Clonal origin of human basophil/mast cells from circulating multipotent hemopoietic progenitors.

The origin of the human basophil/mast cell lineage from a pluripotent hematopoietic stem cell has been surmised but never demonstrated. By examining individual hemopoietic colonies in methylcellulose under inverted microscopy and using histochemical stains in conjunction with single-colony histamine assays, we have previously identified basophil/mast cell progenitors in human peripheral blood. We now report that a large proportion of normal human peripheral blood mixed granuloerythropoietic (GEMM) colonies contain histamine, in contrast to a significantly lower frequency of histamine positivity among normal neutrophil-macrophage, eosinophil, erythroid, macrophage, or megakaryocyte colonies. Morphological observations confirmed the presence of basophil/mast cells in the majority of GEMM colonies. In our work, the clonal derivation of basophils/mast cells from circulating multipotent (CFU-GEMM) hemopoietic stem cells was formally demonstrated, using combined histamine and G6PD isoenzyme analysis of single colonies grown in methylcellulose from a normal G6PD heterozygote.

Basophil/mast cell precursors in human peripheral blood.

Semisolid (methylcellulose) hemopoietic cultures revealed the presence of histamine-containing granulocyte colonies derived from precursors (CFU-C) present in human peripheral blood. Light microscopy and histochemical studies of cells in individual histamine-containing colonies demonstrated homogeneous populations of metachromatic basophil/mast cells (BMC) at various stages of maturation. By inverted microscopy, pure BMC colonies were more often found to have the overall appearance of the previously described "eosinophil" (type II), rather than "neutrophil-macrophage" (type I), colony type. Histamine-positive colonies constituted 58% (50/86) of all (type I and type II) granulocyte colonies in repeated cultures from a patient with systemic mastocytosis (SM), and 19% (13/67) of colonies in cultures from 8 patients with chronic myeloid leukemia (CML); this was in contrast to 8% (12/153) of colonies in cultures from 4 patients with urticaria pigmentosa (UP) and 6 normal controls (p less than 0.0001). Calculated frequency of BMC CFU-C was approximately 1 per 2 X 10(6) in normal and 1 per 2 X 10(5) nucleated cells in SM peripheral blood. Taking colony size into account, histamine content per cell in histamine-positive type II colonies in SM cultures was 1.1 +/- 0.19 pg, compared to 0.29 +/- 0.08 pg in CML and less than or equal to 0.10 in normals and UP. Electron microscopy (EM) of individual colonies revealed electron-dense granules with ultrastructural features of BMC in histamine-positive, but not histamine-negative, colonies. Use of these methods may help to further clarify the nature of BMC precursors and the regulation of their proliferation in bone marrow disorders and allergic states.

Increased numbers of circulating basophil progenitors in atopic patients.

Recruitment of basophils to sites of homocytotropic antibody-mediated hypersensitivity reactions has been well documented in both experimental and clinical situations. Mechanisms underlying tissue basophil accumulation, however, remain unclear and may involve chemotaxis, cell proliferation, or both. We have recently reported the presence in human blood of circulating basophil/mast cell progenitors on the basis of histamine content of granulocyte colonies grown in methylcellulose. In the current studies we have analyzed the peripheral blood of 30 patients with atopy and 25 comparable control subjects for frequency of basophil/mast cell progenitors by analysis of the histamine content of individual granulocyte colonies. Forty percent of granulocyte colonies in cultures of atopic patients contained histamine in comparison to only 11% in cultures of control subjects (p less than 0.001). Histamine content per colony as well as mean histamine per cell in each colony was higher in granulocyte colonies of atopic subjects and could not be related to colony size or culture conditions. Granulocyte colony growth was enhanced by antigen-stimulated, peripheral blood lymphomononuclear cell--conditioned media of atopic patients. Histamine-positive colonies were found more frequently in active versus quiescent atopic disease (p less than 0.05). These results are consistent with the hypothesis that basophils accumulate at sites of allergic reactions at least in part by recruitment of progenitors from circulation and subsequent differentiation in situ in response to lymphokines. Further studies by use of hemopoietic assays could elucidate the contribution of basophil production to the development of allergic conditions.

Basophil/mast cell precursors in mast cell proliferative disorders.

