membrane-bound 233-aa form into a soluble 157-aa form (1
). Because this proteolytic cleavage must take place prior to release of the soluble form of TNF-α, a possible reason for the observed difference may be associated with the enzyme TACE. Solomon et al. (24
) studied the conversion of pro-TNF-α (233 aa) to soluble TNF-α (157 aa) using LPS stimulation of human monocytes and found that the addition of a hydroxamic acid-based metalloprotease inhibitor led to an almost complete inhibition of TNF-α release by the monocytes. If the glycosylation of TACE, or its insertion into the membrane, is delayed by the mechanism proposed for the delay observed with CD69 expression in STM-BFA cells, the membrane-bound form of TNF-α could not be cleaved. With regard to IFN-γ, the human form is a heterogeneously glycosylated protein (3
). Although the amounts detected inside the cells of the STM-BFA and STM-MN groups are equivalent, the amount secreted into the supernatant from the STM-BFA cells is 10% of that detected from the STM-MN cells. This again may be attributed to a delay in processing, specifically in glycosylation, of the protein in the STM-BFA cells, as opposed to processing in the STM-MN cells.
The data presented here confirm previous reports [14
; Chu et al., Hotlines (Pharmingen) 3
:9-10, 1997] that show that differences in lymphocyte marker and intracellular cytokine expression may be dependent on the choice of protein transport inhibitor used in conjunction with intracellular staining. Our data also underscore the fact that although there is great interest in using this technique as a tool for the clinical immunology laboratory, unexpected factors such as those explored here indicate that it may be necessary to further evaluate reagent choices to ensure some measure of reproducibility among laboratories. When considering all the factors that were analyzed, it appears that BFA may be a better choice than MN for detecting intracellular cytokines. From our results, we observed that the advantages of using BFA rather than MN are greater lymphocyte viability following prolonged stimulation, easier analysis of the CD4 subset, and more-efficient intracellular trapping of specific proteins. However, the mechanisms by which BFA and MN affect the measurement of other proteins analyzed with this assay must be further assessed before firm recommendations as to the choice of a protein transport inhibitor can be conclusively stated.