Combined corticosteroid/granulocyte colony-stimulating factor (G-CSF) therapy in the treatment of severe congenital neutropenia unresponsive to G-CSF: Activated glucocorticoid receptors synergize with G-CSF signals.

More than 90% of patients with severe congenital neutropenia (SCN) respond to granulocyte colony-stimulating factor (G-CSF) therapy. The basis for the refractory state in the remaining patients is unknown. To address this issue, we studied a child with SCN who was totally unresponsive to G-CSF and had a novel point mutation in the extracellular domain of the G-CSF receptor (GCSF-R). Marrow stromal support of granulopoiesis was evaluated by plating CD34(+) cells on preformed stromal layers. Nonadherent cells were harvested and assayed in clonogenic assays for granulocytic colony production. The in vitro effect of G-CSF and corticosteroids on granulopoiesis was evaluated in clonogenic assays of marrow mononuclear cells, by proliferation studies of the murine myeloid cell line 32D expressing the patient's mutated G-CSFR, and by measuring STAT5 activation in nuclear extracts from stimulated cells.Patient's stroma supported granulopoiesis derived from control marrow CD34(+) cells in a normal manner. Normal stroma, however, failed to induce granulopoiesis from patient's CD34(+) cells. Clonogenic assays of the patient's marrow mononuclear cells incorporating either G-CSF or hydrocortisone produced little neutrophil growth. In contrast, inclusion of both G-CSF and hydrocortisone in the cytokine "cocktail" markedly increased the neutrophil numbers. Proliferation of 32D cells expressing the mutated receptor and STAT5 activation were improved by a combination of G-CSF and dexamethasone. When small daily doses of oral prednisone were then administered to the patient with conventional doses of subcutaneous G-CSF, the patient responded with increased neutrophil numbers and with a complete reversal of the infectious problems. These data provide insight into SCN unresponsive to standard G-CSF treatment and to the potential corrective action of combined treatment with G-CSF and corticosteroids through synergistic activation of STAT5.