Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells

Journal of Immunological Methods 249 (2001) 207–222 www.elsevier.nl / locate / jim

Quantitative single cell methods that identify cytokine and chemokine expression in dendritic cells a,b ¨ Karin Lore´ a , *, Anna-Lena Spetz b , Thomas E. Fehniger b , Anders Sonnerborg , c b Alan L. Landay , Jan Andersson a

Department of Immunology, Microbiology, and Pathology, Division of Clinical Virology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Stockholm, Sweden b Department of Medicine, Center for Infectious Medicine, Karolinska Institutet, Huddinge University Hospital, Huddinge, Stockholm, Sweden c Department of Immunology /Microbiology, Rush–Presbyterian St. Luke’ s Medical Center, Chicago, IL, USA Received 28 August 2000; received in revised form 30 October 2000; accepted 11 December 2000

Abstract Two techniques based upon flow cytometry (FCM) and in situ image analysis were developed for quantification of intracellular cytokine and chemokine protein expression at the single cell level in dendritic cells (DCs). The qualitative and quantitative differences between the two methods were evaluated. In vitro differentiated DCs were stimulated with lipopolysaccaride (LPS) and thereafter stained for either IL-8, which is secreted through the Golgi-organelle, or IL-1ra, which localises diffusely in the cytoplasm. Microscopic examination, both for fluorophore and enzymatically stained cells, showed that DCs expressed IL-8 and IL-1ra with two different staining patterns. FCM analysis showed high frequencies of IL-1ra producing cells (76613%), which was similar to the frequency obtained by in situ imaging. However, in contrast to IL-1ra, the incidence of IL-8 expressing DCs showed high variability between the donors. The numbers of positive cells were 19619% as measured by FCM. The detection of IL-8 analysed by in situ imaging revealed higher frequencies (26614%). The addition of brefeldin-A, leading to cytoplasmic accumulation of proteins secreted through the Golgi endoplasmatic route, generated a significantly increased signal intensity and incidence of producer cells, resulting in similar frequencies for both methods. FCM has the advantage of being less time consuming than image analysis and is also able to facilitate multiple colour analysis. However, FCM is less accurate in detecting and quantifying cytokines and chemokines with a preserved juxtanuclear staining pattern. The correct choice of detection technique therefore depends on the study question.  2001 Elsevier Science B.V. All rights reserved. Keywords: Dendritic cells; Flow cytometry; In situ imaging analysis; Cytokines; Chemokines

1. Introduction Abbreviations: IL, Interleukin; TNF, Tumour necrosis factor; GM-CSF, Granulocyte / macrophage-colony stimulating factor; PMA, Phorbol 12-myristate-13-acetate *Corresponding author. Tel.: 146-8-5858-1363; fax: 146-87467-637. ´ E-mail address: [email protected] (K. Lore).

Recent findings indicate that dendritic cells (DCs) together with CD4 1 T cells have the capacity to direct immune responses into the Th1 or Th2 type of cytokine pathway (Rissoan et al., 1999). Activation of DCs results in their migration from peripheral

0022-1759 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S0022-1759( 01 )00293-9

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locations into lymphoid tissues. At these sites DCs are involved in the induction of both B and T cell mediated immune responses. DCs can induce naive lymphocytes into specific response patterns which are dependent upon the type of antigen presented and the co-stimulatory molecules expressed by the DCs, as well as the local cytokine and chemokine environment, to which DCs also contribute. Several in vitro studies of DCs have indicated that these cells are capable of expressing numerous immunoregulatory cytokines at the mRNA level (Zhou and Tedder, 1995). However, model studies suggest that specific mRNA levels are not predictive of steady state protein levels within similar cell types, nor between different cell types alone (Anderson and Seilhamer, 1997; Anderson and Anderson, 1998; Gygi et al., 1999). As such, direct quantitative measurements of intracellular cytokine and chemokine expression in DCs at the protein level may provide stronger correlates of potential biological activities than mRNA measurements. We have developed two methods for quantifying intracellular cytokine and chemokine protein expression in DCs at the single cell level based on immunohistochemistry and immunofluorescence followed by computerised in situ image analysis (Bjork et al., 1996) or flow cytometry (FCM) (Andersson et al., 1988), respectively. The aim of this study was to evaluate the relative strengths and weaknesses of these two methods. The techniques were therefore compared with respect to their sensitivity and specificity. Here, the expression of IL-8 and IL-1ra (IL-1 receptor antagonist) was studied in order to investigate proteins secreted through the Golgi– endoplasmatic or microtubuli route in in vitro monocyte derived DCs. To provide a control for global cytokine assessments by FCM, we used freshly isolated peripheral blood mononuclear cells (PBMCs) that were activated with PMA and ionomycin and assessed for IFN-g expression according to previous reports (Prussin and Metcalfe, 1995; Assenmacher et al., 1996; Krouwels et al., 1997; Maino and Picker, 1998; Suni et al., 1998).

