A flow cytometric immune function assay for human peripheral blood

A flow cytometric immune function assay for human peripheral blood dendritic cells Kerstin Willmann and John F. Dunne Becton Dickinson Immunocytometry Systems, San Jose, California

Abstract: CD11cⴙ and CD11cⴚ (CD123ⴙ) dendritic cells (DCs) have been described in blood. Both cell types express high levels of HLA-DR and lack the lineage markers CD3, CD14, CD19, CD20, CD16, and CD56. These immunophenotypic properties were used along with analysis of activation-related surface antigens and intracellular staining of cytokines to characterize functional responses of these DC subsets to stimuli in whole human blood (WB). Samples from healthy donors were activated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate plus ionomycin (PMAⴙI). The only distinct response in CD11cⴚ DCs was the expression of CD25 upon PMAⴙI activation. CD11cⴙ cells responded to LPS stimulation by producing high levels of interleukin-1␤ (IL-1␤) and tumor necrosis factor ␣ (TNF-␣), and lower levels of IL-6, IL-1Ra, and IL-8 and an increased expression of accessory molecules (CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ). PMAⴙI activation of CD11cⴙ cells resulted in high levels of IL-1␤ and lower levels of IL-8, IL-1Ra, and TNF-␣ and up-regulation of CD80, CD86, HLADR, and HLA-DQ. Our data support prior observations of functional differences between peripheral blood DC subsets and demonstrate the power of multiparameter flow cytometry to characterize the pleiotropic responses of these cells to various stimuli. J. Leukoc. Biol. 67: 536–544; 2000. Key Words: cytokines · lipopolysaccharide · phorbol myristate acetate · ionomycin

INTRODUCTION Dendritic cells (DCs) are found as a rare population in peripheral blood and non-lymphoid and lymphoid tissues. They play a central role in the onset of cellular immunity and more recently have been ascribed a role in the induction of tolerance [1]. DCs capture, process, and present antigen to memory and naive T cells [1, 2]. The function of DCs can be characterized by their cytokine expression patterns and by the dynamic regulation of differentiation/activation markers (CMRF-44, CMRF-56, CD83, CD25), of co-stimulatory molecules (CD40, CD80, CD86), and of class II major histocompatibility complexes (MHC class II) [3–10]. Additional changes in adhesion 536

Journal of Leukocyte Biology Volume 67, April 2000

molecules follow signaling events that lead to changes in DC trafficking and intercellular activity [1, 2]. Freshly isolated peripheral blood DCs have a resting phenotype characterized by a lack of dendrite-like morphology, low levels of accessory molecules, and lower immunostimulatory capabilities [1, 2, 8, 11]. Two peripheral blood DC subsets have been described and are distinguished based on their ability or inability to express CD11c [11–14]. The CD11c-negative (CD11c⫺) DCs have been recently reported to express high levels of CD123 [interleukin (IL)-3R␣] [15]. Moreover, functional differences between CD11c-positive (CD11c⫹) and CD11c⫺ DCs have been described. CD11c⫹ DCs are more potent in T cell-stimulating activity, express CD45RO, and have higher levels of MHC class II and adhesion molecules [11, 14]. Studies of DCs have been hampered because of their low frequency and their lack of specific DC markers [1–3]. To date, studies of cytokine production by peripheral blood DCs have been restricted to highly purified populations obtained from extensive cell enrichment procedures. The few studies available have used phorbol 12-myristate 13-acetate (PMA), HIV-1, or herpes simplex virus (HSV) with activation periods ranging from 4 to 24 h [16, 17]. Other groups investigated the cytokine expression in monocyte-derived DCs, which can be generated in substantial numbers by culturing monocytes in granulocytemacrophage colony-stimulating factors (GM-CSF) and IL-4 for a few weeks [7]. At present, the detailed relationship between monocyte-derived DCs and CD11c⫹ and CD11c⫺ peripheral blood DCs is not clear. Here, we describe the results of a whole-blood flowcytometric method to measure cytokine production in CD11c⫹ and CD11c⫺ DCs. The assay is derived with modifications from methods that have been described earlier to measure the cytokine secretion in T cells after generic or antigen-specific stimuli and in monocytes challenged with lipopolysaccharide (LPS) [18–21]. For the detection of cytokines in peripheral blood DCs, LPS or PMA⫹ionomycin (PMA⫹I) were used in a 4-h stimulation protocol. Our investigation showed that LPS-activated CD11c⫹ DCs express IL-1␤, IL-1Ra, IL-6, IL-8, and tumor necrosis factor ␣ (TNF-␣), whereas PMA⫹I-activation lead to the synthesis of IL-1␤, IL-1Ra, IL-8, and TNF-␣ protein. In the CD11c⫺ DC subset we did not detect cytokines in response to either

Correspondence: Kerstin Willmann, 2350 Qume Drive, San Jose, CA 95131. E-mail: [email protected] Received July 12, 1999; revised September 27, 1999; revised December 2, 1999; accepted December 3, 1999.

