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Identification of CD68⁺lin^- Peripheral Blood Cells with Dendritic Precursor Characteristics¹

Herbert Strobl^2,*, Clemens Scheinecker^*,, Elisabeth Riedl^*, Bettina Csmarits, Concha Bello-Fernandez^*, Winfried F. Pickl, Otto Majdic and Walter Knapp^*,

^* Institute of Immunology, Vienna International Research Cooperation Center, Novartis Forschungsinstitut; Institute of Immunology; and Department of Internal Medicine III, Division of Rheumatology, University of Vienna, Vienna, Austria

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of CD68 (macrosialin) in the absence of surface and lysosomal lineage marker molecules is a characteristic feature of T zone-associated plasmacytoid monocytes, which were recently shown to represent precursors of dendritic cells (DC). We demonstrate here a minor population of strongly CD68-positive (CD68^bright) blood cells that lack all analyzed myeloid surface (CD14^-, CD33^-, CD13^-, CD11b^-, CD11c^-) and lysosomal (myeloperoxidase, MPO^- and lysozyme, LZ^-) marker molecules (0.4 ± 2% of the total mononuclear cells). These CD68^bright, lineage marker-negative (lin^-) cells can be induced to proliferate in the presence of IL-3. They do not acquire myeloid features even upon stimulation with granulocyte-macrophage CSF plus IL-1, IL-3, and IL-6. Instead, these cells develop typical DC characteristics upon culture. Furthermore, these CD68^brightlin^- DC precursors acquire mature DC characteristics (CD86⁺, CD83⁺, CD54^bright) upon stimulation with CD40 ligand plus IL-3. A second subset of DC precursor-like blood cells was found to weakly express CD68 (0.3 ± 0.2% of the total mononuclear cells) and to coexpress several myeloid lineage associated molecules (LZ⁺, CD11c⁺, CD33⁺, CD13⁺). Cells of this second subset resemble both previously described myeloid-related peripheral blood DC and germinal center DC. Analysis of peripheral blood leukocytes for CD68 thus revealed the existence of two cell subsets that phenotypically resemble lymphoid tissue-associated DC. The unique phenotype CD68^brightlin^- is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68^brightlin^- blood leukocytes also functionally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68^brightlin^- blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The recently cloned lysosomal/endosomal molecule CD68/macrosialin (1, 2, 3) represents a type I integral membrane protein with significant sequence homology of the membrane proximal and cytoplasmic domains to a family of lysosomal/plasma membrane shuttling proteins known as the lamp/lgp family (4, 5, 6, 7, 8). CD68/macrosialin is heavily O-glycosylated and is classified as a mucin-like membrane protein. Oxidized low density lipoprotein has recently been identified as a putative ligand of CD68 (9, 10).

CD68 represents a classical and widely used immunohistologic marker molecule for cells of the monocyte/macrophage and dendritic cell (DC)³ system (11, 12, 13, 14, 15, 16, 17, 18). Flow cytometric analysis of CD68 expression was only recently introduced by us and allowed us to identify a small population of about 2% of PBMC that express intracellular CD68, but lack the monocyte marker CD14 and are negative for the lineage-associated marker molecules CD3 (T cells), CD19 (B cells), and CD16 (NK cells/neutrophils) (19). This absence of lineage-associated marker molecules together with the expression of the mucin-like lysosomal membrane protein CD68 reminded us of DC found in lymph nodes and skin (12, 13, 14).

Minute numbers of cells with DC precursor characteristics have been demonstrated previously in MNC fractions of peripheral blood (20, 21, 22, 23, 24, 25, 26, 27, 28, 29). They were described as HLA-DR⁺CD4⁺lin^- leukocytes that comprise at least two phenotypically and functionally differing subsets. One subset coexpresses the granulomonocyte-associated molecules CD33, CD13, and CD11c. Phenotypically very similar cells have very recently been demonstrated in germinal centers of lymphoid tissues (30). In contrast, the other subset is negatively defined by the absence of or very weak expression of CD13, CD33, and CD11c and phenotypically resembles a recently identified DC precursor population in T cell areas (31) of lymphoid tissues. Thus, DC populations in lymphoid tissues might be repopulated by the observed peripheral blood DC precursors. HLA-DR⁺lin^- peripheral blood cells also include various other important leukocyte progenitor/precursor populations, such as CD34⁺ circulating hemopoietic progenitor cells or putative CD4⁺ lymphoid precursors, which are difficult to identify using current procedures (32).