described (Romani et al., 1994; Sallusto and Lanziavecchia, 1994). In brief, PBMCs obtained from healthy blood donors were isolated from EDTAblood on Ficoll Hypaque density gradient (Pharmacia, Uppsala, Sweden), resuspended in culture medium (RPMI 1640 with 2 mM L-glutamine (Gibco, Paisley, UK), supplemented with 10% fetal bovine serum (FBS) (Gibco) plus 1% streptomycin and penicillin (Gibco) and were then allowed to adhere for 1.5 h at 378C. The cell culture flasks were washed three times with phosphate buffered saline (PBS). The adherent cells were cultured in medium containing human recombinant IL-4 (6.5 ng / ml, R&D Systems, Minneapolis, MN, USA) and GMCSF (250 ng / ml, Leucomax, Schering-Plough, France). Half of the medium was changed every third day with fresh media containing IL-4 and GM-CSF. Contaminating cells were characterised by FCM and found to be mainly CD4 1 or CD8 1 T cells and never to represent more than 5% of the total cell population. Cultured cells were used for subsequent experiments around days 8–11. The control experiments with stimulated T cells were carried out with freshly isolated PBMCs from healthy blood donors. These cells were also separated on Ficoll Hypaque density gradients.

2.2. Cell stimulation A total of 5310 5 DCs / ml obtained from seven healthy blood donors were stimulated in duplicate by LPS (100 ng / ml, purified from E. coli BR 055:35, Department of Bacteriology, Karolinska institute, Stockholm, Sweden) for 3 h. For the T cell experiments, 2310 6 freshly separated PBMCs / ml were stimulated by PMA (5 ng / ml, Sigma, St. Louis, MS, USA) and ionomycin (0.5 mM, Calbiochem Novabiochem, La Jolla, CA, USA) and cultured in culture media for 6 h in duplicate flasks. Brefeldin-A (10 mg / ml, Sigma) was added to one of the duplicate cell flasks 2 h after addition of PMA and ionomycin. Unstimulated PBMCs were used as controls.

2.3. Fixation and permeabilization of cells 2. Materials and methods

2.1. In vitro differentiation of dendritic cells DCs were generated from PBMCs as earlier

Cultured cells were harvested after the indicated stimulation periods, proteins were washed away and cell fixation was performed in 2% formaldehyde in PBS at pH 7.4 for 15 min. Cells were divided into

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two aliquots. One half was transferred to adhesion slides (BioRad Lab, Munich, Germany) to adhere for 15 min. Excessive cells were washed away. We have previously determined that these adhesion slides bind all viable cells but not dead or necrotic cells (Andersson et al., 1992). The other portion of the cells was placed in tubes for immunofluorescent staining for FCM.

2.4. Cytokine /chemokine-specific antibodies The antigen specific antibodies used for detection of IL-1ra and IL-8 have been described previously (Lore et al., 1998) for direct intracellular cytokine and chemokine staining and included the biotinylated cytokine / chemokine affinity purified polyclonal antibodies; goat anti-human IL-1ra (BAF 280) and IL-8 (BAF 208) (R&D Systems). The same lots of IL-1ra and IL-8 specific primary antibodies were used in both detection methods. Biotinylated monoclonal antibodies for IFN-g (7B6-1, Mabtech AB, Nacka, Sweden) and fluorescein isothiocyanate (FITC) conjugated anti-IFN-g (25723.11, Becton Dickinson, San Jose, CA, USA) were used for cytokine detection in stimulated PBMCs. In initial experiments the specificity of the anti-cytokine / chemokine antibodies were elucidated through pre-incubation with 103 molar excess of the corresponding recombinant cytokine, which significantly abolished the specific staining signal. The antibodies from R&D systems were also evaluated in Cos cells transfected with the appropriate cytokine / chemokine gene by Dr. J. Abrams, DNAX, Palo Alto, CA, USA.

2.5. Cell marker-specific antibodies The following monoclanal antibodies (mAbs) were used for cell surface characterisation by flow cytometric analysis; phycoerythrin (PE)-conjugated mouse anti-human CD14, CD86, CD69 (Becton Dickinson), CD1a (Dako, Glostrup, Denmark), FITC labelled anti-human CD83 (PharMingen, San Diego, CA, USA) and PerCP-conjugated mouse anti-human CD3, HLA-DR (Becton Dickinson).

