Studies on the pathogenesis of refractory anemia.

Nine patients with refractory anemia were studied using the soft agar marrow culture assay (CFU-c) to identify granulocyte-monocyte progenitor cells. Patients' marrows were then cocultured with normal marrow to identify suppressor cells that inhibit normal colony formation. Three of nine patients had low colony formation and no suppression in coculture. These patients may have a defect intrinsic to the marrow granulocyte-monocyte progenitor cell, termed type I. Three of nine patients had normal colony formation and no suppression in coculture, possibly representing a type II defect in the hemopoietic environment. Three of nine patients had low colony formation in the CFU-c assay and their marrow contained cells that suppressed colony formation by normal marrow in coculture. This defect, termed type III, may result from suppressor cells. Thus, refractory anemia may be a syndrome resulting from at least three different pathogenetic mechanisms involving defects in (1) stem cells, (2) the marrow environment or (3) suppressor cells. This may represent one end of the spectrum of pancytopenia with diminished cellularity (aplastic anemia) or normal cellularity (refractory anemia) resulting from similar mechanisms.

Studies on the pathogenesis of aplastic anemia.

Three assays were used to study myelopoiesis in 14 patients with aplastic anemia: (1) the soft agar colony assay for granulocyte-monocyte progenitors (CFU-c); (2) coculture of marrow from patients with normal marrows in the CFU-c assay; and (3) culture of marrow pretreated with antithymocyte globulin (ATG) in the CFU-c assay. Marrow from five patients gave low colony counts when cultured alone and suppressed colony formation by normal marrow cells in coculture. Suppressor cells may have caused the aplasia in these patients. Eight patients had low colony formation and no suppression in coculture. These patients may have absent or defective stem cells. Marrow from one patient produced normal colony formation, did not contain suppressor cells and may have a defective hematopoietic environment. Aplastic anemia thus may result from at least three different defects involving (1) the stem cells, (2) the hematopoietic environment or (3) suppressor cells.

Early stages of human marrow lymphocyte differentiation: induction in vitro by thymopoietin and ubiquitin.

To study early stages of human lymphocyte differentiation, bone marrow cells were physically separated according to their density and size by gradient centrifugation and then velocity sedimentation. The isolated cell fractions were incubated with putative inducing agents and then assayed for their expression of an array of surface differentiation markers. The inducing agents used were two polypeptides, thymopoietin (Tp) and ubiquitin (Ub), and the cyclic nucleotide, dibutyril cyclic 3'5' adenosine monophosphate (cAMP). Tp, Ub, and cAMP each induced the ability to form sheep erythrocyte rosettes by small lymphocytes, which may thus represent T cell precursors. Ub and Tp induced rosette formation with mouse erythrocytes on lymphocytes of more heterogenous size, which may be "early" B cell precursors. Ub alone could induce surface IgM expression on small lymphocytes, which might be "late" B cell precursors. Both Tp and Ub induced Fc receptors on small lymphocytes. Complement receptors could not be induced on marrow lymphocytes by Tp, Ub, or cAMP. A number of lymphocyte precursors can thus be identified by their physical characteristics and their ability to respond to particular soluble factors with the expression of new differentiation markers.

Heterogeneity of stem cells in severe combined immunodeficiency.

Two patients with severe combined immunodeficiency disease (SCID) having variable B-cell development have been shown to have marrow precursors of lymphoid cells which can be induced in vitro by thymic factors to express certain T-cell surface characteristics (HTLA+ phenotypes). Their marrow cells could not, however, be induced by these same factors to develop the E-rosette marker or functional activities of T lymphocytes. The marrow of these children also showed, when compared to that of normal adults, a different distribution of cellular elements on density gradient fractionation. The findings support the view that the disorder under study has a different pathogenesis from other forms of SCID previously analysed.

Induction of human granulocyte differentiation in vitro by ubiquitin and thymopoietin.

Human bone marrow cells were separated according to density by centrifugation on Ficoll-Hypaque gradients and then according to size by velocity sedimentation. This procedure resulted in fractions enriched for immature granulocytes, mature granulocytes, and lymphocytes. Cells in these fractions were analyzed for their expression of certain surface and functional differentiation markers and for their ability to respond to thymopoietin and ubiquitin with the expression of additional differentiation markers. A higher percentage of band form and segmented granulocytes than of more immature granulocytes expressed complement receptors on their surfaces. Thymopoietin and ubiquitin induced a significant percentage of the cells in the immature granulocyte fraction to express this marker. These data suggested that the complement receptor may be viewed as a differentiation marker on human granulocytes, the expression of which can be induced in vitro by thymopoietin and ubiquitin. Furthermore, fractions containing predominantly band form granulocytes were induced by ubiquitin (but not thymopoietin) to develop the capacity to respond to chemotactic agents, and cell fractions containing predominantly myelocytes and metamyelocytes were induced by thymopoietin and ubiquitin to develop the capacity to phagocytose latex particles. These findings indicated that thymopoietin and ubiquitin, two agents known to induce a number of stages of human and mouse lymphocyte differentiation, are also capable of inducing some stages of human granulocyte differentiation in vitro.

Aplastic anemia: presence in human bone marrow of cells that suppress myelopoiesis.

Bone marrow from a patient with aplastic anemia was shown by multiple criteria to have a block in early myeloid differentiation. This block was overcome in vitro by elimination of marrow lymphocytes. Furthermore, this differentiation block was transferred in vitro to normal marrow by coculturing with the patient's marrow. We suggest that some cases of aplastic anemia may be due to an immunologically based suppression of marrow cell differentiation rather than to a defect in stem cells or their necessary inductive environment.