Given the enormous functional importance of DC in the induction and regulation of immune responses and our limited knowledge of in vivo DC development, differentiation, and migration, we considered it of substantial interest to analyze the observed CD68⁺lin^- candidate DC precursor population in more detail. CD68⁺ peripheral blood leukocytes indeed include two DC precursor subsets, and both resemble DC populations in lymphoid tissues. CD68^brightlin^- cells phenotypically and functionally resemble plasmacytoid monocytes. The second CD68^dim DC precursor-like population resembles myeloid-related cells and germinal center DC.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Antibodies

Murine mAbs of the following specificities were used in our study: CD68 (clone Ki-M7, IgG1), human myeloperoxidase (clone H-43-5, IgG1), human lysozyme (clone LZ-1, IgG1), CD3 (clone UCHT1, IgG1), and CD14 (clone MEM18, IgG1) obtained from An der Grub (Kaumberg, Austria); CD19 (clone HD37, IgG1) provided by Dr. B. Dörken (Berlin, Germany); HLA-DR (clone L243, IgG2a), CD34 (clone HPCA2, IgG1) and CD11c (clone Leu 12, IgG1) obtained from Becton Dickinson (San Jose, CA); CD45RA (clone MEM93, IgG1) provided by Dr. W. Horejsi (Prague, Czech Republic); EMBP (clone AHE-2, IgG1) provided by Dr. K.M. Skubitz (Minneapolis, MN); CD33 (clone WM-54, IgG1) obtained from Dako (Glostrup, Denmark); Ki-67 (clone MIB-1, IgG1) obtained from Dianova (Hamburg, Germany); CD16 (clone 3G8, IgG1) obtained from Caltag (San Francisco, CA); and CD13 (clone My7, IgG1) obtained from Coulter (Hialeah, FL). Abs specific for CD3 (clone VIT3b, IgG1), CD4 (clone VIT4, IgG2a), CD5 (clone CD5-5D7, IgG1), CD11b (clone LM-2, IgG1), and CD7 (clone CD7-6B7, IgG2a) were produced in our laboratory. CD54 (clone HA58) and CD86 (clone IT2.2.) were obtained from PharMingen (San Diego, CA). CD83 (clone HB15a) was obtained from Immunotech (Marseille, France).

Immunofluorescence staining

Membrane staining. For membrane staining, 50 µl of isolated MNC (10⁷/ml) were incubated for 15 min at 0 to 4°C with 20 µl of conjugated mAb. Triple stainings were performed by first incubating cells with a mixture of biotinylated Abs specific for the lineage molecules CD14, CD3, CD19, and CD16 together with phycoerythrin (PE)-labeled Abs, then washed twice and subsequently incubated with the second step reagent streptavidin PerCP (Becton Dickinson). Afterward, cells were submitted to intracellular staining.

Intracellular staining. For suspension stainings of intracellular Ags we used the commercially available reagent combination Fix&Perm from An der Grub and followed the proposed procedure. In short, cells were first fixed for 15 min at room temperature (50 µl of cells plus 100 µl of formaldehyde-based fixation medium). After one washing with PBS, pH 7.2, cells were resuspended in 50 µl of PBS and mixed with 100 µl of permeabilization medium plus 20 µl of fluorochrome-labeled Ab. After a further incubation for 15 min at room temperature, cells were washed again and analyzed.

Indirect immunofluorescence stainings for the proliferation-associated nuclear Ag Ki-67 were performed on cytocentrifuged cells using a Cytospin-2 centrifuge (Shandon Southern Products, Astmoor, U.K.). Fixation and permeabilization were performed as described previously (33).

Flow cytometry

Flow cytometric analyses were performed with a FACScan flow cytometer (Becton Dickinson) equipped with a single laser emitting at 488 nm. For analysis of CD68 expression in lineage Ag-negative MNC, data for at least 60,000 cells were acquired and stored in list-mode files. FACS sortings were performed with a FACS Vantage flow cytometer (Becton Dickinson).