2.6. Immunohistochemical staining and enzymatic detection of cytokine production Our staining methodology was performed as previ-

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ously described (Sander et al., 1991) with specific modifications for staining of DCs (Lore et al., 1998). Cells were permeabilized throughout the staining procedure with 0.1% saponin (Riedel-de Haen AG, Seelze, Germany) dissolved in 13 Earl’s balanced salt solution (BSS) (Gibco) supplemented with 0.01 M HEPES buffer (Gibco) to allow the intracellular access of reagents. Endogenous peroxidase was blocked with 1% H 2 O 2 . Incubation with 2% FBS and 1% normal mouse serum for 30 min at 378C was performed in order to reduce the possibility of risks for non-specific antibody hydrophobic Fc-receptor interactions. An additional incubation with Blocking kit (Vector Laboratories, Burlingame, CA, USA) was performed to block endogenous biotin and biotinbinding proteins. The cytokine / chemokine affinity purified biotinylated polyclonal Abs (5–15 mg / ml) were diluted in BSS–saponin and incubated for 1 h at 378C. After washes the cells were incubated with an avidin–biotin horseradish peroxidase complex (Vectastain, Vector Laboratories) for 30 min at room temperature. A colour reaction was developed by 39-diaminobenzidine tetrahydrochloride (DAB) (Vector Laboratories) in the dark and stopped after 2–10 min by washes in BSS. The cells were counterstained with hematoxylin and the slides were left to dry before mounting in buffered glycerol.

2.7. Identification of cytokine producing cells by computerised image analysis The enzymatically-stained cells were examined in a Leica RXM microscope (Leica, Wetzlar, Germany) equipped with a colour three chip CCD camera (DXC-950p, Sony Corporation, Tokyo, Japan) providing red, green and blue (RGB) signals. Cells were analysed microscopically at 4003 original magnification, and images were transferred in RGB mode to a Quantiment 550 IW image analyser (Leica Cambridge Ltd., Cambridge, UK). The image processor was directed by a PC program with a special software routine developed for this application (Bjork et al., 1996). The total cells were enumerated by the image analysis system using the blue colour and morphology of the hematoxylin counterstained cells as a standard. RGB detection (using red, green, blue channels each with 256 levels) permitted the separation of over 16 million different colours. Threshold values were initially defined for the

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determination of cytokine and chemokine producing cells as well as for non-producing cells. The software program combined cell morphological, densitometric and staining specific colour values in order to separate cytokine and chemokine expressing cells from non-producing cells. Cells judged positive for cytokine or chemokine production further fulfilled the minimum inclusion criteria for specific RGB threshold colour values in ten adjacent pixels. This allowed the separation of positive and negative cells even in populations of cells with similar hues of blue intensity. Thus, no gating was required and entire populations of single cells were evaluated in the program. The frequencies of cytokine and chemokine expressing cells were assessed by the examination of $1000 cells in at least 20 fields. The acquired data from the image analysis were imported into a Microsoft Excel dedicated macro set-up [IMAGE2XL, developed by Dr. T.E. Fehniger (Bjork et al., 1996)], which provided statistical analysis using the Astute program (University of Leeds, Leeds, UK) and provided histograms for displaying frequency distribution of the intensity values of the staining signal of the DCs for the entire population evaluated from each of the microscope slide wells. The values of intensity units per individual DC, which are presented, represent the cumulative sums of the blue channel RGB values of all adjacent pixels in each individual cell fulfilling the inclusion criteria. In principle, larger cells with frequent and neighbouring stained pixels will present with the highest intensity scores. Since all cells were stained with hematoxylin, the cells which were negative for the brown DAB reaction product were measured, identified and scored for cumulative pixel intensity.

2.8. Intracellular staining and detection of cytokine /chemokine production by flow cytometry The permeabilization of the cell membrane was achieved by using the presence of 0.1% saponin in PBS supplemented with 0.01 M HEPES buffer throughout all incubations and washes in the staining procedure. A total of 2310 5 cells were initially blocked with 10% human AB-serum. The cells were thereafter exposed to the biotinylated cytokine / chemokine specific antibodies for 30 min at 48C and

then washed. FITC labeled streptavidin (Dako) was used to visualize the positively stained cells. In the experiments done with PBMCs the cell marker specific monoclonal antibodies (mAbs) were added together with FITC labeled streptavidin. The cells were analyzed in a Becton Dickinson FACS Calibur flow cytometer using CellQuest software. Analyses of results were performed with at least 10,000 cells per sample. The results were expressed as the log of fluorescence intensity. In addition, the cells were transferred to adhesion slides (BioRad Lab) for manual evaluation of staining characteristics under the UV–light microscope (Leica RXM microscope). Phenotypic characterisation of cells was also performed using a FACScan flow cytometer. Results were expressed as the log of fluorescence intensity.

2.9. Statistical evaluation The difference in frequencies of IL-8 / IL-1ra expressing DCs obtained using the two methods were evaluated by Student’s paired t-test and was considered to be significant at a two tailed P-value of ,0.05.

3. Results

3.1. Cell phenotyping of in vitro derived dendritic cells The generation of DCs from adherent blood monocytes treated with IL-4 and GM-CSF was found to be less laborious than traditional isolation techniques of authentic DCs from biopsies. High yields of non-adherent CD3 2 , CD1a 1 , CD14 2 cells with a morphology characteristic of DCs were developed from CD14 1 CD1a 2 monocytes after 6–8 days in culture. The in vitro derived immature DCs expressed HLA-DR and low expression of CD86 and CD83 as detected by FCM (Fig. 1), as previously described (Lore et al., 1998).