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stimulus. It is interesting that monocytes demonstrated relatively similar, but much higher, intracellular cytokine levels compared to CD11c⫹ DCs after LPS stimulation. Cell surface expression of CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ was also investigated. The activation of CD11c⫹ DCs is reflected in an altered immunophenotype showing predominantly the up-regulation of co-stimulatory molecules. CD25 was up-regulated on CD11c⫺ DCs under PMA⫹I stimulation and was the only clear indication of activation on this DC subset recognized in this study. We conclude that functional differences between CD11c⫹ and CD11c⫺ DCs can be assessed in great detail by flow cytometric measurements in a whole-blood format.

MATERIALS AND METHODS Cell activation assay Venous blood of normal donors was collected in sodium heparin Vacutainer tubes. For activation with LPS (Sigma, St. Louis, MO), whole blood was incubated with 1 µg/mL LPS. Before the activation using PMA⫹I (Sigma), whole blood was diluted one-to-one with RPMI medium (BioWhittaker, Walkersville, MD). PMA⫹I were then added to the diluted blood at a final concentration of 5 ng/mL and 1 µg/mL, respectively. For the detection of intracellular cytokines, the activation was performed, as above, in the presence of the secretion inhibitor brefeldin A (BFA; Sigma). Resting control samples were incubated with 10 µg/mL BFA in the absence of stimulus. For the detection of changes in surface antigen expression, the samples were incubated with the DC activators, as above, but in the absence of BFA. All samples were incubated for 4 h at 37°C humidified atmosphere and 5% CO2 in polypropylene tubes (12 ⫻ 75 mm).

Cytokine kinetic assay Whole blood was stimulated with 1 µg/mL LPS. One hour before sample harvest BFA was added at 10 µg/mL. Samples were processed every hour from 0 to 8 h after stimulation. The incubation was performed at 37°C humidified atmosphere and 5% CO2 in polypropylene tubes (12 ⫻ 75 mm).

Immunofluorescence staining for intracellular cytokines Monoclonal antibody (mAb) reagents lineage cocktail 1 fluorescein isothiocyanate (lin 1 FITC), anti-HLA-DR Peridinin Chlorophyll Protein (PerCP), CD123 phycoerythrin (PE), CD11c Allophycocyanin (APC), anti-IFN-␥ PE, anti-TNF-␣ PE, anti-IL-1␣ PE, anti-IL-1␤ PE, anti-IL-1Ra PE, anti-IL-2 PE, anti-IL-4 PE, anti-IL-6 PE, anti-IL-8 PE, anti-IL-13 PE, and mouse IgG1 PE (intracellular formulation) were obtained from Becton Dickinson Immunocytometry Systems (BDIS, San Jose, CA). Lin 1 FITC is an antibody cocktail combining CD3, CD14, CD16, CD19, CD20, and CD56 in one vial. Anti-IL-12 PE and anti-IL-10 PE were obtained from PharMingen (San Diego, CA). Dulbecco’s phosphate-buffered saline (PBS) was obtained from GIBCO-BRL (Grand Island, NY), and fetal calf serum (FCS) was obtained from Sigma. Before staining, PMA⫹I-treated blood samples were reduced in volume to 50% by centrifugation and removal of supernatant. All cell preparation was done at room temperature (RT) in polypropylene tubes. A volume of 1 mL blood, incubated with stimulus plus BFA or BFA alone, was added to a 50-mL centrifuge tube containing DC-distinguishing antibodies (lin 1 FITC, anti-HLA-DR PerCP, CD11c APC). The blood was then incubated for 15 min. After incubation, red blood cells were lysed for 10 min by applying 40 mL FACS lysing solution (BDIS). Next, the cells were collected by centrifugation at 500 g. The surface staining and red cell lysis was followed by the permeabilization of leukocytes with the use of 3 mL FACS permeabilizing solution (BDIS). The permeabilization was stopped after 10 min incubation by adding 40 mL of wash buffer (PBS 1⫻, 0.5% FCS). The permeabilized cells

were centrifuged at 500 g and resuspended in the supernatant that was retained after decanting (approximate volume 500 µL). A 50-µL aliquot of the bulk surface-stained, lysed, and permeabilized cells was added to a polypropylene tube (12 ⫻ 75 mm) containing cytokine-specific antibody. For intracellular staining, the cells were incubated for 30 min, washed, and resuspended in 250 µL wash buffer for acquisition. The samples were acquired immediately or kept maximally for 1 h at 4°C.