Cells

Peripheral blood samples were obtained from healthy volunteers and immediately processed. MNC were isolated by flotation on Ficoll/Hypaque (Pharmacia, Uppsala, Sweden). Lineage marker-negative (lin^-) MNC were obtained by first removing rosette-forming cells with neuraminidase-treated sheep erythrocytes and then by immunomagnetic depletion, as previously described (34), of all cells reactive to a mixture of CD14 (clone MEM18), CD11b (clone LM-2), CD3 (clone VIT3b), and CD19 (clone HD-37) Abs. CD4⁺lin^- cells were obtained by FACS sorting of cells double stained for CD4 (FITC; clone VIT4) and lineage molecules (PE; CD14, CD3, CD19, CD16) using a FACS Vantage flow cytometer (Becton Dickinson). For subsorting on the basis of CD45RA expression, purified CD4⁺lin^- cells were stained for CD45RA (PE) and FACS sorted into CD4⁺lin^-CD45RA^bright and CD4⁺lin^-CD45RA^-/dim cells. The purity of all cell populations obtained by sorting was determined by reanalysis by FACS and was >95%. Monocyte-derived DC (mdDC) were generated in the presence of GM-CSF plus IL-4 with or without TNF- as described previously (35, 36).

Cultivation of MNC subsets

Purified subsets of lin^- MNC were cultured as described previously (37) for up to 7 days at 37°C in a humidified atmosphere and in the presence of 5% CO₂ in RPMI 1640 medium supplemented with L-glutamine (2.5 mM), penicillin (125 IU/ml), streptomycin (125 µg/ml), and human plasma (10%) and in the presence or the absence of the following recombinant human (rh) cytokines: rhGM-CSF (100 ng/ml; Novartis, Basel, Switzerland), rhIL-1 (100 U/ml; Novartis), rhIL-6 (10 ng/ml; Novartis) and rhIL-3 (100 U/ml; Behring, Marburg, Germany), trimeric human CD40 ligand (CD40L) fusion protein (200 ng/ml; provided by Dr. S. D. Lyman, Immunex, Seattle, WA).

Morphologic analysis

Both freshly isolated and cultured cells were morphologically analyzed in culture vessels using phase contrast microscopy or were cytocentrifuged on microscope slides (2 x 10⁴ cells/slide) using a Cytospin-2 centrifuge (Shandon, Pittsburgh, PA), stained with May-Grunwald-Giemsa, and then analyzed by light microscopy.

Thymidine incorporation assay

Thymidine incorporation by cultures of purified subsets of lin^- MNC (5 x 10³ cells/well) was measured after a total culture period of 72 h in the presence or the absence of the above-mentioned cytokines. [³H]thymidine (Amersham, Aylesbury, U.K.) was added to cultures 16 h before harvesting. Incorporated radioactivity was measured using a Top-Count microscintillation counter (Packard, Meriden, CT).

Mixed leukocyte reaction

Graded numbers of irradiated (30 Gy; ¹³⁷Cs source) stimulator cells (subsets of lin^- MNC) were added to constant numbers (5 x 10⁴/well) of purified (>98%) allogeneic T cells in round-bottom 96-well tissue culture plates (Costar, Cambridge, MA). Stimulation of responding T cells was monitored by measuring [³H]thymidine incorporation on day 5 of culture as described above.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Identification of CD68⁺lin^- MNC

Double staining of PBMC for intracellular CD68 (x-axis) vs lineage marker molecules (mixture of CD14, CD3, CD19, and CD16 Ab conjugates; y-axis) clearly resolved a small population of CD68⁺lin^- cells (Fig. 1). The size of this population varied between 1.5 and 2.6% (mean, 1.9 ± 0.5%; n = 5) of all MNC.

View larger version (29K): [in this window] [in a new window]   FIGURE 1. Intracellular CD68 expression in lin- MNC. Freshly isolated MNC were stained with a mixture of biotinylated Abs specific for the lineage molecules CD14, CD3, CD19, and CD16 (LIN) followed by streptavidin PerCP. After subsequent fixation and permeabilization, cells were stained for intracellular CD68 expression (FITC). Dot plots show CD68 (x-axis) against lineage molecule expression (LIN, y-axis) or negative control of ungated MNC. Sixty thousand cells were analyzed.

