lo Cells, CD14+ Monocytes, and CD16− Dendritic Cells

Clinical Immunology Vol. 100, No. 3, September, pp. 325–338, 2001 doi:10.1006/clim.2001.5072, available online at http://www.idealibrary.com on

Comparative Analysis of the Morphological, Cytochemical, Immunophenotypical, and Functional Characteristics of Normal Human Peripheral Blood Lineage ⫺/CD16 ⫹/HLA-DR ⫹/CD14 ⫺/lo Cells, CD14 ⫹ Monocytes, and CD16 ⫺ Dendritic Cells Julia Almeida,* ,† Clara Bueno,* ,† M a Carmen Alguero´,* ,† M a Luz Sanchez,* ,† M a de Santiago,* † Luis Escribano,‡ Beatriz Dı´az-Agustı´n,‡ Jose Miguel Vaquero,* ,† F. Javier Laso,§ Jesus F. San Miguel,† ,¶ and Alberto Orfao* ,† *Servicio General de Citometrı´a y Departamento de Medicina and †Centro de Investigacio´n del Ca´ncer, Universidad de Salamanca, Salamanca; ‡Servicio de Hematologı´a, Hospital Ramo´n y Cajal, Madrid; and §Servicio de Medicina Interna and ¶Servicio de Hematologı´a, Hospital Universitario de Salamanca, 37007 Salamanca, Spain

lo

ⴙ

Human peripheral blood (PB) CD14 /HLA-DR cells were initially described as a subset of mature monocytes. Recently, it has been suggested that these represent a part of a new subset of dendritic cells (DC), characterized by the coexpression of MDC-8/HLA-DR/ CD16. The aim of the present paper was to analyze the morphological, cytochemical, phenotypical, and functional characteristics of PB CD16 ⴙ/HLA-DR ⴙ cells compared to both PB CD14 ⴙ monocytes and CD16 ⴚ DC. In contrast to CD14 ⴙ monocytes, purified CD16 ⴙ/ HLA-DR ⴙ cells displayed cytoplasmic veils and lacked cytoplasmic myeloperoxidase and ␣-naphthyl acetate esterase. Normal human PB CD16 ⴙ/HLA-DR ⴙ cells also displayed phenotypic characteristics different from those of CD14 ⴙ monocytes: they lacked the CD64 Fc␥ receptor, showed lower levels of CD32, and expressed higher amounts of CD16 compared to CD14 ⴙ monocytes. They also displayed a different pattern of expression of other antigens, including CD14, HLA-DR, CD45RA, CD45RO, complement receptors and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors, among others. When compared to CD16 ⴚ DC, CD16 ⴙ/HLA-DR ⴙ cells showed reactivity for CD16, dim positivity for CD14, higher expression of both Ig- and complementreceptors and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86). The CD16 ⴙ/HLA-DR ⴙ cell subset displayed a higher Ig/complement-mediated phagocytic/oxidative activity than CD16 ⴚ DC, although this activity was significantly lower than that of mature monocytes. Regarding cytokine production at the single cell level, LPS plus IFN-␥-stimulated PB CD16 ⴙ/HLA-DR ⴙ cells produced significant amounts of IL1␤, IL6, IL12, TNF␣, and IL8; however, the percentage of cytokine-producing cells and the amount of cytokine/cell were lower in CD16 ⴙ/HLA-DR ⴙ cells than in CD14 ⴙ monocytes. In ad-

dition, upon comparing CD16 ⴙ/HLA-DR ⴙ cells with CD33 ⴙⴙⴙ/CD16 ⴚ DC, we found that the percentage of cytokine-producing cells and the amount of cytokine/ cell were significantly different in both cell subsets. In summary, our results show that CD16 ⴙ/HLA-DR ⴙ cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to both mature monocytes and CD16 ⴚ DC. Interestingly, these cells are more frequent than other DC in normal human adult PB and cord blood samples, while they are less represented in normal bone marrow. © 2001 Academic Press Key Words: human dendritic cell subsets; flow cytometry; immunophenotype; phagocytic/oxidative burst activity; cytokines.

INTRODUCTION

Dendritic cells (DC) are highly specialized APC of the immune system (1). In contrast to other types of APC, DC are potent activators of naive T cells and, therefore, important initiators of primary specific immune responses (2–5). The ability of mature DC to activate naive T lymphocytes has been related to their high expression of MHC, adhesion and costimulatory molecules, as well as to immunomodulatory effects through cytokine production (6 –11). The potent immunostimulatory function of DC makes them attractive tools for manipulating immune responses (12). Accordingly, it has been suggested that in vitro expanded DC (13–17) could be used for immunotherapeutic purposes. However, distinct DC subsets can be found in human tissues (18 –21). Accordingly, at present it is well established that DC are not homogeneous and at least two different DC subsets have been described in normal human peripheral blood (PB) (18 –25). In addition, a third subset of human DC has recently been

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1521-6616/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

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described in PB, which can be specifically identified by its reactivity with the M-DC8 monoclonal antibody (26). Interestingly, Scha¨kel et al. (26) show that one of the major phenotypic features displayed by M-DC8 ⫹ DC compared to the other two PB DC subpopulations is a high expression of Fc␥RIII (CD16), which is present in M-DC8 ⫹ DC while absent in the other two DC subpopulations. It should be noted that Thomas and Lipsky (20) and Ziegler-Heitbrock et al. (27, 28) have previously described the existence of a minor population of PB mononuclear cells which were CD16 ⫹, HLA-DR ⫹, CD4 ⫹ and displayed a weak reactivity for the CD14 antigen, but it was not associated with a DC origin. Apparently, these CD16 ⫹/CD14 lo cells displayed a relatively low activity as APC in comparison to other peripheral blood DC and were considered a minor subpopulation of monocytes (20, 27, 28). Despite this, in their recent paper, Scha¨kel et al. (26) clearly show that these M-DC8 ⫹/CD16 ⫹/HLA-DR ⫹ cells display a high ability to generate in vitro activation of naive T cells. The aim of the present study is to comparatively analyze the morphological, cytochemical, phenotypical, and functional characteristics of CD16 ⫹/HLA-DR ⫹/ CD14 ⫺/lo cells compared to CD14 ⫹ monocytes and CD16 ⫺ DC. For that purpose, a total of 67 normal adult PB, 3 cord blood (CB), and 10 normal BM samples were analyzed. In each sample an extensive phenotypic characterisation of these cell subsets was performed. In addition, sorting experiments were performed to specifically evaluate the morphological and cytochemical characteristics of these cell subsets. From the functional point of view, our goal was to comparatively explore in these three cell subsets both the Ig/complement-mediated phagocytosis and the oxidative burst activity as well as their ability to produce cytokines. MATERIAL AND METHODS

Samples PB samples from a total of 67 healthy adult subjects from the University Hospital of Salamanca Blood Bank were analyzed. The mean age of these individuals was 31 ⫾ 8 years (range: 19 to 54 years; median: 32 years); of them, 41 (61%) were males and 26 (39%) were females. In addition, 3 CB specimens from healthy fullterm new-borns, as well as 10 BM samples from normal individuals who underwent orthopaedic surgery, with no evidence of systemic disorders were also included in the study. Samples were collected in EDTA anticoagulant and were obtained after informed consent. For the evaluation of phagocytic/oxidative activity and cytokine production by PB DC, heparinized whole blood samples were collected from groups of 5 and 26 of the above-mentioned healthy subjects, respectively.