3.2. Characteristics of intracellular staining of cytokines and chemokines in dendritic cells In vitro cultured unstimulated DCs were shown in our previous study to spontaneously express IL-1ra

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Fig. 1. Cell surface marker expression on in vitro IL-4 and GM-CSF derived DCs from monocytes after 7 days of culture. The histograms derived from flow cytometry show (A) lack of CD14 expression, (B) low expression of CD83 and (C) high expression of CD1a as well as of (D) HLA-DR. (E) shows the intermediate expression of the co-stimulatory receptor CD86. The DC culture is one representative example from seven donors. The isotype-matched negative staining controls overlie the histograms for the cell marker staining.

and IL-1a (mean 25 and 10% of the cells, respectively) but not other cytokines (IL-1b, TNF-a, IL-6, IL-10, IL-12) or chemokines (IL-8, MIP-1a / b, RANTES). However, stimulation with LPS generated a significant induction of all these cytokines and chemokines after 0.5–3 h. Therefore, an LPS stimulation period of 3 h was selected in the present study. The cells were stimulated, fixed and divided into two aliquots for immunohistochemical staining followed by image analysis and immunofluorescence staining evaluated by FCM. Microscopic examination, both for enzymatically stained cells and cells stained with the fluorophore, revealed that numerous LPS stimulated DCs showed a characteristic staining pattern. The expression of IL-8 protein was localised to the Golgi reticular complex resulting in a very distinct juxtanuclear positive signal in the producer cells (Fig. 2a and b). We have previously shown cytokine co-localization to the Golgi organelle using in situ hybridisation for cytokine mRNA detection and immunohistochemistry for protein detection. The cells expressing protein with the juxtanuclear locali-

sation were mRNA positive for the same cytokine (Raqib et al., 1996). In contrast, the IL-1ra proteins were found diffusely distributed throughout the cytoplasm and in close proximity to the nuclear membrane (Fig. 2c and d). The secretory pathway of cytokines belonging to the IL-1 family differs from many cytokines due in part to the lack of a signal peptide sequence targeting the rough endoplasmic reticulum. As such, the staining intensity signal of IL-1ra expressing cells was much greater than IL-8 expressing cells. (Fig. 2). In order to increase the sensitivity for FCM based cytokine / chemokine detection the cells were treated with brefeldin-A. Brefeldin-A uncouples the secretory pathway, which leads to the inability to secrete proteins from the cell via the Golgi-endoplasmatic route. The localisation of proteins in the Golgiorganelle was thereby disrupted by brefeldin-A. The proteins were instead accumulated within the cytoplasm of the cells, which promoted an increased intensity in the fluorochrome staining signal (Fig. 2e and f).

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Fig. 2. Characteristics of intracellular IL-1ra and IL-8 expression in DCs found by immunohistochemical and immunofluorescent staining, respectively. The DCs were stimulated for 3 h with LPS and subsequently analysed for IL-1ra and IL-8 expression. The dot-like feature of the IL-8 expression localised to the Golgi–endoplasmatic reticulum complex in the cells is shown in (A) and (B). IL-1ra (C,D) proteins are not sectreted via the Golgi and are therefore diffusely distributed in the cell cytoplasm. The addition of brefeldin-A in the cell culture (E,F) blocks secretion via the Golgi and the IL-8 proteins are thus accumulated in the cell cytoplasm and a different staining signal is generated.

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3.3. Intensity distribution profiles from in situ image analysis of cytokine positive and negative DCs Evaluation of the immunohistochemically stained DCs by the computerised in situ image analysis system permitted enumeration of cytokine / chemokine expressing cells based on the threshold criteria but also made it possible to formulate histograms of the accumulated intensity values of the staining signals based on each individual DC measured. The cumulative pixel intensity values from each positive or negative cell were intergrated and each cell’s total intensity value was distributed into bins of linear steps and then plotted as a histogram. The IL-1ra / IL-8 expressing DCs (positive) in the sample were plotted in separate bars from the nonexpressing DCs (negative). The positively stained DCs within the total population showed a wide distribution of staining intensity. The mean intensity values of LPS stimulated IL-1ra expressing DCs (mean 227,500, 95% CI 220,100–234,900) were well separated from the non-IL-1ra expressing DCs (mean 67,800, 95% CI 61,800–73,800) (Fig. 3a). This was also seen for the IL-8 expressing DCs (mean 224,700, 95% CI 214,100–235,400) compared to the non-IL-8 expressing DCs (mean 67,200, 95% CI 63,700–70,600) (Fig. 3f). The DCs from cultures exposed to both brefeldin-A and LPS showed a comparable mean intensity signal for the DCs positive for IL-1ra (mean 232,500, 95% CI 225,400– 239,600) (Fig. 3d) and for the DCs expressing IL-8 (mean 227,709, 95% CI 218,500–236,900) (Fig. 3h). The first peak in the positive graph represents small sized cells, probably apoptotic cells, with intact IL1ra or IL-8 expression. Over 90% of the analysed DCs, both IL-1ra / IL-8 expressing and non-expressing DCs, were found to range in size between 75 and 100 m2 .