To further characterize this small CD68⁺lin^- cell population, we performed triple staining experiments. For this purpose, one fluorochrome (PerCP) was used for the combined exclusion of the lineage-associated molecules CD3, CD19, CD16, and CD14 (as shown in Fig. 1), allowing the use of FITC and PE to analyze lin^- cells for their expression of intracellular CD68 (FITC) and a panel of informative marker molecules (PE). Figure 2 shows a representative phenotypic analysis of such gated CD68⁺lin^- cells. In total, five individual samples were tested.

View larger version (67K): [in this window] [in a new window]   FIGURE 2. Phenotypic analysis of CD68+lin- normal adult PBMC. Freshly isolated MNC were combined stained for lineage molecules (CD14, CD3, CD19, and CD16; PerCP), intracellular CD68 (FITC), and several informative molecules (PE). Lin- cells were gated according to the marker settings shown in Figure 1. Gated lin- cells are shown. Diagrams represent analyses for CD68 (FITC; x-axes) vs various marker molecules (PE; y-axes). Fluorescence distributions are representative of five experiments. Markers were set according to isotype-matched negative control stainings. At least 60,000 cells were analyzed.

As shown in Figure 2, CD68⁺lin^- cells contain three distinct populations in terms of HLA-DR and CD68 expression. Two populations, clearly distinct in their CD68 expression density, coexpress HLA-DR. The strongly CD68-positive (termed the CD68^brightlin^- subset) and the second subset with clearly lower CD68 expression density (termed the CD68^dimlin^- subset) also differ in their HLA-DR expression intensity. CD68^brightlin^- cells show considerably weaker HLA-DR staining than CD68^dimlin^- cells (Fig. 2). The CD68^brightlin^- and CD68^dimlin^- subsets represent, on the average (n = 5), 0.4 ± 0.2 and 0.3 ± 0.2%, respectively, of adult blood MNC (Table I). The third CD68⁺ subset clearly distinguishable in this staining profile differs in two respects from the other two subsets. It lacks HLA-DR and has a CD68 staining intensity that lies between those of the two CD68⁺/HLA-DR⁺ populations. This subset represents 1.2 ± 0.3% of all MNC.

The three above-described subsets are also heterogeneous in terms of their CD4 expression pattern. The CD68^brightlin^- subset is strongly positive for CD4 (Fig. 2). The CD68^dimlin^- subset also expresses CD4, but the intensity is slightly lower. CD68⁺lin^- cells with intermediate CD68 staining intensity lack CD4 expression. The proportions of CD68⁺lin^- cells coexpressing CD4 and HLA-DR are virtually identical.

CD45RA molecule

In all five experiments the CD68^brightlin^- subset was found to strongly express CD45RA, whereas the CD68^dimlin^- subset was CD45RA negative to only weakly positive.

CD33 and CD13 molecules

Similar expression patterns were observed for the two GM-associated marker molecules CD33 and CD13. The CD68^brightlin^- subset consistently lacks CD33 and CD13; the CD68^dimlin^- and the CD68 intermediate density subsets are positive for both molecules. The highest CD33 expression density was observed in all experiments for the CD68^dimlin^- subset.

CD11b and CD11c molecules

The ß₂ integrin molecule CD11b is absent from both, CD68^brightlin^- and CD68^dimlin^- subsets. Cells with intermediate CD68 density coexpress CD11b. A somewhat different staining pattern was found for CD11c. The CD68^brightlin^- subset lacks CD11c, whereas the CD68^dimlin^- subset is strongly CD11c positive. Cells with intermediate CD68 expression density are CD11c weakly positive to negative.

CD5 and CD7 molecules

Within the population of CD68⁺lin^- cells, only CD68^dimlin^- cells coexpress CD5. The pan T/NK cell marker molecule CD7 is absent from CD68⁺lin^- cells.

CD34 molecule

CD34 expression was, in all five experiments, restricted to CD68^- cells.

Lysosomal protein expression

Virtually all CD68⁺lin^- cells lack the highly selective pan-granulomonocytic lysosomal marker molecule MPO (38). The intracellular marker molecule of basophils and eosinophils, eosinophil major basic protein (39) is expressed, but is restricted to CD68⁺Lin^- cells with intermediate CD68 density.

Together these data show that the two identified subsets CD68^brightlin^- and CD68^dimlin^- share phenotypic characteristics with DC.