Immunophenotypic Studies All PB and BM samples were immediately stained (approximately 0.5–1 ⫻ 10 6 cells in 100 ␮l/test) after they were obtained using a stain-and-then-lyse direct immunofluorescence technique, as previously described in detail (22). In all cases, four-color stainings of monoclonal antibodies (MoAb) directly conjugated with fluorescein isothiocyanate (FITC), phycoerythrin (PE), peridin chlorophyll protein (PerCP), and allophycocyanin (APC) were used. In order to specifically identify and enumerate the CD16 ⫹/HLA-DR ⫹ cells, as well as CD14 ⫹ monocytes, a tube containing the following combination of MoAb (all obtained from Becton/ Dickinson (BD), San Jose, CA) was analyzed: CD14FITC (Leu-M3), CD16-PE (Leu-11c), anti-HLA-DRPerCP (L243), and CD4-APC (Leu-3). Further phenotypical analyzes of both cell subsets were performed using a mixture of either FITC- or PE-conjugated lineage specific [CD3 (Leu-4, BD), CD19 (Leu-12, BD), CD56 (C5.9, Imico, Madrid, Spain), and CD14 (Leu-M3, BD)] MoAb combined with HLA-DR-PerCP and CD33-APC (Leu-M9, BD), as described in a previous paper (22). In all CB and BM samples CD34-FITC or -PE (8G12, BD) was added to the cocktail of MoAb. The identification of the different cell populations under study was made as follows: CD16 ⫹/ HLA-DR ⫹ cells were identified by their positivity for the two markers; in addition, they displayed intermediate FSC and SSC characteristics between lymphocytes and monocytes (Fig. 1A), lacked expression of CD3, CD19, and CD56 as well as CD34 —in CB and BM samples— but expressed HLA-DR and intermediate levels of CD33; mature monocytes were identified as being CD16 ⫺/HLADR ⫹/CD33 ⫹⫹/CD14 ⫹ by their typical light scatter pattern (intermediate/high FSC and SSC). Other subsets of DC were identified as previously described in detail (CD3 ⫺/CD19 ⫺/CD56 ⫺/CD14 ⫺/CD34 ⫺/CD16 ⫺/ HLA-DR ⫹/CD4 ⫹) (22). The complete characterization of the immunophenotype of the different cell subsets under study was performed using a large panel of MoAb whose source and specificity are shown in detail in Table 1. In order to improve the sensitivity of the analysis of cell populations present at low frequency, data acquisition was performed in two consecutive steps on a FACScalibur flow cytometer (BD), as described in detail in Ref. (29). First, 2 ⫻ 10 4 events/test, corresponding to all nucleated cells present in the sample, were collected; in a second step, information was stored exclusively for those cells included in a HLADR ⫹⫹ live gate. In this latter step, a minimum of 3 ⫻ 10 5 events from the total cellularity were measured. For data analysis, the Paint-A-Gate PRO software program (BD) was used. Enumeration of the different cell subsets present in each sample was performed by means of calculating their proportion among all nucle-

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TABLE 1 Monoclonal Antibodies Used for the Analysis of Surface and Intracellular Protein Expression Specificity MHC

Adhesion molecules

Costimulatory molecules

Ig Fc receptors

Complement receptors and regulatory proteins

Myeloid-associated antigens

DC-associated antigens T-cell-associated antigens B-cell-associated antigens

NK-cell related antigens Megakaryocyte-related antigens

CD45 antigens

Cytokine receptors

Miscellaneous

1

MoAb conjugates

Clone

Source

HLA-ABC-PE HLA-DP-FITC HLA-DQ-FITC CD11a-FITC CD29-FITC CD54-FITC CD58-PE CD62L CD103-FITC CD106-PE CD138-FITC CD2-PE CD5-PE CD28-PE CD72-FITC CD80-PE CD86-PE CD152-PE CD154-PE CD23-PE CD16-PE CD32-FITC CD64-PE Anti-RFc⑀2-FITCanti-IgE C11b-PE CD11c-PE CD21-FITC CD35-FITC CD88-FITC CD55-PE CD59-PE CD13-PE CD33-PE MPO-PE Lysozyme-FITC CD1a-PE CD83-PE CD7-FITC CD8-PE CD10-PE CD20-PE CD22-PE CD24-PE CD161-PE CD41-FITC CD42b-FITC CD61-FITC CD45-FITC CD45RA-FITC CD45RO-PE CD25-PE CD116-FITC CD117-PE CD122-FITC CD123-PE CD126-PE CD127-PE CD135-PE CD38-PE CD69-PE CD87-PE CD99-FITC AC133-PE

W6.32 HI43 TU169 LFA-1/2,TB-133 4B4 15.2 Anti-LFA-3 Leu-8 B-Ly7 1.G11B1 B-B4 T11-RD1 Leu-1 Leu-28 3F3 Anti-B7 1T2.2 BNI3 TRAP1 Leu-20 Leu-11c AT10 10.1

Cymbus Bioscience 1 Pharmingen 2 Pharmingen 2 CLB 3 Immunotech 4 CLB 3 BD 5 BD 5 Immunoquality 6 Cymbus Bioscience 1 Imico 7 Coulter 8 BD 5 BD 5 Imico 7 BD 5 Pharmingen 2 Pharmingen 2 Dakopatts 9 BD 5 BD 5 Imico 7 Caltag 10 The Binding Site 11 BD 5 BD 5 Dakopatts 9 CLB 3 Serotec 12 Cymbus Bioscience 1 Cymbus Bioscience 1 BD 5 BD 5 Dakopatts 9 Caltag 10 Caltag 10 Caltag 10 BD 5 BD 5 Coulter 8 BD 5 BD 5 Immunotech 4 BD 5 Dakopatts 9 Dakopatts 9 Dakopatts 9 Caltag 10 BD 5 BD 5 BD 5 Pharmingen 2 Immunotech 4 CLB 3 Pharmingen 2 Immunotech 4 Immunotech 4 Immunotech 4 BD 5 BD 5 Pharmingen 2 Pharmingen 2 Myltenyi 13

Cymbus Bioscience, Southampton, UK. Pharmingen, San Diego, CA. 3 CLB, Amsterdam, The Netherlands. 4 Immunotech, Marseille, France. 5 Becton/Dickinson (BD), San Jose, CA. 6 Immunoquality Products, Gro¨ningen, The Netherlands. 7 Imico, Madrid, Spain. 8 Coulter Corp., Miami, FL. 9 Dakopatts A/S, Glostrup, Denmark. 10 Caltag Laboratories, San Francisco, CA. 11 The Binding Site, Birmingham, UK. 12 Serotec, Oxford, UK. 13 Myltenyi, Bergisch Gladbach, Germany. 2