Immunofluorescence staining for surface antigens Before staining, PMA⫹I-treated blood samples were reduced in volume to 50% by centrifugation and removal of supernatant. All cell preparation was done at RT in polypropylene tubes. A volume of 150 µL activated (stimulus) or resting blood was added to staining tubes containing a mixture of monoclonal fluorophore-conjugated antibodies. This amount of blood was sufficient for one test on peripheral blood DCs to identify changes in surface antigen expression. The mixture of mAbs included DC-distinguishing antibodies (lin 1 FITC, anti-HLA-DR PerCP, CD11c APC) and one PE-conjugated surface marker (CD25, CD40, CD80, CD86, or HLA-DQ). Next, red blood cells were lysed for 10 min by adding 3 mL of FACS lysing solution. The surface-stained leukocytes were then collected by centrifugation and washed with 3 mL wash buffer. For acquisition, the stained cells were resuspended in 250 µL wash buffer. If acquisition was delayed, samples were kept for a maximum of 1 h at 4°C. Monoclonal antibody (mAb) reagents CD80 PE, CD25 PE, and anti-HLA-DQ PE were obtained from BDIS. Anti-HLA-DQ is a BDIS clone and was custom conjugated to PE. CD86 PE and anti-IL-12 PE were obtained from PharMingen. CD40 pure mAb, clone 89, was obtained from DNAX Research Institute (Palo Alto, CA) and conjugated to PE at BDIS.

Flow cytometric analysis The samples were analyzed on a FACSCalibur dual laser flow cytometer (BDIS). The instrument was set up using automated FACSCompy 4.0 software and four-color Calibritey beads according to mean fluorescence intensities (BDIS). Events were acquired with a FSC threshold and a live gate on HLA-DR-positive events (Fig. 1). The data files were analyzed with PAINT-A-GATE Proy software. The side scatter (SSC) signal was transformed using a polynomial function to enhance the cluster resolution of leukocytes in multidimensional flow cytometric analysis [22]. Paint-A-Gate was chosen because it allows the enlargement of events, which helps in the analysis and display of rare event populations.

Gating strategy (CD11c⫹ DCs, CD11c⫺ DCs, monocytes) The resolution of DCs in a whole-blood format is complex and requires the combination of multiple gates. First, high light scatter events originating from neutrophils and eosinophils were identified in a SSC/FSC plot and excluded from further analysis by removal from the display. In addition, low light scatter events appearing as debris were removed. For CD11c⫹ DCs, the analysis is initiated by the identification of SSC low/CD11c bright events, capturing mostly CD11c⫹ DCs. Subsequently, these events are displayed in an HLA-DR/lin 1 dot plot where CD11c⫹ DCs can be further distinguished from most monocytes due to their low lin 1 staining. To check for monocytes in the CD11c⫹ DC subset, the events are examined in an HLA-DR/CD11c dot plot. Here, contaminating monocytes would appear as individual events with comparatively lower HLA-DR and CD11c antigen expression distinct from the cluster of the CD11c⫹ DC population. CD11c⫹ DCs are thus CD11c bright, lin 1 dim, and SSC low (Fig. 1, green events). Following the above gating strategy, CD11c⫺ DCs are excluded from the analysis in the first step improving the resolution of CD11c⫹ DCs from lin 1-positive events in the HLA-DR/lin 1 dot plot (Fig. 1, red events). CD11c⫺ DCs are also identified by the use of multiple regions. The analysis is initiated by gating on HLA-DR bright/lin 1 dim events in an HLA-DR/lin 1 distribution, mostly capturing CD11c⫹ and CD11c⫺ DCs. Then these events are displayed in an HLA-DR/CD11c dot plot and CD11c⫺ DCs can be identified by their lack of the CD11c antigen. CD11c⫺ DCs are thus HLA-DR bright, lin 1 dim, and CD11c negative (Fig. 1, red events).

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Fig. 1. Identification of DCs in activated whole blood. DCs are defined by low lin 1 levels (includes mAb for CD3, CD14, CD16, CD19, CD20, CD56; all FITC conjugated), by high HLA-DR levels and low scatter characteristics (FSC, transformed SSC). Subsequently, two DC subsets can be distinguished by their differential expression of CD11c. CD11c⫹ DCs are shown in green and CD11c⫺ (CD123⫹) DCs are shown in red. The CD123⫹ DCs can be indirectly identified by their lack of expression of CD11c. Monocytes, shown as blue events, are identified by their scatter characteristic, lin 1, and CD11c staining. All displayed events are restricted by FSC threshold, by low to intermediate SSC and a live gate on HLA-DR⫹ events. Approximately 11,000 events are viewed. The fluorescence parameters are displayed in a four-decade logarithmic scale, the FSC parameter is displayed in a 0–1024 linear scale and SSC in a 0–1024 transformed linear scale.