Purification of CD68^brightlin^- and CD68^dimlin^- subsets

To further investigate the nature of the CD68^brightlin^- and CD68^dimlin^- subsets we purified these two populations. Because the detection of intracellular CD68 requires fixation and precludes sorting of viable cells, an alternative strategy had to be followed for purification of viable CD68^brightlin^- and CD68^dimlin^- cells. The best way appeared to be FACS sorting of lin^- MNC into CD4⁺CD45RA^bright and CD4⁺CD45RA^dim/- cell fractions. This strategy is based on our observation (see Fig. 2) that CD68^brightlin^- and CD68^dimlin^- cells both coexpress CD4, but differ in CD45RA expression. CD68^brightlin^- cells are also CD45RA^bright; CD68^dimlin^- cells are CD45RA dim to negative. Figure 3 shows an example of the sort window settings used in these experiments. Pre-enriched MNC (see Materials and Methods) were first sorted for CD4⁺lin^- cells (SORT I). Sorted CD4⁺lin^- cells were then stained for CD45RA and sorted again for CD45RA^bright and CD45RA^dim/- cells (SORT II). Triple stainings for CD68, CD4, and lin molecules, performed in parallel, confirmed that all CD4⁺lin^- cells were CD68⁺ and subdivided into CD68^bright and CD68^dim subsets (data not shown).

View larger version (73K): [in this window] [in a new window]   FIGURE 3. Isolation of CD68brightlin- and CD68dimlin- subsets. The two subsets were isolated from pre-enriched MNC using a sequential two-step flow-sorting procedure (see Materials and Methods). Representative sort window settings are shown. CD4+lin- cells were isolated (SORT I). Purified CD4+lin- cells were stained for CD45RA expression and resorted (SORT II) into CD45RAbright and CD45RAdim fractions, which were identical with CD68brightlin- and CD68dimlin- subsets, respectively. Cytospin preparations of freshly isolated CD68brightlin- and CD68dimlin- subsets stained with May-Grunwald-Giemsa are shown.

The two subsets of CD68⁺lin^- MNC clearly differ in their morphologic appearance (see cytospin preparations stained with May-Grunwald-Giemsa in Fig. 3). CD68^brightlin^- cells are round, with round or lobulated nuclei and abundant cytoplasm. CD68^dimlin^- cells are of similar size, but differ from CD68^brightlin^- cells in that they have a more ruffled cell shape with irregularly shaped and multilobed nuclei. In addition, cells of the CD68^brightlin^- subset were more vacuolated than CD68^dimlin^- cells.

Growth characteristics of CD68^brightlin^- and CD68^dimlin^- cells

To further analyze the stage of differentiation and lineage restriction of the two subsets, we tested their in vitro proliferation and differentiation capacities in the presence of cytokines. We first analyzed the myelopoietic differentiation potential of isolated CD68^brightlin^- and CD68^dimlin^- subsets using the cytokine combination GM-CSF, IL-1, IL-3, and IL-6, which represents a powerful stimulus for growth and myeloid differentiation of hemopoietic progenitors (37). The following profound changes were observed when stimulating purified CD68^brightlin^- cells with this growth combination. Within 48 h large aggregates were formed (Fig. 4A). This was followed on day 5 by the development of long thin DC projections (Fig. 4B). In addition, substantial proportions of cultured cells stimulated with GM-CSF, IL-1, IL-3, and IL-6 expressed the proliferation-associated nucleoprotein Ki-67 (21 and 26% in two experiments on day 4, respectively; Fig. 4C). These observations prompted us to analyze the effects of individual cytokines. We observed that addition of IL-3 alone induces cell cycling of CD68^brightlin^- cells. These results were confirmed using [³H]thymidine incorporation experiments (Fig. 4D). Total cell numbers stayed approximately constant over the analyzed 7-day culture period and were equivalent in cultures supplemented with GM-CSF, IL-1, IL-3, and IL-6 or with IL-3 alone (data not shown). The second, CD68^dimlin^- subset did not show growth factor dependency similar to that observed for CD68^brightlin^- cells and did not proliferate. Most of these cells rapidly lost viability, and individual cytokines or cytokine combinations did not enhance viability. In comparison, cultures of CD68^brightlin^- cells set up in parallel clearly showed higher percentages of viable cells, and viability was dependent on whether the culture medium was supplemented with IL-3 (data not shown).