Leu-15 Leu-M5 1F8 E11 W17/1 143-30 MEM43 Leu-M7 Leu-M9 MPO-7 LZ-1 VIT6B HB15 Leu-9 Leu-2a J5-RD1 Leu-16 Leu-14 ALB9 DX12 5B12 AN51 Y2/51 HI30 Leu-18 Leu-45RO 2A3 M5D12 95C3 MIK-B1 9F5 M91 R34.34 SF1.340 Leu-17 Leu-23 VIM5 TU12 AC-133

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ated cells acquired, after excluding cell debris (29). For the quantitation of positivity for each of the markers tested, the mean fluorescence intensity (MFI) obtained as assessed by the mean fluorescence channel (arbitrary relative linear units scaled from 0 to 10,000) was used. Based on the isotype-matched negative controls, those markers which displayed MFI values higher than 5 were considered positive. In order to check the potential day-to-day variability, a CD3-FITC/CD4-PE/ CD8PerCP stained normal PB sample was analyzed in parallel with each set of samples. Functional Analysis of both Phagocytic and Oxidative Burst Cell Activities In five heparinized PB samples from healthy adult subjects, both phagocytic and oxidative burst activities of CD16 ⫹/HLA-DR ⫹ cells, CD14 ⫹ monocytes, and CD16 ⫺ DC were comparatively analyzed. This functional analysis was performed according to the Phagotest and Bursttest kit protocols (Orpegen Pharma, Heidelberg, Germany) for analyzing the Ig/ complement-mediated phagocytic activity and the oxidative burst activity, respectively. In parallel, a control sample was processed (negative control). However, the last step of both Phagotest and Bursttest kits (staining of DNA with propidium iodide) was not performed, and instead, cells were incubated (30 min at 4°C, in the dark) with 10 ␮l of CD33-PE (Leu-M9, BD), HLA-DRPerCP, and [CD14 plus CD3]-APC MoAb in order to stain the nucleated cells for the identification of the different subsets of PB DC and monocytes under analysis. For data acquisition and analysis, a FACScalibur flow cytometer was used following the protocol described above. Evaluation of phagocytic/oxidative activities for each cell population was based on both the measurement of the percentage of phagocyting/oxidating cells (green fluorescence positive cells, using the control sample as reference) and their MFI, the latter correlating with the number of bacteria/oxidation levels per individual cell. Bacteria were excluded from the analysis based on their negativity for HLA-DR. Analysis of Cytokine Production by PB Stimulated DC and Mature Monocytes In a subset of 26 normal adult individuals, cytokine production by PB CD16 ⫹/HLA-DR ⫹ cells, CD14 ⫹ monocytes, and CD16 ⫺ DC was comparatively analyzed. The production of IL1␤, IL6, IL12, TNF␣, and IL8 was investigated at the cytoplasmic level by flow cytometry after stimulation with 100 ng/ml of LPS (from Escherichia coli, serotype 055:B5, Sigma, St. Louis, MO) and 10 ng/ml of human recombinant IFN-␥ (Promega, Madison, WI) (30) for 6 h (37°C, 5% CO 2, 95% humidity and

sterile environment) in the presence of 10 ␮g/ml of brefeldin A (BFA, Sigma), according to a previously described technique (22). An unstimulated sample, also containing BFA and processed in parallel in an identical way, was used in each case as a negative control. In order to analyze the cytokine production by each DC subset, we performed simultaneous stainings for intracytoplasmic cytokines and surface markers. The source and specificities of the monoclonal antibody reagents used to detect cytokines and surface antigens are detailed in Ref. (22). For data acquisition and analysis, a FACScalibur flow cytometer was used following the protocol described above. Evaluation of cytokine production for each cell population was based on both the measurement of the percentage of positive cells and their MFI, after subtracting the MFI of the negative control. Isolation of PB CD16 ⫹/HLA-DR ⫹/CD14 ⫺/dim⫹ Cells and HLA-DR ⫹/CD14 ⫹⫹/CD16 ⫺ Monocytes A total of 500 ⫻ 10 6 nucleated cells were obtained from two buffy-coat samples according to the conventional procedure of the Blood Bank of the University Hospital of Salamanca. Mononuclear cells (MNC) isolated using a Ficoll–Hypaque density gradient centrifugation were washed in PBS (5 min, 540g) and resuspended in PBS containing 0.5% paraformaldehyde. MNC were then stained by direct immunofluorescence with the following MoAb: HLA-DR-FITC/CD16-PE/ CD14-PECy5 (clone TUK4, Caltag Laboratories, San Francisco, CA). Cell-sorting experiments were performed on a FACSvantage flow cytometer (BD). The purity of both the CD16 ⫹/HLA-DR ⫹/CD14 ⫺/lo and the CD16 ⫺/HLA-DR ⫹/CD14 ⫹⫹ sorted cell populations ranged between 85 and 96%, respectively. For the morphological and cytochemical examination under a light microscopy (Olympus, BX40), the sorted cell fractions were placed onto separated slides (10 4– 10 5 cells/slide) using a cytocentrifuge (Shandon, Southern Products, Sewickly, UK). Four slides for each sorted cell subset were stained with May–Gru¨nwald– Giemsa for further morphological analysis. On the remaining slides with purified CD16 ⫹/HLA-DR ⫹ cells or CD14 ⫹ monocytes, cytochemical stainings were performed for the monocytic-associated and monocyticspecific myeloperoxidase (MPO) (following the Sato– Selkiya technique) and ␣-naphthyl acetate esterase (with sodium fluoride inhibition; Sigma Diagnostics) enzymes, respectively. In addition, ultrastructural analysis of both sorted cell subsets was performed using conventional methods and a Jeol 100CX microscope.

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Statistical Analysis Mean values and their standard deviation, as well as range and median, were calculated for each variable using the SPSS software program (SPSS 6.1.2, Inc., Chicago, IL). The statistical significance of the differences observed between groups was assessed using the nonparametric Mann–Whitney U test. P values of less than 0.05 were considered associated with statistically significant differences between groups. RESULTS

Immunophenotypical Identification and Enumeration of CD16 ⫹/HLA-DR ⫹ Cells by Flow Cytometry in Normal Human Adult PB, CB, and BM Samples In all PB samples analyzed, a minor population of CD16 ⫹/HLA-DR ⫹ cells was present, which displayed a typical light scatter pattern (in the region where DC are usually located), with FSC/SSC values higher than lymphocytes and lower than monocytes (Fig. 1A). In addition, these cells displayed a strong HLA-DR expression and they were either CD14 ⫺ or CD14 lo. On a multidimensional basis FSC, SSC, CD14, CD16, HLA-DR, and CD4 expression was simultaneously considered and they were found to cluster in a space which was far away from and not connected to that occupied by the CD14 ⫹⫹ monocytes present in the same sample (Fig. 1B). All other DC present in the samples analyzed were constantly negative for CD16. As shown in Table 2, the overall frequency of the CD16 ⫹/HLA-DR ⫹/CD14 ⫺/lo cell subset in normal adult PB samples was 0.72% ⫾ 0.35% of all nucleated cells, significantly lower (P ⫽ 0.0001) than that of monocytes and higher (P ⫽ 0.0001) than that of the remaining lineage ⫺/HLA-DR ⫹ DC. The mean absolute number of CD16 ⫹/HLA-DR ⫹ cells/␮l in normal adult PB was similar (P ⬎ 0.05) to that found in CB samples (39.9 ⫾ 17.4 vs 42.4 ⫾ 8.7 cells/␮l, respectively) (Table 2). Interestingly, in the BM samples analyzed few CD16 ⫹/ HLA-DR ⫹ cells were detected (median of 0.19%). Morphological and Cytochemical Characterization of Human PB CD16 ⫹/HLA-DR ⫹ Cells As shown in Fig. 2A, when analyzed under a light microscope, most of the purified CD16 ⫹/HLA-DR ⫹ cells sorted by FACS displayed cytoplasmic veils and showed an irregular nucleus, in contrast to FACS sorted pure CD14 ⫹ cells, which showed a typical monocytic morphology (Fig. 2B). Moreover, the sorted CD16 ⫹/HLA-DR ⫹ cells lacked cytoplasmatic MPO and ␣-naphthyl acetate esterase (Figs. 2C and 2E). As ex-