Monocytes are identified by their positive lin 1 (CD3, CD14, CD16, CD19, CD20, CD56), their positive CD11c and HLA-DR staining, and their characteristic light scatter properties. Monocyte detection is initiated by gating on CD11c-high events with a monocyte scatter profile in a SSC/CD11c dot plot. Then these events are displayed in a lin 1/HLA-DR plot. Here, lin 1-positive and HLA-DR-positive events are identified as monocytes. The analyzed monocytes are thus SSC higher than lymphocytes, lin 1-positive, CD11cpositive, and HLA-DR-positive (Fig. 1, blue events). CD14 was not used for the identification of monocytes, because all four fluorescence parameters are already taken (lin 1 FITC, cytokine or surface marker PE, HLA-DR PerCP, CD11c APC). This method was chosen because it allowed the identification of CD11c⫹ DCs, CD11c⫺ DCs, and most monocytes in the same sample. In healthy individuals HLA-DR-negative monocytes compose a small subpopulation that were neglected using the above gating strategy. This approach is not suitable in samples of diseased patients in which the HLA-DR-negative monocyte subset could be more relevant and should not be excluded from the analysis. Also, few CD11c-positive natural killer cells with low expression levels of HLA-DR could potentially contaminate the monocyte population. Finally, all three populations were displayed in a SSC/FSC plot to allow the exclusion of remaining debris, which would appear as events with low scatter characteristic distinct from the cell clusters of interest. The identification of CD11c⫹ DCs, CD11c⫺ DCs, and the monocyte population was performed sequentially because Paint-A-Gate has the capability to memorize the analyzed events. For display the populations of interest were recalled.

RESULTS Flow cytometric assay The flow cytometric assay is based on single-cell identification using four fluorescence parameters and cell-specific forward 538


(FSC) and orthogonal light scatter (SSC) characteristics. FSC is a measure of cell size and SSC is an indicator of cell granularity. Three fluorescence parameters are used to identify and to distinguish CD11c⫺ (CD123⫹) and CD11c⫹ DCs. Both subsets of peripheral blood DCs demonstrate bright staining with anti-HLA-DR PerCP, dim or negative staining with a lineage cocktail (lin 1) FITC and differential staining with CD11c APC (Fig. 1). Lin 1 is a monoclonal antibody cocktail containing CD3, CD14, CD16, CD19, CD20, and CD56, each of which is conjugated to FITC. This lineage cocktail is used as an exclusion parameter, staining leukocytes other than DCs and basophils. Low FSC and SSC was used as an additional criterion for the identification of peripheral blood DCs (Fig. 1). As demonstrated in Figure 1, CD123⫹ DCs can be identified indirectly by their lack of the CD11c antigen, allowing the immune function to be assessed with the remaining PE fluorescence parameter [11, 15]. Then the changes in CD25, CD40, CD80, CD86, HLA-DQ expression or cytokine secretion were measured upon cell stimulation (Figs. 2–4). All data of CD11c⫺ DCs were generated with the indirect identification method, allowing the evaluation of both CD11c⫹ and CD11c⫺ DC subsets in the same sample. Monocytes were identified based on their bright lin 1 FITC, anti-HLA-DR PerCP, CD11c APC staining, and their scatter characteristics (Fig. 1). Because CD14 and CD16 are both present in lin 1, this reagent also captures a CD14 dim leukocyte subset which is CD16 bright. These cells have been described as monocytes and more recently characterized as a novel peripheral blood DC subset [23, 24]. The cytokine expression of monocytes was evaluated in the same samples as well. To assess the cytokine profile upon stimulation, BFA was added to whole blood in the presence of stimulus. BFA is a drug that disrupts the Golgi-transport of proteins, leading to an intracellular accumulation of cytokines during cell activation, thus increasing the intracellular signal detected by immunofluorescence [20, 25]. The functional studies of surface molecules were performed in the absence of BFA to enable transport of newly synthesized molecules to the cell membrane. The resting control samples were incubated in the absence of stimulus with or without BFA as required.