3.4. Intracellular detection of IL-1 ra and IL-8 in DCs by flow cytometry Induction of IL-1ra and IL-8 production in DCs by LPS stimulation resulted in a hetergeneous staining pattern as seen by FCM, ranging from completely negative cells to highly positive cytokine producing cells. Additionally, DCs cultured for several days

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gain autofluorescence, which changes the FCM scatter properties compared to PBMCs and thus complicates analyses based upon immunofluorescence staining. Therefore, the spectrum of fluorescent signals that was gained from the positive staining by stimulated DC cultures partly overlapped the spectrum for the negative unstained DC control cultures (Fig. 4a). We therefore set the threshhold defining positive / negative cells based upon the irrelevant antibody isotype control. Further controls testing the specificity of the anti-IL-1ra / IL-8 specific antibodies were performed using competitive inhibition with matched recombinant cytokine / chemokine protein and resulted in nearly complete quenching of the positive signals above the cut off threshold detected by FCM. The evaluation of IL-8 and IL-1ra by FCM was acceptable when the incidence was high ($20% of total cells), but in low incidence cases, (#1–5% of total cells) it was difficult to separate a positive signal from the background signal due to the low staining-to-noise ratio. The presence of brefeldin-A in the cell culture during the LPS stimulation period increased the intensity of the IL-8 staining from geometric mean 13.5 (95% CI 4.4–23.4) of fluorescence spectrum to 24.0 (95% CI 3.4–44.6). Although brefeldin-A improved the shift of positive DCs, it rarely led to a separation of discrete cell populations (Fig. 4c). Expression of IL-1 protein in the cytoplasm gave rise to a staining signal that was more easily detected by FCM. Nonetheless, the addition of brefeldin-A resulted in a demonstrative difference in intensity (geometric mean 65.4, 95% CI 21.8–109.0 to 102.8, 95% CI 21.7–181.9) of the positive staining signal (Fig. 4b and d).

3.5. Comparison of IL-1 ra /IL-8 assessment by image analysis and flow cytometric analysis IL-1ra and IL-8 expression in in vitro derived LPS stimulated DCs from seven different healthy donors were analysed by image analysis and FCM. The frequencies of positive DCs obtained by the two methods were compared. The frequency of IL-1ra expressing DCs after 3 h of LPS stimulation was 56–85% (mean575612%) of the DCs as revealed by image analysis and 55– 88% (mean576613%) as revealed by FCM (Fig.

214 K. Lore´ et al. / Journal of Immunological Methods 249 (2001) 207 – 222 Fig. 3. Histograms revealed from the in situ imaging program showing the accumulated values of intensity units per each individual DC. The IL-1ra / IL-8 expressing DCs in the populations based on the threshold criteria were plotted into separate bars (coloured dark grey and marked as positive in the graph) from the non-expressing DCs (light grey bars marked as negative). (A,B) shows IL-1ra staining on LPS stimulated DCs and (C,D) represent the IL-1ra expression after LPS and brefeldin-A stimulation. (E,F) and G,H) illustrate the IL-8 staining in DCs with and without brefeldin-A, respectively. Both the IL-1ra and IL-8 expressing DCs had significantly higher intensities than non-expressing DCs.

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Fig. 4. Histograms revealed from FCM of stimulated DCs. (A) IL-8 expression in DCs after LPS stimulation for 3 h. The negative staining control is overlaid. The frequency of IL-8 expressing DCs was assessed by measuring the frequency of cells with the fluorescence displayed in the histogram above the fluorescence emitted from the negative control. A clear separation of positive and negative DCs was not found. The IL-8 expressing DCs showed a shift in fluorescence from the non-expressing DCs and the negative unstained control. (A) shows approximately 35–45% positive IL-8 expressing DCs. (B) LPS stimulation showed that IL-1ra was expressed in a higher number (55–65%) of the DCs. The evaluation of the incidence of IL-1ra expressing cells was simplified due to a better separation between positive and negative populations. (C) DCs cultured with LPS and brefeldin-A. The accumulation of IL-8 protein in the cells enhanced the staining signal and improved the separation of the negative and positive populations. The IL-8 expressing DC population in this donor after treatment with brefeldin-A was increased to approximately 55–65%. (D) The numbers of IL-1ra expressing DCs were also increased to approximately 85–90%. The histograms revealed by FCM for this DC culture are derived from one representative experiment out of seven.