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pected, both enzymes were present in relatively high amounts in the purified CD14 ⫹ monocytes (Figs. 2D and 2F), in which ␣-naphthyl acetate esterase reaction was inhibited by sodium fluoride (Fig. 2H). Figure 3 shows the ultrastructural characteristics of FACS sorted CD16 ⫹/HLA-DR ⫹ cells, which display clear cytoplasmic veils and pinocytic vesicles, without dense azurophilic granules, in contrast to purified mature CD14 ⫹ monocytes. Immunophenotypical Characterization of Normal Human PB CD16 ⫹/HLA-DR ⫹ Cells From the phenotypical point of view, normal human PB CD16 ⫹/HLA-DR ⫹ cells showed significant differences from both CD14 ⫹ monocytes and CD16 ⫺ DC. Accordingly, as shown in Table 3, the most prominent differences between CD16 ⫹/HLA-DR ⫹ cells and CD14 ⫹ monocytes were that the former cells displayed strong expression of Fc␥RIII (CD16) but lacked expression of Fc␥RI (CD64), cytoplasmic MPO, and lysozyme, which were present in CD14 ⫹ monocytes. In addition, in comparison to CD14 ⫹ monocytes, CD16 ⫹/HLA-DR ⫹ cells showed lower levels of the CD11b and CD35 complement receptors and of the CD55 complement regulatory molecule. Expression of CD14, Fc␥RII (CD32), CD33, and CD38 was also lower. By contrast, the expression of the CD11a, CD54, and CD86 adhesion and/or costimulatory molecules was higher, while that of CD29 was lower. Moreover, CD16 ⫹/HLA-DR ⫹ cells showed significantly higher expression of HLA-DR, CD45, CD45RA, and the cytokine receptors CD116 and CD123. CD62L, CD87, and CD45RO antigens were negative in CD16 ⫹/HLA-DR ⫹ cells, while positive in monocytes (Table 3). No significant differences were found between CD16 ⫹/HLA-DR ⫹ cells and CD14 ⫹ monocytes for the remaining antigens explored (Table 3). In Fig. 4, representative histograms of the most prominent phenotypic differences between both cell populations are displayed. Based on the expression of CD16, a clear distinction could be made between the CD16 ⫹/HLA-DR ⫹ cells and the two CD16 ⫺ PB DC subsets (CD33 strong⫹/CD123 dim⫹ and CD123 strong⫹/CD33 dim⫹) (22). Interestingly, for these two latter markers CD16 ⫹/HLA-DR ⫹ cells also displayed unique phenotypic features (CD123 dim⫹/CD33 ⫹). A clear distinction between CD16 ⫹/HLA-DR ⫹ cells and CD16 ⫺ DC could also be found regarding the expression of other surface molecules: part of the CD16 ⫹/ HLA-DR ⫹ cells were dimly positive for CD14; they showed a significantly lower expression of the CD4, CD38, CD126, and HLA-DR antigens, together with a significantly greater reactivity for the CD11a, CD11c, CD32, CD45, CD55, and CD86 surface molecules. In addition, CD16 ⫹/HLA-DR ⫹ cells were CD58 dim⫹ and

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FIG. 1. Light scatter characteristics (FSC/SSC) of CD16 ⫹/HLA-DR ⫹ cells and CD14 ⫹ monocytes (A). On a multidimensional representation (B), CD16 ⫹/HLA-DR ⫹ cells cluster in a space not connected to that occupied by CD14 ⫹ monocytes.

CD88 ⫹ and negative for CD2, CD5, CD22, and CD62L, in contrast to CD16 ⫺ DC (Table 3). Ig/Complement-Mediated Phagocytic and Oxidative Activity of Normal Human PB CD16 ⫹/ HLA-DR ⫹ Cells Table 4 shows the results of the Ig/complement-mediated phagocytic and oxidative burst activities of PB CD16 ⫹/HLA-DR ⫹ cells, compared to that of both mature CD14 ⫹ monocytes and PB CD16 ⫺ DC. Within the monocytic subpopulation, most cells (87.1 ⫾ 5.1%) phagocyted E. coli; moreover, mature monocytes sig-

nificantly showed the highest number of bacteria internalized per cell (MFI of 920.3 ⫾ 258.9) (Table 4). The CD16 ⫹/HLA-DR ⫹ cell subset showed an intermediate position (45.8 ⫾ 11.7% phagocyting cells, with a MFI of 660 ⫾ 122), while CD16 ⫺ DC showed a significantly lower (P ⱕ 0.016) Ig/complement-mediated phagocytic activity, in terms of both percentage of phagocyting cells and number of bacteria per cell (Table 4). Similar results were observed upon analyzing the oxidative burst activity against E.coli (Table 4): CD16 ⫹/HLA-DR ⫹ cells showed intermediate values (62.9 ⫾ 23% of oxidating cells, MFI of 66.8 ⫾ 8.4) between mature monocytes (all mature monocytes

TABLE 2 Frequency of CD16 ⫹/HLA-DR ⫹ Cells, CD16 ⫺ DC, and CD14 ⫹ Monocytes in Human Normal Adult PB, CB, and BM Samples Peripheral blood (n ⫽ 67) CD16 ⫹/HLA-DR ⫹ cells % of NC Cells/␮l* CD16 ⫺ DC % of NC Cells/␮l* CD14 ⫹ monocytes % of NC Cells/␮l*

Cord blood (n ⫽ 3)

Bone marrow (n ⫽ 10)

0.72 ⫾ 0.35 (0.23–1.72) 0.65 a 39.9 ⫾ 17.4 (14.8–88.2) 34.3 a

0.35 ⫾ 0.17 (0.23–0.54) 0.28 d 42.4 ⫾ 8.7 (33–50.2) 43.9 d

0.19 ⫾ 0.15 (0–0.42) 0.19 —

0.30 ⫾ 0.09 (0.17–0.56) 0.27 b 16.8 ⫾ 5.0 (8.8–28.14) 16.1 b

0.13 ⫾ 0.04 (0.08–0.16) 0.15 e 18.7 ⫾ 10.9 (7.6–29.4) 19.6 e

0.18 ⫾ 0.07 (0.11–0.24) 0.20 —

4.27 ⫾ 1.6 (2.62–5.82) 4.37 f 609 ⫾ 437.9 (309.2–1111.6) 406.4 f

2.54 ⫾ 1.02 (1.82–4.03) 2.16 —

5.33 ⫾ 1.42 (2.65–9.5) 5.31 c 307.8 ⫾ 114.2 (129.8–624.8) 283.8 c

Note. Results are expressed as means ⫾ SD (range) median. NC, nucleated cells. * Absolute number of each cell subset is expressed as mean ⫾ SD (range) median cells/␮l. N.D., not detected (sensitivity level: 10 ⫺4). a vs bP ⫽ 0.0001. d vs fP ⫽ 0.04. d vs eP ⫽ 0.05. Significant statistical differences were not found either in the relative distribution of the different DC subsets/total DC or in the absolute number of DC subsets between peripheral blood and cord blood samples.