Immune function of DCs and monocytes in whole blood: cytokine responses PMA⫹I and LPS stimulation induced a cytokine response in CD11c⫹ DCs. The cytokine expression is displayed as PE mean fluorescence intensity (MFI) in two-parameter distributions of CD11c APC/cytokine PE (Fig. 2). LPS-activated samples were tested for IL-1␣ and ␤, IL-1Ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, TNF-␣, and IFN-␥ expression (Table 1). CD11c⫹ DCs showed clear up-regulation of TNF-␣, IL-1␤, IL-1Ra, IL-6, and IL-8. In addition, two out of three investigated donors synthesized IL-12 minimally. IL-1␣, IL-2, IL-10, IL-13, and IFN-␥ were not detected in CD11c⫹ DCs (data not shown). PMA⫹I activated samples were tested for IL-1␣ and -␤, IL-1Ra, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, and TNF-␣ expression (Table 1). CD11c⫹ DCs showed responses for IL-8, IL-1␤, IL-1Ra, and TNF-␣ protein. IL-1␣ was detected at very low levels. IL-2, IL-6, IL-10, IL-12, and http://www.jleukbio.org

Fig. 2. Intracellular cytokine expression of DCs in activated whole blood. CD11c⫹ DCs are displayed green and CD11c⫺ DCs are displayed red. CD11c⫹ DCs express detectable TNF-␣, IL-1␤, IL-1Ra, IL-6, IL-8, IL-1␣, and IL-12 protein upon activation using LPS⫹BFA (A) or PMA⫹I⫹BFA (B). CD11c⫺ DCs did not express detectable levels of the cytokines tested. The resting control samples failed to express cytokines, as expected (data not shown). CD11c⫹ gray events represent mostly monocytes. All events displayed in the bivariates meet light scatter properties characteristic of mononuclear cells and are HLA-DR⫹. Peripheral blood DCs and monocytes are defined as described in Figure 1. The above distributions show one representative out of three experiments. The fluorescence parameters are displayed in a four-decade logarithmic scale.

IL-13 were not detected in CD11c⫹ DCs upon PMA⫹I stimulation (data not shown). TNF-␣ secretion was greater in LPS-activated DCs compared with PMA⫹I-activated DCs. This observation is reversed for IL-8 secretion. Here, IL-8 was secreted to a greater extent upon PMA⫹I stimulation (Fig. 3). Unstimulated blood did not produce detectable cytokines (data not shown). Monocytes and CD11c⫹ DCs showed generally similar patterns of cytokine expression, although LPS stimulation triggered much more cytokine expression in monocytes (Figs. 2 and 4). CD11c⫺ DCs did not produce detectable cytokine levels under our experimental conditions (Fig. 2).

Immune function of DCs and monocytes in whole blood: surface molecule responses Changes in the expression of CD25, CD40, CD80, CD86, HLA-DR, and HLA-DQ was evaluated as the mean fluorescence intensity (MFI) ratio of stimulated to unstimulated samples cultured in parallel (Table 1). In CD11c⫹ DCs, using LPS stimulation, CD80 was most dramatically up-regu-

lated and followed by CD86 and CD40 (Fig. 5). CD25, HLA-DR, and HLA-DQ were up-regulated to the least extent of all six investigated surface markers. For PMA⫹I activation, CD86 and CD80 were predominantly up-regulated in CD11c⫹ DCs. Some enhanced levels of HLA-DR and HLA-DQ were easily detected, whereas changes in CD40 and CD25 expression were negligible. CD80 was more up-regulated than CD86 in LPS-activated DCs compared with PMA⫹I activation. Here, CD86 was expressed to a greater extent than CD80 (Fig. 3). CD11c⫺ DCs up-regulated the activation marker CD25 (IL-2R␣) substantially after PMA⫹I activation and only minimally after LPS stimulation (Fig. 5). Some enhanced CD40 levels were observed after LPS activation. HLA-DR, HLA-DQ, CD80, and CD86 increases were minimal.

Kinetics of TNF-␣, IL-1␤, IL-6, and CD80 in LPS-activated CD11c⫹ DCs The expression of IL-1␤, IL-6, TNF-␣, and CD80 were investigated over a time course of 8 h (Fig. 6). In the cytokine experiments BFA Willmann and Dunne Cytokines in dendritic cells

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Fig. 3. Comparison of TNF-␣, IL-8, CD80, and CD86 production in CD11c⫹ DCs through the use of LPS and PMA⫹I-activated whole blood. Two donors are displayed. The expression pattern of cytokines and co-stimulatory molecules varies between different stimuli and is consistent between donors. IL-8 and CD86 are the dominant signal in PMA⫹I stimulation. In LPS-activated samples, TNF-␣ and CD80 are produced to a greater extent. See Figures 1 and 2 for gating strategies on DCs.

was added only for the last hour of the indicated stimulation period. TNF-␣ synthesis was detected first (1 h), followed by IL-1␤ (ⱖ1 h) and IL-6 (ⱖ3 h) and finally by CD80 (ⱖ4 h). Intracellular TNF-␣ protein levels declined nearly to baseline at 2 h of activation, whereas the expression of IL-6 was more prolonged and declined nearly to baseline at 7 h. IL-1␤ did not decline to baseline during the time course of the experiment, but showed significantly decreased protein levels at 7 h of activation. In contrast, the CD80 co-stimulatory molecule was detected at ⱖ4 h and constantly increased over the entire time course of the study.