View larger version (94K): [in this window] [in a new window]   FIGURE 4. Analyses of cultured CD68bright lin- cells. Flow-sorted CD68brightlin- cells were cultured in the presence of cytokines as described in Materials and Methods. Representative cultures are shown (n = 4). Phase contrast photomicrographs (A, low; B, high magnification) of cells cultured for 7 days in the presence of IL-1, IL-3, IL-6, and GM-CSF are shown. C, Fluorescence microscopy of Ki-67 stainings of cultured cells on day 4 in the presence of IL-1, IL-3, IL-6, and GM-CSF. D, Analysis of the effect of IL-3 or combinations of cytokines on cell proliferation. CD68brightlin- cells were stimulated with the indicated cytokines for 72 h. [3H]thymidine incorporation was determined as described in Materials and Methods after a final 16-h pulse labeling. Data from one representative experiment are shown.

Given the DC-like features of in vitro cultured CD68^brightlin^- cells, we analyzed their capacity to induce allogeneic T cell proliferation. As shown in Figure 5, after culture for 7 days in above-described GM-CSF-, IL-1-, IL-3-, and IL-6-supplemented medium, CD68^brightlin^- cells significantly induce allogeneic T cell proliferation. They are similar in relative potency in the MLR to DC generated from autologous CD14⁺ monocytes in the presence of GM-CSF plus IL-4 stimulation (mdDC), but are less efficient inducers of T cell proliferation than autologous mdDC generated in parallel in the presence of GM-CSF, IL-4, and TNF-.

View larger version (23K): [in this window] [in a new window]   FIGURE 5. Immunostimulatory capacity of in vitro cultured CD68brightlin- cells. Purified allogeneic T cells (5 x 104/well) were cultured with graded numbers of irradiated CD68brightlin- stimulator cells. Stimulator cells are CD68brightlin- cells after culture for 7 days in the presence of IL-1, IL-3, IL-6, and GM-CSF. DC generated in parallel from autologous CD14+ monocytes (mdDC) in the presence of GM-CSF plus IL-4 or in the presence of GM-CSF plus IL-4 and TNF- are compared. Negative controls represent unstimulated T cell-depleted autologous MNC (freshly isolated and frozen).

Freshly isolated CD68^brightlin^- cells, in marked contrast to CD68^dimlin^- cells, show no specific features of granulomonocytic cells. They express neither MPO (Fig. 2), a hallmark molecule of granulomonocytic differentiation (38), nor LZ (Fig. 6A), a molecule constituitively expressed by granulomonocytic cells that is up-regulated in monocytes and macrophages upon activation (40, 41, 42, 43). Even upon culture of CD68^brightlin^- cells with the granulomonopoietic growth combination GM-CSF plus IL-1, IL-3, and IL-6, we were unable to detect LZ or MPO in these cells (Fig. 6B). Similarly, no induction of expression of the monocyte-associated surface molecules CD14, CD11c, and CD33 occurred (Fig. 6B). The molecular features of in vitro cultured (differentiated) CD68^brightlin^- cells, therefore, are clearly different from those of freshly isolated CD68^dimlin^- cells. The CD68^dimlin^- population expresses LZ and is positive for CD11c and CD33 (Figs. 2 and 6A).

View larger version (32K): [in this window] [in a new window]   FIGURE 6. Analysis of intracellular LZ and other GM-associated molecules. A, LZ expression in fresh CD68brightlin- and CD68dimlin- subsets. MNC were pre-enriched by sheep erythrocyte rosetting and immunomagnetic depletion as described in Materials and Methods. Lin-depleted fractions were stained for CD68 (FITC), LZ (PE), and the lineage Aga CD3, CD19, CD14, and CD16 (PerCP). Dot plots show gated lin- cells analyzed for CD68 vs LZ expression. B, Expression of LZ-, MPO-, and GM-associated surface molecules by cultured CD68brightlin- cells. CD68brightlin- cells were purified by two-step flow sorting and were cultured for 7 days in the presence of IL-1, IL-3, IL-6, and GM-CSF as described in Materials and Methods. Histograms show the expression of the lysosomal proteins LZ, MPO, and CD68 as well as the surface membrane molecules CD14, CD11c, and CD33.

Both CD68^brightlin^- and CD68^dimlin^- cell subsets lack significant expression of the T cell costimulatory molecule CD86, but they are clearly CD54 positive. Furthermore, both subsets lack expression of the mature DC marker molecule CD83 (see Fig. 7).