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FIG. 2. Morphological and cytochemical characteristics of purified CD16 ⫹/HLA-DR ⫹ cells (left) and CD14 ⫹ monocytes (right). A and B show the morphological characteristics of sorted CD16 ⫹/HLA-DR ⫹ cells (A) and CD14 ⫹ monocytes (B) stained with MGG: CD16 ⫹/HLA-DR ⫹ cells display cytoplasmic veils and an irregular nucleus, in contrast to FACS sorted pure CD14 ⫹ cells. The sorted CD16 ⫹/HLA-DR ⫹ cells lack cytoplasmatic MPO (C) and ␣-naphthyl acetate esterase (E), while both enzymes are present in purified CD14 ⫹ monocytes (D and F). G and H show the ␣-naphthyl acetate esterase staining with sodium fluoride inhibition. Original magnification, ⫻1000.

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FIG. 3. Ultrastructural features of purified CD16 ⫹/HLA-DR ⫹ cells and CD14 ⫹ monocytes: Mature monocyte (A) showing an eccentric and indented nucleus, with an abnormal chromatin pattern, displaying apoptotic features. Dense low-size azurophilic granules, most of them located near the Golgi area, can be seen. Sorted CD16 ⫹/HLA-DR ⫹ DC are shown in B, C, and D. Cell depicted in B exhibits an oval nucleus, with apoptotic features, and two cytoplasmic veils resembling lamellipodia (arrowheads). The CD16 ⫹/HLA-DR ⫹ cell depicted in C possesses short cytoplasmic processes, and at high magnification (D), pinocytic vesicles are clearly seen (arrows). Original magnification: ⫻7500 (A), ⫻7500 (B), ⫻6600 (C), ⫻16,000 (D).

were able to develop oxidative burst activity, with a MFI value of 81.6 ⫾ 12.1) and CD16 ⫺ DC (33.7 ⫾ 14.9% of oxidating cells, MFI of 54.5 ⫾ 6.4). Cytokine Production by Normal Human PB CD16 ⫹/ HLA-DR ⫹ Cells Table 5 shows the results for the production of inflammatory cytokines and the IL8 chemokine illustrated for TNF␣ in Fig. 5. As shown in this table, overall PB CD16 ⫹/HLA-DR ⫹ cells showed a pattern of cytokine production similar to that of both CD14 ⫹ monocytes and CD16 ⫺ DC. However, a careful analysis of the results shows that the proportion of CD16 ⫹/ HLA-DR ⫹ cells which produced detectable levels of IL1␤, IL6, IL12, TNF-␣, and IL8 was lower than that of CD14 ⫹ monocytes (Table 5). Furthermore, the mean fluorescence intensity (MFI) of IL1␤ ⫹, IL6 ⫹, IL12 ⫹, TNF␣ ⫹, and IL8 ⫹ cells was lower among CD16 ⫹/HLADR ⫹ cells compared to CD14 ⫹ monocytes, with statistically significant differences for the TNF␣ and IL8 producing cells (P ⫽ 0.05 and P ⫽ 0.0005, respectively) (Table 5). Moreover, lower percentages of IL1␤ ⫹,

IL6 ⫹, IL12 ⫹, TNF␣ ⫹, and IL8 ⫹ cells were found within the CD16 ⫹/HLA-DR ⫹ cell subset than in CD16 ⫺/ CD33 ⫹⫹DC. However, the MFI of CD16 ⫹/HLA-DR ⫹ cells which produced IL1␤, IL6, and TNF␣ was significantly higher than for CD16 ⫺ DC (Table 5). Figure 5 shows representative dot plots displaying the TNF␣ production by CD14 ⫹ monocytes, CD16 ⫹/HLA-DR ⫹ cells and CD16 ⫺/CD33 ⫹⫹ DC. DISCUSSION

Recently, a new subset of DC has been described by Scha¨kel et al. (26) in normal human PB, identified as M-DC8 ⫹/CD16 ⫹/HLA-DR ⫹ cells; this cell subset would represent around one-third of the total CD16 ⫹/HLADR ⫹ present in PB (31). The existence of a minor subset of PB CD16 ⫹/CD14 ⫹lo cells has previously been reported (20, 27, 28), but it was initially ascribed to the monocytic lineage, on the basis of its ability to produce reactive oxygen species and expression of cytoplasmic esterase (20). However, Scha¨kel et al. (26) clearly showed that M-DC8 ⫹/CD16 ⫹ cells functionally behave as APC, since these cells present antigens to naive T

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TABLE 3 Phenotypic Differences between CD16 /HLA-DR ⫹ Cells and both CD14 ⫹ Monocytes and CD16 ⫺ DC ⫹

Antigen

CD16 ⫹/HLA-DR ⫹ cells

CD14 ⫹ monocytes

P value a

CD16 ⫺ DC

P value b

CD14 CD4 HLA-DR CD11a CD29 CD54 CD58 CD62L CD2 CD5 CD86 CD16 CD32 CD64 CD11b CD11c CD35 CD88 CD55 CD33 cy-MPO cy-Lysozyme CD22 CD45 CD45RA CD45RO CD116 CD123 CD126 CD38 CD87

7.7 ⫾ 1.2 (7.6) 97.5 ⫾ 48.5 (89.8) 103.9 ⫾ 50 (97.3) 252 ⫾ 26 (249.9) 54.9 ⫾ 7.9 (54.8) 77.6 ⫾ 23 (79.6) 9.9 ⫾ 3.5 (10.3) 5 ⫾ 4 (3.2) 5 ⫾ 3 (3.5) 1.5 ⫾ 0.3 (1.4) 147.6 ⫾ 48.8 (136) 210.1 ⫾ 108.2 (195.1) 68.6 ⫾ 10 (64.5) 4.0 ⫾ 0.6 (4.2) 9.1 ⫾ 6.11 (6.2) 560.3 ⫾ 77 (594.8) 13.9 ⫾ 3.8 (13.7) 11.5 ⫾ 3.8 (10.8) 72.3 ⫾ 12.5 (68) 73.9 ⫾ 42.2 (62.6) 4.4 ⫾ 1.3 (4.3) 1.5 ⫾ 0.5 (1.6) 1.3 ⫾ 0.8 (1.1) 476 ⫾ 47.7 (477.5) 63.5 ⫾ 23 (60.4) 4.1 ⫾ 0.8 (4.1) 11.7 ⫾ 3.1 (10.6) 142.4 ⫾ 111.6 (109) 8.8 ⫾ 2.6 (9.2) 12.6 ⫾ 5.1 (11.8) 4.7 ⫾ 0.7 (4.8)