Data readout The intracellular expression of the investigated cytokines has a uniform baseline in all resting samples and therefore MFI was chosen for the intracellular protein quantification. In contrast, surface antigens show a heterogeneous baseline expression. For example, CD86 is constitutively expressed on resting CD11c⫹ DCs and is up-regulated in activated CD11c⫹ DCs, whereas CD80 is not expressed in resting and is up-regulated in activated samples. Because the antigen density of CD80 is much less than that of CD86, an evaluation of surface antigens was chosen as a ratio of MFI (MFI activated/MFI resting) instead of MFI, because the latter would provide unrepresentative results.

DISCUSSION Resolution of DCs from monocytes is difficult conceptually and technically, and the use of the lin 1 antibody cocktail is problematic for the identification of DCs in an activated whole 540


blood environment. As seen in Figure 1, and in contrast to resting blood from healthy donors (our unpublished data), activated preparations include some leukocytes that are lin 1 dim and therefore difficult to distinguish from DCs. These cells are further characterized by their comparatively high light scatter, low HLA-DR, and low CD11c expression. Monocytes have been reported to shed CD14 upon activation, which could explain a decreased lin 1 staining [26]. It is interesting that these same lin 1 dim cells have especially high expression levels of cytokines, typical of monocytes in LPS⫹BFAactivated samples, but not in PMA⫹I⫹BFA-treated blood (data not shown). Generally speaking, the analysis of DCs from PMA⫹I-activated samples was technically more challenging than from LPS-stimulated samples. PMA⫹I activation was associated with a higher cell loss and with a decreased lin 1 cocktail staining. We have chosen a very conservative definition of lin 1-dim cells, and experimental conditions in which monocytes are maximally excluded from our DC analysis. The goal of this study was to survey responses to pharmacological (PMA⫹I) and biological (LPS) stimuli in peripheral blood DCs while they were in their native environment, in order to minimize artifacts associated with cell enrichment procedures or long-term culture. Our observations demonstrated the pleiotropic responses of CD11c⫹ DCs under PMA⫹I and LPS stimulation as well as functional differences between CD11c⫹ and CD11c⫺ DCs. The CD11c⫹ DCs responded to LPS by first making TNF-␣, then IL-1␤ and IL-6, then CD80 (Fig. 6). In our investigations, activated CD11c⫹ DCs strongly upregulated co-stimulatory molecules in contrast to CD11c⫺ DCs (Fig. 5). MHC class II molecules were minimally to moderately enhanced in both DC subsets using PMA⫹I or LPS stimulation. The up-regulation of MHC class II and co-stimulatory molhttp://www.jleukbio.org

Fig. 4. Comparison of cytokine expression between monocytes (shaded bars) and CD11c⫹ DCs (black bars) in activated whole blood. (A) In LPS⫹BFA-stimulated samples, monocytes express cytokines at higher levels than CD11c⫹ DCs. (B) Upon PMA⫹I⫹BFA activation, the cytokine secretion of CD11c⫹ DCs and monocytes is equivalent and much less compared with LPSactivated samples. Intracellular protein secretion is evaluated as PE MFI. See Figures 1 and 2 for gating strategies on DCs. Monocytes were identified based on high HLA-DR, high CD11c, high lin 1 expression, and monocyte specific scatter characteristics. One representative experiment out of three is shown.

ecules in CD11c⫹ DCs after activation is in agreement with the literature because these signals are widely discussed as a measure of DC activation/maturation [1, 2]. Zhou and Tedder assessed cytokine gene expression levels in peripheral blood DCs by reverse transcriptase-polymerase chain reaction (RT-PCR) [17]. Their data were generated with TABLE 1. Functional Responses of CD11c⫹ and CD11c⫺ DCs in Whole Blood After Stimulation with LPS and PMA ⫹ I

IL-1␣ IL-1␤ IL-Ra IL-2 IL-4 IL-6 IL-8 IL-10 IL-12 IL-13 TNF-␣ IFN-␥ CD25 CD40 CD80 CD86 HLA-DQ HLA-DR

LPS CD11c⫹ DCs

PMA ⫹ I CD11c⫹ DCs

LPS CD11c⫺ DCs

PMA ⫹ I CD11c⫺ DCs

⫺ ⫹ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺ ⫺/⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺/⫹ ⫹ ⫹ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫹ N.A. ⫺/⫹ ⫹ ⫹ ⫹ ⫹ ⫹