View larger version (40K): [in this window] [in a new window]   FIGURE 7. Analysis of costimulatory molecules and CD83. MNC were pre-enriched by sheep erythrocyte rosetting and immunomagnetic depletion as described in Figure 6A. Lin-depleted fractions were triple stained for CD68 (FITC), CD86, CD83, CD54, or isotype-negative control (PE) and the lineage Ags CD3, CD19, CD14, and CD16 (PerCP). Dot plots show gated lin- cells analyzed for CD68 vs isotype-negative control, CD86, CD83, or CD54. Data are representative of four experiments.

Grouard et al. (31) recently demonstrated that isolated tonsil plasmacytoid monocytes can be induced by CD40L plus IL-3 stimulation to acquire features of mature DC. CD68^brightlin^- peripheral blood leukocytes identified in our study share unique immunophenotypic (CD68^bright lin^-, MPO^-, LZ^-) and functional features (IL-3-dependent growth) with plasmacytoid monocytes, suggesting that they may represent immediate precursors of plasmacytoid monocytes. Therefore, we analyzed the effect of CD40L costimulation on the acquisition of mature DC features by CD68^brightlin^- cells. As shown in Figure 8, stimulation of CD68^brightlin^- cells for 7 days with IL-3 plus CD40L significantly induces up-regulation of CD86 and CD54 molecules, and most cells become positive for the mature DC marker molecule CD83. In contrast, in the presence of IL-3 alone, the majority of cells remain CD86 negative, and virtually all cells remain CD83 negative.

View larger version (29K): [in this window] [in a new window]   FIGURE 8. CD40L up-regulates costimulatory molecules and induces CD83 expression on CD68brightlin- cells. CD68brightlin- cells were purified by sequential immunomagnetic depletion and flow sorting as described in Materials and Methods. Freshly isolated cells were stimulated for 7 days in the presence of IL-3 or IL-3 plus CD40L and then analyzed for the expression of CD86, CD83, and CD54 molecules as described in Materials and Methods. Dotted lines represent isotype-matched controls; faint lines and bold lines show stainings with specific Abs as indicated. Data are representative of three experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The most striking finding of our study is that this CD68^brightlin^- DC subset clearly differs from granulomonopoietic cells and from the other, CD68^dimlin^- DC subset based on the lack of expression of all analyzed granulomonocyte-associated intracellular (MPO^-, LZ^-) and cell surface (CD33^-, CD13^-, CD11c^-) molecules. Furthermore, CD68^brightlin^- DC are functionally distinguishable from granulomonopoietic precursors in that they fail to acquire MPO, LZ, CD33, CD14, and CD11c expression when stimulated in the presence of cytokines (37). We further observed that cells with this unique myeloid marker-negative (my^-) CD68^brightlin^- phenotype can survive and enter cell cycling if they are stimulated in vitro with IL-3. Other cytokines or cytokine combinations previously shown to be important for in vitro DC development from progenitors or monocytes, including GM-CSF, IL-1, IL-4, IL-6, TNF-, and TGF-ß, do not replace IL-3 in this function (data not shown). Thus, on the basis of a bright intracellular CD68 expression pattern, we describe here a distinct subset of IL-3-responsive DC precursors that lack all analyzed lineage features of granulomonopoietic cells, suggesting that they arise from a nonmyeloid progenitor cell differentiation pathway.

We show that the absence of expression of the intracellular lysosomal protein LZ clearly distinguishes CD68^brightlin^- cells from granulomonocytic cells as well as from the other CD68^dimlin^- subset. The distribution of LZ is of particular relevance for our study, since LZ is known as a highly sensitive and specific intracellular lineage marker molecule for granulomonopoietic cells (40, 42). Using a detection method identical with described in this study, we previously observed that LZ protein expression is rapidly induced during monopoietic differentiation and even precedes acquisition of (pro)monocyte morphology by hemopoietic progenitors (37). The observed distribution of LZ among the two subsets of DC included in the CD68⁺lin^- population is interesting, since it shows a striking correlation with the distribution of the surface marker molecules CD33, CD13, and CD11c. Only CD68^dimlin^- peripheral blood DC express these myeloid-related molecules. Further analysis of these my⁺CD68^dimlin^- peripheral blood DC showed additional features (i.e., CD4⁺, CD5⁺, HLA-DR^bright, CD45RA dim to neg, and ruffled cell shapes) previously described characteristic of myeloid peripheral blood DC precursors (23, 25, 46) and germinal center DC (30). The expression pattern of LZ thus further supports the concept that these DC are myeloid in origin (22).