5a). The addition of brefeldin-A resulted in a relatively unchanged increased number of positive DCs, 63–89% (mean578610%) by image analysis and 71–92% (mean58268%) by FCM. Hence, a good correlation was found between the two methods for the detection of IL-1ra producing cells. There was no

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Fig. 5. The bars showing the max, min and median values and standard deviations of the percentage of IL-1ra (A) and IL-8 expressing (B) DCs analysed by the different methods, namely, in situ image analysis and FCM. The DCs were stimulated 3 h with LPS, with or without brefeldin-A. Data were generated from seven different donors.

significant difference between the two methods (P$ 0.082) The immunohistochemical method followed by image analysis showed significantly higher frequencies of IL-8 expressing DCs than were detected by FCM. LPS stimulation resulted in IL-8 expression in 5–47% (mean526614%) of the cells measured by image analysis while FCM assessment revealed 0– 46% (mean519619%) of IL-8 producing DCs (P5 0.047). The most remarkable difference was seen in two donors with the lowest IL-8 expressing cells (5 and 18%, respectively) as enumerated by image

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Fig. 6. Experiments were performed to study IFN-g expression in freshly purified PBMCs after PMA and ionomycin stimulation for 6 h. Two different antibodies were used for IFN-g detection; biotinylated anti-IFN-g and directly FITC-labelled anti-IFN-g. The cells were triple-stained for CD69 and CD3 simultaneously. The scatter properties of PBMCs of side scatter (SSC) and forward scatter (FSC) were changed after stimulation (A). The CD3 1 T cells were gated (B) and analysed. (C) Unstimulated T cells did not show any CD69 or IFN-g expression. Brefeldin-A only induced a low expression of CD69. PMA–ionomycin stimulation generated a vast activation of T cells as indicated by the high expression of CD69. The IFN-g expression seen in the Golgi-compartment of the cells resulted in a low number of positive cells detected by FCM. The addition of brefeldin-A significantly enhanced the staining signals for IFN-g expressing cells and a high number of positive cells were detected. Top panel in (c) shows IFN-y staining with biotinylated anti-IFNy mAbs. Lower panel shows directly FITC conjugated anti-IFN-y.

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analysis but with undetectable expression by FCM. Manual examination under the UV–light microscope of the DCs that had been analysed by FCM clearly verified that all donors indeed exhibited IL-8 expression with the typical juxtanuclear signal. The immunohistochemical method and evaluation by image analysis permitted the identification of one positive DC with IL-8 localised to the Golgi complex out of 1000 negative DCs. Conversely, FCM did not show accurate frequencies of DCs expressing IL-8 in the Golgi particularly when the incidence of positive cells was low. In contrast, detection by FCM of low numbers of DCs that were expressing IL-1ra was accurately detected by FCM and the in situ imaging method showed similar frequencies. DCs stimulated with LPS in the presence of brefeldin-A showed significantly higher incidences of IL-8 expression compared to DCs not exposed to brefeldin-A as detected both by in situ imaging (P50.024) and FCM (P50.009) analysis. The frequency of IL-8 expressing DCs after LPS stimulation supplemented with brefeldin-A was found to be 11– 65% (mean538617%) by FCM and 5–56% (mean532619%) of the DCs were positive by image analysis. Thus, the addition of brefeldin-A was associated with a comparable sensitivity for the detection of IL-8 expressing cells in both methods and no significant difference was found (P50.249) (Fig. 5b). The incidence of IL-8 expressing DCs showed a large variation among the seven donors as detected by both methods. In contrast, IL-1ra was more uniformly expressed in a large number of cells (Fig. 5).

3.6. Quantification of IFN-g in CD3 1 T cells In order to obtain additional data from to the FCM studies performed on cultured DCs, the frequencies of IFN-g producing cells were analysed in PMA and ionomycin stimulated PBMCs with and without the presence of brefeldin-A. In these experiments the negative control cells were unstimulated PBMCs from the same donors cultured in the presence or absence of brefeldin-A. Cells were triple stained for IFN-g CD69 and CD3 expression. The problem of autofluorescence frequently observed with DCs was not evident in the cultured PBMCs. Thus, the

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separation of positive and negative populations was enhanced. The size and the granularity of the cells were dramatically changed in the PMA-ionomycin stimulated PBMCs compared to the unstimulated cells (Fig. 6a and b). Unstimulated PBMCs did not show any expression of IFN-g or the early activation marker CD69 (Fig. 6c). Brefeldin-A without any additional stimuli resulted in a small but obvious upregulation of CD69 on the CD3 1 T cells. However, brefeldin-A did not appear to induce IFN-g production. Furthermore, stimulation by PMA and ionomycin led to a high upregulation of CD69 and a proportion of IFN-g expressing cells. Brefeldin-A amplified the positive signal, and the incidence of IFN-g expressing cells was readily detected. The FITC-conjugated IFN-g mAbs was found to be more sensitive and was detected at higher frequencies (19–41%) of IFN-g expressing cells than the biotinylated IFN-g mAbs in combination with FITC– streptavidin (9–20%). Evaluation of the cells analysed by FCM under the UV–light microscope showed a distinct juxtanuclear staining signals in the IFN-g expressing cells and diffuse intracellular signals in the cells cultured with brefeldin-A.

