Identification of CD68+lin- peripheral blood cells with dendritic precursor characteristics.

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 (CD68bright) 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 CD68bright, 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 CD68brightlin- DC precursors acquire mature DC characteristics (CD86+, CD83+, CD54bright) 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 CD68brightlin- is highly reminiscent of T zone-associated plasmacytoid monocytes. CD68brightlin- blood leukocytes also functionally resemble plasmacytoid monocytes. The lack of all analyzed myeloid features by CD68brightlin- blood leukocytes suggests that these cells arise from a novel nonmyeloid human DC differentiation pathway.

Granulomonocyte-associated lysosomal protein expression during in vitro expansion and differentiation of CD34+ hematopoietic progenitor cells.

Using an in vitro expansion and differentiation system for human CD34+ cord blood (CB) progenitor cells, we analyzed the induction and expression kinetics of the granulomonocyte associated lysosomal proteins myeloperoxidase (MPO), lysozyme (LZ), lactoferrin (LF), and macrosialin (CD68). Freshly isolated CD34+ CB cells were negative for LZ and LF, and only small proportions expressed MPO (4% +/- 2%) or CD68 (3% +/- 1%). Culturing of CD34+ cells for 14 days with interleukin (IL)-1, IL-3, IL-6, stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF resulted in on average a 1,750-fold amplification of cell number, of which 83% +/- 7% were MPO+. Without addition of GM-CSF and G-CSF, lower increases in total cell numbers (mean, 211-fold) and lower proportions of MPO+ cells (54% +/- 11%) were observed. The proportion of MPO+ cells slightly exceeded but clearly correlated with the proportion of cells positive for the granulomonocyte associated surface molecules CD11b (Mac-1), CD15 (LeX), CD64 (Fc gamma RI) CD66, or CD89 (Fc alpha R). At day 14 MPO+ and LZ+ cells were virtually identical. However, at earlier time points during culture (days 4 and 7), single MPO+ or LZ+ cell populations were also observed, which only later acquired LZ and MPO, respectively. Maturation of cells into the neutrophilic pathway was indicated by the acquisition of MPO, followed by LZ. In contrast, maturation of cells into the monocytic pathway was indicated by the acquisition of LZ followed by MPO and CD14. CD68 was found to be expressed at day 4 by the majority of cells and was not restricted to the granulomonocytic cells, as cells with megakariocytic (CD41+) or erythroid (CD71hi) features were CD68+. LF expression was observed only in GM- plus G-CSF-supplemented cultures, in which only 26% +/- 5% of cells expressed LF by day 14.

Flow cytometric analysis of intracellular CD68 molecule expression in normal and malignant haemopoiesis.

CD68 molecules are heavily glycosylated lysosomal membrane constituents of unknown function with strong expression in monocytes and macrophages. Using flow cytometry, we quantified expression levels of CD68 molecules in normal and malignant haemopoietic cells. CD68 molecules are intensely expressed in the cytoplasm and weakly on the surface of mature CD14+ monocytes. CD68 expression seems to start very early during granulomonopoietic differentiation. Virtually all myeloperoxidase (MPO)+ bone marrow cells coexpress CD68 and similar proportions of CD34+ progenitor cells weakly express CD68 or MPO molecules. During further differentiation, CD68 expression is strongly up-regulated in early MPO+ precursor cells which lack lactoferrin (LF) and CD14 molecules. Compared to these, more mature MPO+LF+ bone marrow and peripheral blood granulocytes express considerable lower levels of CD68. In-line with this broad expression, all investigated acute myeloid leukaemia (AML) cases, classified as FAB M1-M5, were CD68 positive, and compared to normal CD34+ bone marrow cells. CD34+ AML blast cells expressed increased levels. CD68 expression is, however, not restricted to cells of myeloid origin, because a subset (40 +/- 15%, n = 6) of CD19+ peripheral blood B-lymphocytes and 50% of B-ALL are also weakly positive. In contrast, normal CD3+ lymphocytes lack (< 3%, n = 6) CD68 and only low proportions (6 +/- 3%, n = 6) of CD56+ NK cells are CD68+. Also, all investigated T-ALL cases (n = 6) lacked CD68.