The second, my^-CD68^brightlin^- DC precursor-like subset clearly shares characteristics with previously described peripheral blood leukocyte populations. They are phenotypically similar to previously described "immunologically immature" peripheral blood DC precursors (lin^- HLA-DR⁺ CD11c^-, CD33^dim/^-, CD13^dim/^-) (23, 24, 27) and closely resemble recently described CD2^- peripheral blood DC precursors (29). Positive identification based on bright intracellular CD68 expression as shown in our study clearly distinguishes CD68^brightlin^- blood DC from early lymphoid cells (19) and from CD34⁺ circulating hemopoietic progenitor cells, which are both negative or only weakly CD68 positive. We demonstrate that CD34⁺ cells are, on the average, fourfold less frequent among total MNC (on the average, 0.1% of the total MNC) compared with CD68^brightlin^- DC (Table I).

As described above, based on phenotypic and functional criteria, the subset of CD68^brightlin^- DC clearly differs from granulomonopoietic cells. One may speculate, therefore, that these DC originate from a separate nonmyeloid progenitor cell differentiation pathway (47). Evidence for the existence of such a pathway has been presented recently (48). Further studies should analyze whether CD68 represents a useful marker for the identification of putative bone marrow progenitors of CD68^brightlin^- blood DC.

One key finding of our study is that CD68^brightlin^- DC respond in vitro to IL-3 stimulation. We show that IL-3 as a single cytokine maintains viability and induces cycling of CD68^brightlin^- cells. These features independently observed in our study together with phenotypic (lack of CD11c, CD13, and CD33 expression) and morphologic (round cell shape when freshly isolated) characteristics are highly reminiscent of a recently characterized population of DC precursors in T cell areas of human tonsils (31). Identical cells were previously identified in electron microscopy studies as T-associated plasma cells (49).

Apart from this, independent evidence presented in our study strongly supports our assumption that CD68^brightlin^- blood DC represent circulating precursors of T-associated plasma cells. Immunohistology studies previously demonstrated that T zone-associated plasmacytoid cells (plasmacytoid T cells) are discriminable from other lymphoid tissue-associated macrophage or DC populations in that they are brightly CD68⁺ (50, 51, 52) (later renamed plasmacytoid monocytes based on bright CD68 staining (51)), but negative for the classical myelomonocytic lineage marker molecules LZ (52, 53, 54) and MPO (52, 55). This unique lin^-CD68^bright/LZ^-/MPO^- phenotype also defines the small population of CD68^brightlin^- blood DC in our study. Phenotypic resemblance of the CD68^brightlin^- blood DC described here with plasmacytoid monocytes in lymphoid tissues is further supported by coexpression of HLA-DR, CD4, and CD45RA (50, 51, 54, 55).

Typical location of plasmacytoid monocytes around high endothelial venules (56) further argues that blood precursors continuously repopulate these cells. A high turnover rate of plasmacytoid monocytes, probably regulated by the rate of precursor cell immigration, is suggested from the higher frequency of these cells under certain pathologic conditions associated with reactive T cell infiltration or extravasation (50, 57, 58, 59, 60).

	Acknowledgments

 
We are grateful to A. Renner for sorting cells on the FACS Vantage, M. Waclavicek for MLR analyses, and Immunex Corp. (Seattle, WA) for providing CD40 ligand.

	Footnotes

 
¹ This work was supported by Fonds zur Förderung der Wissenschaftlichen Forschung in Österreich.

² Address correspondence and reprint requests to Dr. Herbert Strobl, Institute of Immunology, Vienna International Research Cooperation Center, Novartis Forschungsinstitut, University of Vienna, Brunnerstrasse 59, A-1235 Vienna, Austria. E-mail address:

³ Abbreviations used in this paper: DC, dendritic cells; MNC, mononuclear cells; LZ, lysozyme; PE, phycoerythrin; lin^-, lineage marker negative; mdDC, monocyte-derived dendritic cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; rh, recombinant human; CD40L, CD40 ligand; MPO, myeloperoxidase.

Received for publication August 1, 1997. Accepted for publication March 23, 1998.

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