348.5 ⫾ 101.1 (345.8) 92.9 ⫾ 13.9 (89.6) 56.9 ⫾ 32.7 (52.2) 156.6 ⫾ 25.5 (155.7) 78.1 ⫾ 6.2 (80) 29.2 ⫾ 14.2 (25.2) 10.9 ⫾ 4.5 (10.7) 26.9 ⫾ 12.9 (23) 6.1 ⫾ 2.6 (6.1) 1.6 ⫾ 0.1 (1.7) 79.4 ⫾ 28.3 (72) 1.5 ⫾ 0.4 (1.5) 97.6 ⫾ 7.5 (97) 19.3 ⫾ 4.5 (21) 624.9 ⫾ 245.7 (604) 474.5 ⫾ 56 (460) 77.4 ⫾ 30.6 (68.5) 12.1 ⫾ 4.2 (11.8) 186.2 ⫾ 61.9 (169.7) 324.7 ⫾ 191.8 (306.5) 279.8 ⫾ 345 (137.7) 29.75 ⫾ 10 (29) 1.6 ⫾ 1.2 (1.1) 242.1 ⫾ 40.4 (238) 4.4 ⫾ 1.3 (4.4) 21.9 ⫾ 6.9 (21.6) 3.4 ⫾ 3.9 (2.1) 60.4 ⫾ 57.5 (47.8) 10.6 ⫾ 7.1 (9.9) 236.2 ⫾ 57.9 (232.9) 11.6 ⫾ 5.5 (11.4)

0.002 N.S. 0.00001 0.002 0.002 0.0004 N.S. 0.0027 N.S. N.S. 0.0001 0.00001 0.001 0.001 0.0002 N.S. 0.0008 N.S. 0.0027 0.0001 0.0001 0.001 N.S. 0.0002 0.0027 0.0027 0.001 0.00001 N.S. 0.0006 0.003

5.0 ⫾ 1.5 (5.0) 190.7 ⫾ 89.8 (177) 251.6 ⫾ 121.1 (213.4) 172.1 ⫾ 45.3 (154.2) 56.3 ⫾ 3.9 (55.5) 73.5 ⫾ 13.5 (73.3) 4.5 ⫾ 2.1 (4.5) 73.1 ⫾ 30.7 (59.6) 171 ⫾ 34.6 (159.9) 53.8 ⫾ 11.9 (53.8) 29.2 ⫾ 10.4 (23.6) 2.1 ⫾ 1.3 (1.7) 32.6 ⫾ 8.6 (30.6) 1.9 ⫾ 0.1 (2.0) 8.8 ⫾ 5.6 (8.2) 325 ⫾ 66.3 (318.2) 11.3 ⫾ 1.9 (11.0) 2.2 ⫾ 1.4 (1.9) 42.7 ⫾ 9.3 (41.8) 195.7 ⫾ 122.5 (176.6) 2.6 ⫾ 0.7 (2.4) 3.2 ⫾ 0.9 (3.3) 32.5 ⫾ 4.6 (32.5) 248.9 ⫾ 40 (300.3) 58.7 ⫾ 21.9 (59.0) 4.7 ⫾ 2.3 (4.7) 9.5 ⫾ 0.5 (9.5) 650.7 ⫾ 143.7 (625.4) 22.7 ⫾ 6.8 (22.1) 290.8 ⫾ 45.6 (278.8) 2.2 ⫾ 0.2 (2.2)

0.05 0.00001 0.00001 0.004 N.S. N.S. 0.003 0.0017 0.0002 0.006 0.0001 0.00001 0.0017 N.S. N.S. 0.0017 N.S. 0.05 0.003 0.00001 N.S. N.S. 0.0002 0.0002 N.S. N.S. N.S. 0.00001 0.0001 0.0002 N.S.

Note. N.S., statistically not significant (P ⬎ 0.05). There was no differential expression between CD16 ⫹/HLA-DR ⫹ cells and CD14 ⫹ monocytes for the following antigens, both cell subsets being positive for CD4, CD11c, CD13, CD58, CD88, HLA-ABC, HLA-DP, and HLA-DQ and negative for CD1a, CD5, CD7, CD8, CD20, CD21, CD22, CD23, CD24, CD25, CD28, CD41, CD42b, CD59, CD61, CD69, CD72, CD80, CD83, CD99, CD103, CD106, CD117, CD122, CD126, CD127, CD135, CD138, CD152, CD154, CD161, AC133, and IgE. The CD16 ⫹/HLA-DR ⫹ cell subset and CD16 ⫺ DC showed variable reactivity for the CD11b, CD21, CD29, CD35, CD54, CD99, CD116, CD135, HLA-DP, HLA-DQ, and HLA-ABC antigens, but with no differences among them; all of them were constantly negative for the CD1a, CD7, CD8, CD10, CD20, CD21, CD23, CD24, CD25, CD28, CD41, CD42b, CD45RO, CD59, CD61, CD64, CD69, CD72, CD80, CD83, CD87, CD103, CD106, CD117, CD122, CD127, CD138, CD152, CD154, CD161, AC133, lysozyme, and MPO antigens. Results are expressed as means ⫾ SD (median) of MFI (mean fluorescence intensity). a P value obtained upon comparing CD16 ⫹/HLA-DR ⫹ cells versus CD14 ⫹ monocytes. b P value obtained upon comparing CD16 ⫹/HLA-DR ⫹ cells versus CD16 ⫺ DC.

cells and induce CD8 ⫹ T cells to become alloantigenspecific cytotoxic cells more efficiently than monocytes. However, at present the information on the morphologic, cytochemical, phenotypical, and functional properties of CD16 ⫹/HLA-DR ⫹ PB cells is still scanty. In order to gain further insights into the characteristics of CD16 ⫹/HLA-DR ⫹ PB cells, in the present study we have performed a comprehensive analysis of their characteristics compared to those of normal PB CD14 ⫹ monocytes and CD16 ⫺ DC. In addition, it was our aim to explore the existence of these cells in other samples apart from PB from adults, such as in normal human CB and BM specimens. Interestingly, our results show that, although present in normal CB at similar levels

to those detected in normal human adult PB, CD16 ⫹/ HLA-DR ⫹ cells are less represented in normal BM samples. From the morphological point of view, most of the freshly purified PB CD16 ⫹/HLA-DR ⫹ cells displayed cytoplasmic veils, similar to the “dendritic” processes observed in some CD16 ⫺ DC (22), as well as a more irregular nucleus, compared to sorted mature monocytes. These differences were more evident when examined by electron microscopy. In this sense, freshly purified PB CD16 ⫹/HLA-DR ⫹ cells displayed ultrastructural features similar to those observed in DC, such as cytoplasmic veils and pinocytic vesicles. Moreover, they lacked dense azurophilic granules, which were characteristically present in purified PB CD14 ⫹

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FIG. 4. Illustrative histograms of the most prominent phenotypic differences between the CD16 ⫹/HLA-DR ⫹ cells (shown in black) and CD14 ⫹ monocytes (displayed in gray).