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫹ ⫺/⫹ ⫺/⫹ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ N.A. ⫹ ⫺/⫹ ⫺ ⫺ ⫺/⫹ ⫺/⫹

Cytokine expression is measured as MFI; change in surface molecule expression is measured as a ratio of MFI of activated and control samples. ⫹, positive; ⫹/⫺, minimal; ⫺, no response after cell activation.

purified DCs, obtained after extensive cell enrichment procedures using density gradient centrifugations, overnight culture, and flow cytometric sorting. They identified IL-6, IL-8, IL-10, and TNF-␣ mRNA in resting CD83⫹ DCs and an induction of IL-1␣ and IL-1␤ mRNA after PMA⫹I activation. Further, De Saint-Vis et al. investigated mRNA expression in germinal center DCs (GCDCs), which have been described to be closely related to the peripheral blood CD11c⫹ DCs [13, 27]. They found that IL-10 mRNA was present before activation and elevated in PMA⫹I-activated samples, whereas IL-13 mRNA was only expressed after PMA⫹I activation. Using the same stimulus, our whole blood assay detected IL-1␣, IL-1␤, IL-1Ra, and TNF-␣, but not IL-10 or IL-13 in CD11c⫹ DCs, whereas CD11c⫺ DCs did not secrete any cytokines (Fig. 2B and Table 1). Taking De Saint-Vis’ findings into consideration, it was surprising that CD11c⫹ DCs did not express any IL-10 or IL-13 protein, despite very similar activation conditions. We have restricted our investigations to the measurement of proteins on a single-cell level and therefore cannot address the possible induction of IL-10 or IL-13 mRNA directly. Peripheral blood CD11c⫹ DCs may require an additional signal to induce the translation of IL-10 and IL-13 protein. In a related study, Huang et al. used an enzyme-linked immunospot (ELISPOT) assay on differentially purified blood mononuclear cells to report that DCs are enriched for IL-4, IL-10, and IFN-␥producing cells relative to monocytes or T cells [28]. Although the cells were not intentionally stimulated, their activation status after a 2-day enrichment procedure may be different from the DCs analyzed in this report where these cytokines were not present in detectable levels. Willmann and Dunne Cytokines in dendritic cells

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Fig. 5. Measurement of CD25, co-stimulatory molecules, and MHC class II molecules in activated versus resting peripheral blood DCs. Filled bars, CD11c⫹ DCs; open bars, CD11c⫺ DCs. Change in surface molecule expression is measured as a ratio of MFIs of stimulated and resting control sample. See Figures 1 and 2 for gating strategies. One representative out of three experiments is shown.

The susceptibility of human DCs to LPS stimulation has been reported earlier by Sallusto et al. [29]. These DCs were generated from peripheral blood low-density mononuclear cells in 8–15 days of culture with GM-CSF and IL-4. After LPS stimulation, these cells were found to have a decreased staining for MHC class II protein associated with endosomal compartments, and an increase of MHC class II on the cell surface. As stated earlier, the functional relationship between monocytederived DCs and the peripheral blood DCs is not clear. A cytokine response of DCs generated with similar methods was described by Lo´re et al. on a single-cell level [30]. All monocyte-derived DCs were shown to express IL-1Ra, IL-1␣, and IL-1␤ protein after 3 h of LPS stimulation, and IL-1Ra, IL-1␣, and IL-1␤ were detected in 10–25% of unstimulated DCs. In addition, TNF-␣, IL-6, IL-10, IL-12, and GM-CSF were detected in a small fraction (0–5%) of DCs after stimulation. We have shown that CD11c⫹ DCs did respond to LPS stimulation with the secretion of IL-1␤, TNF-␣, IL-6, IL-1Ra, IL-8, and very low levels of IL-12 (Fig. 2A). IL-1␤ and TNF-␣ were produced at highest levels, as measured by the mean fluorescence intensity of the cytokine staining (Fig. 4). Ghanekar et al. analyzed enriched peripheral blood DCs with up to 24 h culture and detected the secretion of IFN-␥ and IL-6 protein in response to HIV-1 and HSV and IL-1␤ in response to HSV [16]. In addition, in their study, mRNA coding for IFN-␥, IL-1␣, IL-1␤, IL-6, IL-10, IL-12, GM-CSF, and TNF-␣ was detected. Using LPS stimulation, our kinetic study revealed that synthesis of TNF-␣, IL-1␤, and IL-6 occurred before the up-regulation of CD80 (Fig. 6). CD80 may be an unusually significant surface activation signal in that it was the only 542