4. Discussion Cytokines and chemokines regulate the immune system in a paracrine and autocrine fashion. The proteins are often released and act locally. The antigen presenting capacity of DCs and effector functions of T cells are closely linked to their capacity to produce cytokines and chemokines. However, quantitative measurements of cytokine and chemokine protein production in DCs have been limited and new methods for single cell assays may provide further insight into the functional properties of the DCs. There is therefore a demand for accurate methods addressing the quantification of cytokine and chemokine producing cells at the single cell level. During recent years there have been several reports of improvements in the detection of intracellular cytokines in mononuclear cells. Traditional immunohistochemical methods commonly compete with assessments performed by flow cytometric analysis, as the former technique can be more difficult and time consuming to perform and the

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evaluation of the results can be less objective. Here we tried to develop two quantitative techniques for DCs. These cells were stimulated, fixed and divided into two aliquots for blinded comparison of the two methods, namely, immunohistochemical staining on adhesion slides evaluated by image analysis and immunofluorescence staining in cell suspension followed by flow cytometric analysis.

4.1. Methodological considerations Several different conditions of fixation and permeabilization of the cells have successfully been used to detect intracellular cytokines. The detergents n-octyl-beta-D-glucopyranoside (Andersson et al., 1988) and n-octyl-glucoside (Hallden et al., 1989) or lysolectithin in acidic acetate buffer combined with paraformaldehyde fixation (Labalette-Houache et al., 1991), digitonin (Kreft et al., 1992), methanol (Sedlmayr et al., 1996) and Tween 80 have been reported to be useful for intracellular cytokine detection evaluated by flow cytometry. We have previously shown that fixation of the cells in formaldehyde followed by permeabilization with saponin is a suitable technique that preserves cell morphology and facilitates the access of intracellular antibodies to intracellular organelles (Sander et al., 1991). In contrast to Tween 20 or Triton X-100 this combination has also been found not to change the scatter properties of the cells in FCM (Jacob et al., 1991) and has been successfully used by several others (Rigg et al., 1989; Jung et al., 1993; McIntyre et al., 1994; Prussin and Metcalfe, 1995; Grutzkau et al., 1997; Krouwels et al., 1997). We have previously outlined a method for immunohistochemical detection of cytokine and chemokine production in DCs (Lore et al., 1998). We found that blocking with high concentrations of serum in addition to the use of biotin labelled cytokine / chemokine specific antibodies greatly reduced the risks of nonspecific background staining with the DC cultures. We have found that cultured DCs require more blocking considerations than do PBMCs. The expression of pinocytosis as well as macrocytosis and the nonspecific uptake of antibodies can differ considerably depending on the state of activation of DCs.

4.2. Induction of cytokine /chemokine production in DCs The specific patterns of cytokines and chemokines expressed by DCs may be related to their state of maturation, the co-stimulatory molecules that are expressed by the surrounding CD4 1 and CD8 1 T cells and also the mode of activation. LPS from E. coli activates cells through the CD14 molecule but also activates cells that express Toll-like receptors (TLR), particularly TLR 2 and 4 (Kirschning et al., 1998; Yang et al., 1998; Chow et al., 1999). TLR 3 has recently been shown to be selectively expressed on subtypes of DCs (Muzio et al., 2000). We found that LPS is a gentle way to induce production of several cytokines and chemokines in DCs (Lore et al., 1998). In this study, IL-8 and IL-1ra expression were selected to study in DCs. In vivo derived DCs activated directly in freshly drawn whole blood with brefeldin-A and LPS prior to cell separation and staining have also been shown to produce both IL1ra and IL-8 (Willmann and Dunne, 2000). In the absence of brefeldin-A, IL-8 expressing DCs showed a staining signal clearly localised to the Golgi-organelle, while the IL-1ra synthesising DCs showed a positive signal in the whole cytoplasm due to the secretion through the microtubuli of IL-1ra. IL-1ra / IL-8 expressing DCs did not increase in cell size compared to non-expressing DCs and in contrast to IFN-g expressing T cells, which did change in size compared to non-expressing T cells. However, in vitro differentiated DCs often comprise a heterogeneous population both in terms of size, granularity and intensity of cell marker expression. In addition, a large number of DCs were found to constitutively express IL-1ra, while IL-8 synthesis was induced by LPS and showed extensive individual variation.

4.3. Quantification of IFN-g expression in PBMCs To provide a control for our approach to the intracellular assessments of cytokines / chemokines in DCs by FCM, PMA-ionomycin stimulated PBMCs were quantified for IFN-g expression. Detection of IFN-g expression by FCM is an established method used by ourselves (Andersson et al., 1988) and others (Prussin and Metcalfe, 1995; Assenmacher et al.,

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1996; Krouwels et al., 1997; Maino and Picker, 1998; Suni et al., 1998). Freshly isolated PBMCs show less autofluorescence than do cultured DCs and detection based on immunofluorescence is therefore simplified. The possibility to gate on a subgroup of cells also facilitates the separation of positively and negatively stained cells and the addition of brefeldinA resulted in a good separation of the populations. The differences found between the two anti-IFN-g antibodies may be due to the specificity of the different clones or the generation of higher sensitivity with improved separation of negative and positive populations using directly conjugated antibodies. The cytokine / chemokine detection procedure for PBMCs by FCM offers quick, efficient handling of large numbers of T cells, and multiple colour detection facilitates more comprehensive analyses. However, one has to take into account the fact that brefeldin-A alone without any other stimuli induces activation of the cells and upregulation of CD69.