monocytes. The cytochemical findings would also support that CD16 ⫹/HLA-DR ⫹ cells and monocytes belong to different cell subtypes, since CD16 ⫹/HLA-DR ⫹ cells do not show expression of MPO, ␣-naphthyl acetate esterase, and lysozyme. These cytoplasmic enzymes are characteristically present in cells from the monocytic lineage from the earliest stages of monocytic differentiation (32) to macrophages, and the presence of ␣-naphthyl acetate esterase, which is inactivated by sodium fluoride, is classically considered a hallmark of the monocytic lineage (33). However, it should be noted

that previous reports (34 –36) have shown that CD14 lo PB cells (corresponding to a subset of CD16 ⫹/HLA –DR ⫹ cells) might express low levels of peroxidase and nonspecific esterase. Such apparent discrepancies might be related to technical issues including the type and sensitivity of the methods used to measure these enzymes and the exact fraction of PB CD16 ⫹/HLA DR ⫹ cells analyzed (CD14 lo versus CD14 -/lo) in our study and previous ones (34 –36). In this sense, CD14 lo/CD16 ⫹/ HLA-DR ⫹ PB cells could also represent a monocytic cell displaying specific maturation into CD14 ⫺/CD16 ⫹/

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TABLE 4 Ig/Complement-Mediated Phagocytic and Oxidative Burst Activities of CD16 ⫹/HLA-DR ⫹ Cells Compared to Mature CD14 ⫹ Monocytes and CD16 ⫺ DC Present in Normal Human PB (n ⫽ 5)

Ig/complement-mediated phagocytic activity % positive cells MFI of positive cells Oxidative burst activity % positive cells MFI of positive cells

CD16 ⫹/HLA-DR ⫹ cells

CD14 ⫹ monocytes

P value a

CD16 ⫺ DC

P value b

45.8 ⫾ 11.7 (29–60) 48.5 660 ⫾ 122 (489–791) 715.2

87.1 ⫾ 5.1 (82–95) 86 920.3 ⫾ 258.9 (680–1317) 849

0.009 N.S.

27.6 ⫾ 6.1 (22–38) 27 317.5 ⫾ 70.6 (241–413) 290.5

0.016 0.009

62.9 ⫾ 23 (35–87) 60.3 66.8 ⫾ 8.4 (58.5–79) 65.2

100 ⫾ 0 (100–100) 100 81.6 ⫾ 12.1 (63–93) 80.2

0.005 0.047

33.7 ⫾ 14.9 (17–54) 30.1 54.5 ⫾ 6.4 (50–59) 54.5

0.047 N.S.

Note. MFI, mean fluorescence intensity. N.S.: statistically not significant (P ⬎ 0.05). Results are expressed as means ⫾ SD (range) median. a P value obtained upon comparing CD16 ⫹/HLA-DR ⫹ cells versus CD14 ⫹ monocytes. b P value obtained upon comparing CD16 ⫹/HLA-DR ⫹ cells versus CD16 ⫺ DC.

HLA-DR ⫹⫹ antigen presenting cells with intermediate features between a PB monocyte and dendritic cell (36). In accordance with the findings of Scha¨kel et al. (26), we have observed that CD16 ⫹/HLA-DR ⫹ cells have a surface phenotype different from CD14 ⫹ monocytes. In contrast to PB monocytes, fresh PB CD16 ⫹/HLA-DR ⫹ cells lacked the CD64 Fc␥ receptor, showed lower levels of CD32, and expressed higher amounts of CD16. It is well established that the capture of antigens by surface receptors such as Fc␥R allows antigen processing via receptor-mediated endocytosis (37). Since both cell subsets display a distinct pattern of expression of Fc␥R, their role in phagocytosis would be different. Accordingly, the presence of CD16 (low-affinity IgG receptor) and the absence of CD64 (high-affinity IgG receptor) on CD16 ⫹/HLA-DR ⫹ cells would suggest that these cells could play an important role when antigen

concentration is high. In comparison to CD14 ⫹ monocytes, CD16 ⫹/HLA-DR ⫹ cells also displayed a different expression for other antigens, including HLA-DR, CD45RA, CD45RO, complement receptors, and complement regulatory surface proteins, adhesion and costimulatory molecules, and cytokine receptors. The expression of CD16 also makes possible the distinction between CD16 ⫹/HLA-DR ⫹ cells and CD16 ⫺ DC. Up till Scha¨kel et al.’s (26) report, no expression of Fc␥RIII had been described on fresh PB human DC, despite the fact that, in our experience, CD16 ⫹/HLADR ⫹ cells are more frequent than CD16 ⫺ DC in human adult PB and CB samples. This could be due to the rapid down-regulation of CD16 observed after manipulation of cells, such as in vitro culture or Ficoll density gradient centrifugation (26). Moreover, CD16 has frequently been included in the cocktail of monoclonal antibodies used for the immunomagnetic selection of

TABLE 5 Cytokine Production by CD16 ⫹/HLA-DR ⫹ Cells Compared to Mature CD14 ⫹ Monocytes and CD33 strong⫹/CD16 ⫺ DC Present in Normal Adult Human PB (n ⫽ 26) CD16 ⫹ /HLA-DR ⫹ cells IL1␤ % of MFI IL6 % of MFI IL12 % of MFI TNF␣ % of MFI IL8 % of MFI

CD14 ⫹ monocytes

P value a

CD33 strong⫹ /CD16 ⫺ DC

P value b

97.4 ⫾ 2 (92–99) 98 61.5 ⫾ 34.7 (25–158) 51

0.00001 N.S.

88.7 ⫾ 9.2 (61–100) 91 23.5 ⫾ 9.3 (15.2–52.7) 21.6

0.00001 0.0002

⫹ve cells of ⫹ve cells

56.4 ⫾ 15 (25–75) 57 48.4 ⫾ 24.3 (23.3–109.5) 41.3

⫹ve cells of ⫹ve cells

46.8 ⫾ 18.2 (22–83) 43 386.8 ⫾ 272.3 (99–1063) 351.5

91.8 ⫾ 13.6 (59–99) 98 533.9 ⫾ 328.7 (149–1254) 541

0.00001 N.S.

82.5 ⫾ 9.8 (68–97) 83 147.5 ⫾ 83.2 (32.8–361) 149

0.00001 0.009

⫹ve cells of ⫹ve cells

36.7 ⫾ 14 (15–59) 38.5 240.9 ⫾ 199.1 (42.7–675) 150.2

66.5 ⫾ 14.3 (40–98) 66 283.4 ⫾ 278.8 (99–1008) 220.3

0.0002 N.S.

83.2 ⫾ 13.2 (57–100) 84.8 285.8 ⫾ 212 (80.9–784) 232.8

0.00001 N.S.

⫹ve cells of ⫹ve cells

50.3 ⫾ 17.3 (19–79) 54 1247 ⫾ 708 (309–289) 889

92.3 ⫾ 13.9 (57–100) 98 1777 ⫾ 1337 (409–4434) 1493

0.00001 0.05

83.8 ⫾ 16.3 (47–100) 89.5 531.9 ⫾ 372 (149.5–1558) 413

0.0001 0.01

⫹ve cells of ⫹ve cells

56.7 ⫾ 18.7 (23–80) 59.5 7.4 ⫾ 4.4 (5–17.2) 5.6

81.3 ⫾ 12.5 (53–92) 86.5 15.8 ⫾ 7.6 (9.1–33.8) 12.6

0.0004 0.0005

88.4 ⫾ 8.2 (76–100) 88 12 ⫾ 7.4 (6.3–32) 9.9

0.00001 0.002

Note. MFI, mean fluorescence intensity. N.S., statistically, not significant (P ⬎ 0.05). Results are expressed as means ⫾ SD (range) median. a P value: comparison between CD16 ⫹/HLA-DR ⫹ cells and mature monocytes. b P value: comparison between CD16 ⫹/HLA-DR ⫹ cells and CD16 ⫺ DC.