Mast cell proliferative disorders include urticaria pigmentosa, localized to the skin, and systemic mastocytosis, with progression from urticaria pigmentosa to systemic mastocytosis in some adults. We have evaluated the presence of basophil/mast cell precursors in urticaria pigmentosa and systemic mastocytosis using an in vitro assay for cells which form histamine-positive peripheral blood granulocyte colonies in methylcellulose. In 17 cultures from 10 patients with mast cell proliferative disorders (6 urticaria pigmentosa, 4 systemic mastocytosis), the frequency of histamine-positive granulocyte colonies was significantly higher in systemic mastocytosis (40% colonies picked) than in urticaria pigmentosa (15%, p less than 0.002). The mean histamine content per cell of urticaria pigmentosa patient colonies was less than or equal to 0.1 pg compared to 0.7 +/- 0.1 pg in systemic mastocytosis patient colonies. Precursor assays done serially at different times in individual patients appeared to reflect clinical extent of disease. In one patient with urticaria pigmentosa, a repeatedly normal number of histamine-positive colonies paralleled no change in clinical course, while in 2 others (1 systemic mastocytosis, 1 urticaria pigmentosa) increasing skin lesions, leukopenia, increased urinary histamine or refractoriness to therapy was accompanied by an increase in the frequency of basophil/mast cell precursors. Using an index of disease activity, the frequency of histamine-positive colonies was significantly higher in active, versus inactive, mast cell proliferative disorders (p less than 0.0001). These studies confirm the biologic relevance of mast cell proliferation in mast cell proliferative disorders, and suggest that precursor assays using histamine content of granulocyte colonies may be useful in predicting extent of disease.

Basophilic cell progenitors, nasal metachromatic cells, and peripheral blood basophils in ragweed-allergic patients.

Circulating hematopoietic progenitor cells giving rise to colonies containing basophilic cells (basophilic cell colony-forming units in culture [CFU-c]), nasal epithelial metachromatic cells (basophils and/or mast cells) (NMC), and blood basophils were enumerated before, during, and after a ragweed-pollen season in patients with ragweed hayfever and patients with perennial allergic rhinitis who were not ragweed allergic. In the patients with seasonal hay fever, the following was observed: basophilic cell CFU-c, measured as basophilic cell or histamine-containing colonies, were significantly reduced during the ragweed season compared to before (p less than 0.005) or after (p less than 0.025) the season in the ragweed-allergic group only. Conversely, peripheral blood basophils were higher during the ragweed season than before or after (p less than 0.001) in the ragweed-allergic group, whereas the number of NMC was higher during the season than before the season in this group (p less than 0.05). There were no such changes during the season in the group of patients with perennial allergic rhinitis. The observed seasonal changes in both NMC and circulating basophilic cell CFU-c are discussed in the context of lineage relationships among metachromatic cell types.

Heterogeneity of human peripheral blood eosinophil-type colonies: evidence for a common basophil-eosinophil progenitor.

We have recently shown that a proportion of previously designated human eosinophil "(Eo)-type" colonies in methylcellulose contain basophils and histamine (Denburg et al Blood 61:775, 1983). In the present studies, individual Eo-type colonies have been analyzed by cell morphology as well as by biochemical assays for histamine, Charcot-Leyden crystal protein (CLC), and eosinophil granule major basic protein (MBP). Clonal origin of single Eo-type colonies was confirmed by G6PD isoenzyme analysis. Morphological observations of such colonies revealed the existence of two distinct colony types: (1) Eo type containing 100% basophils and (2) Eo type containing mixtures of basophils and eosinophils, including cells with mixed basophil-eosinophil granulation. Histamine was not detected in pure, mature peripheral blood eosinophils. Immunofluorescent studies demonstrated bright staining for CLC and MBP in 95% +/- 3% of cells in Eo-type colonies but only in 5% +/- 4% of cells in GM-type colonies. Radioimmunoassay for MBP was positive in 5/9 Eo-type and 0/10 neutrophil-macrophage ("GM-type") colonies, with a mean level (nanogram/colony) of 11.6 +/- 4.2 per Eo-type colony; four of the latter colonies were doubly positive for both histamine and MBP. These and previous findings point out the morphological and biochemical heterogeneity of peripheral blood Eo-type colonies and provide direct evidence for the existence of a common, circulating basophil-eosinophil progenitor.