co-stimulatory molecule without constitutive expression in CD11c⫹ DCs. Similar kinetics of IL-1␤ and IL-6 were observed earlier in LPS-stimulated monocytes. It is interesting that TNF-␣ was maximally expressed in DCs after 1 h of activation, whereas in monocytes maximal secretion was seen at 4 h of stimulation [J. H. Allaert and J. F. Dunne, unpublished observations]. It was not surprising that CD11c⫺ DCs did not respond to LPS because they lack the CD11c and the CD14 LPS receptor [11, 15]. Ingalls and Golenbock describe CD11c/CD18 to be an independent transmembrane signaling receptor for LPS and hypothesize that CD14, known as the major LPS receptor, binds to LPS, amplifies its responses, but does not actually transduce a signal [31]. Our results are consistent with this hypothesis in that we found that the cytokine secretion by monocytes expressing CD11c and CD14 was much more potent than cytokine expression in CD11c⫹ DCs, which express very low levels of the CD14 antigen (Fig. 4) [11, 14]. The signal transduction by phorbol esters is mediated differentially by various isoforms of protein kinase C (PKC) [32–34]. Activated PKC can phosphorylate a number of downstream signaling molecules, such as nuclear factor-␬B (NF-␬B), which is also induced by the LPS signal transduction pathway [33–36]. Because NF-␬B has been reported to be involved in the gene regulation of IL-1, IL-1Ra, IL-6, IL-8, TNF-␣ proteins, and CD80, it is not surprising that both PMA⫹I and LPS induced a similar cytokine secretion pattern and CD80 expression in CD11c⫹ DCs, but also in monocytes (Figs. 4 and 5) [36–38]. When we investigated changes in surface molecule expreshttp://www.jleukbio.org

phenotyping using HLA-DR and CD14, thus precluding the discrimination of CD11c⫺ and CD11c⫹ DCs. A simple interpretation consistent with the findings of O’Doherty, Vakkila, and the present report is that Vakkila’s enriched cell preparation consisted mostly of the CD11c⫺ subset described here, which did not secrete any cytokines. Summarizing, this and other studies describe the expression of mRNA and/or protein characteristic of inflammatory responses after PMA, LPS, HSV, or HIV-1 stimulation. We would therefore suggest that inflammatory cytokines play an important role in the immune function of DCs. This is evident not only by the use of various stimuli, but also across several DC subsets in unseparated and separated samples as well as fresh and long-term culture-generated DCs. Blockade or knockout studies are called for to characterize the redundant or individual necessity of each of these mediators in DC function. We conclude that pleiotropic and functional responses of individual DC subsets and monocytes can be reproducibly measured in a whole-blood format.

ACKNOWLEDGMENTS The authors would like to thank Dr. Bruce Koppelman and Dr. Tom Frey for thoughtful suggestions and for the critical review of the manuscript.

REFERENCES Fig. 6. Kinetics of TNF-␣, IL-1␤, IL-6, and CD80 in LPS-activated CD11c⫹ DCs. The time course of LPS incubation was 0–8 h. Intracellular cytokines and CD80 expression are measured as PE MFI. TNF-␣ was produced first, then IL-1␤ and IL-6. CD80 was intensely up-regulated beyond 4 h of activation. See Figs. 1 and 2 for gating strategies.

sion, CD11c⫺ DCs showed a distinct up-regulation of the activation marker CD25 (IL-2R␣) in PMA⫹I-activated samples, whereas CD11c⫹ cells did not. Activated PKC mediates signaling pathways other than NF-␬B, which may play a role in the induction of CD25 [33]. Although the gene regulation of CD25 has been well characterized in T cell activation, it has not been broadly reported in dendritic cells. O’Doherty et al. describe an increase of CD25 in human blood DCs after 2 days of culture with monocyte-conditioned medium [8]. The immunophenotype of the purified DCs in the O’Doherty study was very similar to the immunophenotype of the CD11c⫺ DCs (lineage marker⫺, MHC class II⫹ CD45RA⫹, CD45RO⫺) [11, 15]. In a related study, Vakkila et al. investigated IL-1␣, IL-1␤, and IL-6 production in human peripheral blood DCs after LPS stimulation [39]. This group also used extensive enrichment procedures to obtain purified DCs. Their studies conclude that peripheral blood DCs do not express IL-1␣, IL-1␤, or IL-6 upon LPS stimulation. Using the same stimulation conditions, Vakkila et al. demonstrated the cytokine mRNA and protein expression in monocytes, confirming our results (Fig. 4). The purity of the enriched blood DCs was ascertained by immuno-

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