4.4. Flow cytometry versus in situ imaging Quantification of intracellular cytokine expression by FCM has been more extensively used in several applications. Many commercialised products are also available for this purpose today. Quantitative measurements of multiple fluorescence parameters can also be accurately detected by FCM. The large numbers of different fluorophores available give the opportunity to detect simultaneously several molecules. FCM, therefore, allows a more comprehensive analysis of experimental data in a facilitated manner. The ability to gate on cell populations of interest is also a strength in this assay. DCs cultured for several days gain autofluorescence, which can complicate analyses based on immunofluorescence. In this study the high autofluorescence was adjusted by decreasing the photo multiplication tube (PMT) voltage and adjusting the compensation on the FACS Calibur. However, this adjustment may reduce the sensitivity of the detection for weakly expressed molecules. Specifically localised intracellular signals have a low fluorescence intensity in FCM and this may create problems in the separation of positive and negative cell populations. Thus, there is a diminished usefulness in using FCM to determine intracellular expression of cytokines / chemokines which are secreted

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through the Golgi–endoplasmatic route. The histograms generated from in situ imaging and measurements of the integrated pixel intensity of positively stained cells and the negative population, provided evidence that the brown substrate threshold values could be well separated from the blue counter staining. However, the key factor though with this latter technology was to relate the morphometric inclusion / exclusion criteria of minimal limits of adjacently located pixels with correct RGB thresholds for each measured cell. The RGB intensity level of individual cells alone was thus not sufficient to permit designation as a positive cytokine-producing cell. The technique of in situ image analysis was found to reveal significantly higher and more accurate frequencies than FCM in the detection of IL-8 in DCs. This is in line with what has been previously observed in a comparison of the two methods in the quantification of IFN-g and IL-4 in T cell clones (Krouwels et al., 1997). It has been previously shown that cytokine producing cells may downregulate their cytokine production within 30 min if removed from the microenvironment (Swoboda et al., 1991). Therefore methods that enhance the stability of cytokines proteins within cells will probably be more accurate. Treatment with either the carboxylic ionophore monensin (Jung et al., 1993; North et al., 1996) or brefeldin-A (Hallden et al., 1989; Kubota et al., 1998) disrupts the Golgi-organelle network and results in a dispersed cytosolic accumulation of the cytokine / chemokine proteins. This results staining with in significantly higher fluorescence intensity within single cells. Our adoption of this approach has resulted in a significantly improved sensitivity and more clear-cut separation of positive and negative cell populations in FCM. Furthermore, by using brefeldin-A treatment we were able to demonstrate that FCM had a comparable sensitivity to the in situ imaging technique. However, the addition of brefeldin-A requires in vitro culturing of cells prior to assessment and this may affect the assessment of cytokine / chemokine producing cells in vivo. As brefeldin-A modifies the secretion of protein and generates a higher staining signal, it may falsely create a higher incidence of producing cells not corresponding to the true biological situation. This

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Table 1 Comparison of flow cyometry and in situ image analysis Flow cytometry

In situ image analysis

Fast

Time consuming

Easy phenotypic characterisation of cytokine / chemokine expressing cells

Limited measurement of several colours

Requires protein transport inhibitor

Accurate detection of expressing cells due to remained golgi-morphology

Detection of in vivo expressing cells more difficult

High sensitivity

can be an important issue since cytokine production in vivo naturally takes place in a low number of cells. Even at small levels of IL-8 expressing DCs (,1–5%) there was no difficulty in finding positive cytokine / chemokine producing cells by in situ imaging. The direct visualisation of the cells in image analysis also permitted the morphologic assessment of the positive signal, which enhances the accuracy of the evaluation. FCM offers the advantage of a more rapid readout system and the possibility of examining a large number of cells. FCM is less time consuming than image analysis and the use of fluorescent-labelled cytokine / chemokine specific antibodies greatly facilitates the staining procedure. In this study the comparison between the methods was done using the same clones of biotinylated antibodies in order to avoid differences in this step and the respective conjugated streptavidin was subsequently added. However, the use of directly fluorescent-labelled cytokine / chemokine specific antibodies in FCM creates an even more rapid detection procedure and may exhibit higher sensitivity in some applications as the IFN-g experiments in this study showed. Further modifications using an amplification step, such as Tyramide Signal Amplification (TSA, Nen Life Science Products, Zavendem, Belgium) or the development of dyes with a higher fluorescent signal may also make it possible to omit the brefeldin-A incubation step in the detection by FCM. The in situ imaging technique currently has the advantage of not requiring addition in vitro of brefeldin-A. Differences between the two methods are summarised in Table 1. Bearing in mind the quantitative

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