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FIG. 5. TNF␣ production by CD14 ⫹ monocytes, CD16 ⫹/HLA-DR ⫹ cells, and CD16 ⫺/CD33 ⫹⫹ DC. (Right) Representative dot plots of TNF␣ production by PB CD14 ⫹ monocytes (dot plot B), CD16 ⫹/HLA-DR ⫹ cells (gray dots in plot D), and CD16 ⫺/CD33 ⫹⫹ DC (black dots in plot D) after stimulation for 6 h in the presence of brefeldin A with LPS plus IFN-␥. (Left) TNF␣ production by PB CD14 ⫹ monocytes (dot plot A), CD16 ⫹/HLA-DR ⫹ cells (gray dots in plot C), and CD16 ⫺/CD33 ⫹⫹ DC (black dots in plot C) from the same PB sample in the absence of LPS plus IFN-␥ stimuli (negative control).

DC, in order to exclude NK cells (21, 25), so that CD16 ⫹/HLA-DR ⫹ cells have usually not been considered within the analysis of DC. In addition, CD16 ⫹/ HLA-DR ⫹ cells and CD16 ⫺ DC are different in their reactivity for other markers. Accordingly, CD16 ⫹/HLADR ⫹ cells showed dim positivity for CD14, higher expression of both Ig- and complement-receptors, and lower reactivity for HLA-DR, adhesion, and costimulatory molecules (with the exception of CD86), compared to CD16 ⫺ DC. Based on the unique pattern of expression of both Fc␥ and complement receptor, in the present paper we have also explored the Ig/complement-mediated phagocytic/oxidative activity of CD16 ⫹/ HLA-DR ⫹ cells. Our results show that these cells displayed a pronounced phagocytic/oxidative activity, although the percentage of phagocyting/oxidating cells, as well as both the amount of bacteria internalised and the oxidative activity per cell, was lower than that observed for the CD14 ⫹ monocytes present in the same sample. Since CD16 ⫹/HLA-DR ⫹ cells lacked the expression of the MPO, ␣-naphthyl acetate esterase, and lysozyme cytoplasmic enzymes, which are involved in the processing of phagocytosed particles in monocytes,

it could be speculated that CD16 ⫹/HLA-DR ⫹ cells use different pathways for processing internalized particles, as yet not fully understood. Upon comparing the Ig/complement-mediated phagocytic/oxidative activity of CD16 ⫹/HLA-DR ⫹ cells and CD16 ⫺ DC, we found that the former cells displayed a higher phagocytic/oxidative activity, as reflected by a higher percentage of phagocytosing/oxidating cells, greater numbers of internalized bacteria/cell, and a higher oxidative activity per cell. Several reports have shown that fresh PB CD16 ⫺ DC efficiently uptake antigens (38 – 40). However, we have found that Ig/complement-mediated phagocytic/oxidative activity can only be seen (at relatively low levels) among the CD33 ⫹⫹⫹/CD16 ⫺ DC subpopulation, whereas the CD123 ⫹⫹⫹/CD16 ⫺ DC subset has a very limited phagocytic/oxidative activity (data not shown). These results, together with the observation of abundant pinocytic vesicles in CD16 ⫹/HLA-DR ⫹ cells, would suggest that, despite their efficient uptake of antigens by pinocytosis, they may also use Ig/complement-mediated phagocytosis for further antigen processing.

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Finally, we have investigated by flow cytometry the production at the cytoplasmic level of the IL1␤, IL6, IL12, and TNF␣ inflammatory cytokines and the IL8 chemokine. CD16 ⫹/HLA-DR ⫹ cells showed a pattern of cytokine production typical of inflammatory cells, since significant amounts of IL1␤, IL6, IL12, TNF␣, and IL8 were detected in their cytoplasm. These results suggest that CD16 ⫹/HLA-DR ⫹ cells do not represent immature monocytic-lineage cells, since they are able to produce significant amounts of cytokines after short-term in vitro stimulation, which is typical of more differentiated cells. Although, like CD14 ⫹ monocytes and CD33 ⫹⫹⫹/CD16 ⫺ DC, CD16 ⫹/HLA-DR ⫹ cells produced inflammatory cytokines, the percentage of cytokinepositive cells, as well as the amount of cytokine/cell, was different in CD16 ⫹/HLA-DR ⫹ cells. Thus, the percentage of cytokine-producing cells and the amount of cytokine/cell was lower in CD16 ⫹/HLA-DR ⫹ cells, compared to CD14 ⫹ monocytes. Upon comparing cytokine production by CD16 ⫹/HLA-DR ⫹ cells with that of CD33 ⫹⫹⫹/CD16 ⫺ DC, we found that, while the percentage of cytokine-producing cells is higher in the latter cell subset, the amount of cytokine/cell is, in general, greater for the CD16 ⫹/HLA-DR ⫹ population. These results suggest that both subsets are differentiated antigen-presenting cells and could play a different role in priming T cell response. By contrast, as previously reported by our group, the CD123 ⫹⫹⫹/CD16 ⫺ DC did not produce any of the cytokines explored (22), suggesting that it could either correspond to a more immature DC subpopulation or follow different activation pathways, as recently suggested by Rissoan et al. (24). In summary, our results show that CD16 ⫹/HLA-DR ⫹ cells clearly display different morphologic, cytochemical, immunophenotypical, and functional characteristics compared to mature monocytes. Interestingly, these cells are more frequent than other DC in normal human adult PB and CB samples, while they are less represented in normal BM. In addition, our results show that they display different phenotypic and functional characteristics from other DC. Further studies are necessary in order to establish the precise origin of these CD16 ⫹/HLA-DR ⫹ PB cells and their exact role in the immune response. ACKNOWLEDGMENTS This work has been partially supported by grants (PM 97-0161 and 1FD97-2189) from the Direccio´n General de Ensen˜anza Superior (DGES) from the Ministerio de Educacio´n y Cultura (Madrid, Spain) and a grant from the Fondo de Investigaciones Sanitarias de la Seguridad Social (FIS) del Ministerio de Sanidad y Consumo (FIS 99/1239). C. Bueno is supported by a grant from the Direccio´n General de Universidades e Investigacio´n (Consejerı´a de Educacio´n y Cultura, Junta de Castilla y Leo´n; Valladolid, Spain). M. L. Sa´nchez and J. M. Vaquero are supported by a grant from Fondos Feder (1FD97-0451) from the DGES, Madrid, Spain.

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Received January 31, 2001; accepted with revision May 22, 2001; published online July 25, 2001