Functional characteristics of mature granulocytes in a patient with acute promyelocytic leukemia treated with all-trans retinoic acid.

All-trans retinoic acid (ATRA) is a differentiating agent that has been successfully used in the treatment of patients with acute promyelocytic leukemia (APL). Functional properties of peripheral blood neutrophils from a patient with APL during treatment with ATRA have been studied. Wright stain of patient neutrophils showed hypogranulation and loose nuclear chromatin when compared with normal neutrophils. These cells were of lower density than normal neutrophils and separated on density gradient centrifugation with mononuclear cells. Surface antigen expression by FACS distinguished these cells from lymphocytes. The histograms showed a population of larger cells expressing CD18 and CD11b, distinct from the smaller cells which did not express CD11b. fMLP caused an increase in intracellular calcium (measured spectrophotometrically) that was inhibited by the calcium chelator BAPTA. Actin polymerization following cell activation was measured using NBD-phallacidin staining and FACS. Both IL-8 and fMLP caused rapid increases using F-actin content (2.5-3.0 fold), which were of greater magnitude than generally seen with normal neutrophils. Treatment with BAPTA before activation with fMLP did not blunt the actin responses, despite complete inhibition of an intracellular calcium increase. In summary, neutrophils derives from differentiated APL cells express CD18/CD11b, and exhibit a similar degree of actin polymerization in response to fMLP and IL-8, independent of an increase in intracellular calcium. Although the actin responses are greater than normal neutrophils, most properties are similar, supporting the contention that these cells can protect the host. The exaggerated actin response to inflammatory mediators, however, may play a role in the 'retinoic acid syndrome'.

Functional properties of HL60 cells matured with all-trans-retinoic acid and DMSO: differences in response to interleukin-8 and fMLP.

All-trans-retinoic acid (ATRA) causes granulocyte differentiation in patients with acute promyelocytic leukemia. HL60 cells are frequently used as an in vitro model for studying granulocytes during maturation. We have previously studied actin polymerization in response to fMLP in HL60 cells undergoing DMSO induced maturation, and reported that IL-8 causes actin polymerization in neutrophils in a manner similar to fMLP. We now compare chemotaxis and actin polymerization in response to IL-8 and fMLP, and nitroblue tetrazolium (NBT) reduction in HL60 cells matured with ATRA and DMSO. Cells cultured for 4 days with ATRA and DMSO showed morphologic evidence of maturation. NBD-phallacidin staining and flow cytometry were used to measure changes in F-actin content in response to IL-8 and fMLP. Uninduced cells were not capable of actin polymerization or chemotaxis. Cells matured with ATRA exhibited a 2.6-fold increase in F-actin content in response to IL-8, but only a 1.2-fold increase in response to fMLP. Cells matured with DMSO responded to both IL-8 and fMLP in an equal manner with 1.6-fold increases in F-actin. The 2 h migration for ATRA induced cells was 124 microns in response to IL-8, 107 microns with fMLP, and 105 microns in buffer. DMSO induced cells migrated 89 microns in response to IL-8, 106 microns with fMLP, and 66 microns in buffer. With maturation, 65% of the ATRA induced cells reduced NBT compared with only 15% of the DMSO induced cells. In summary, HL60 cells cultured in ATRA develop greater functional maturity than those cultured in DMSO, and a greater responsiveness to IL-8 than fMLP, a finding distinct from previously reported work in neutrophils.

The effect of dexamethasone on functional properties of HL60 cells during all-trans retinoic acid induced differentiation. Are there implications for the retinoic acid syndrome?

Differentiation therapy for acute promyelocytic leukemia (APL) using all-trans-retinoic acid (ATRA) has improved the prognosis of the disease. ATRA therapy also causes a newly recognized clinical syndrome, the "retinoic acid syndrome" (RAS), which can be successfully managed with dexamethasone. Because aberrant function of maturing leukemic granulocytes may cause this syndrome, and because dexamethasone is useful clinically, we studied functional properties of maturing HL60 cells cultured in the presence and absence of dexamethasone. HL60 cells were cultured for 4 days with ATRA and studied daily to determine acquisition of mature neutrophil-like properties including phagocytosis, NBT reduction, actin polymerization, chemotaxis and adhesion molecule expression. Undifferentiated HL60 cells could not polymerize actin or reduce NBT, and exhibited only a minimal ability to undergo chemotaxis or ingest latex beads. Following 4 days of maturation with ATRA, the cells would increase F-actin content in response to interleukin-8, ingest latex beads, migrate in a chemotaxis chamber, reduce NBT, and express CD11b. When dexamethasone was added to the cells in culture, there was no major enhancement or suppression of these properties. We also studied the effect of dexamethasone on functional properties of normal neutrophils and found minimal if any effect on their function. Overall, these studies suggest that in vitro, dexamethasone has little effect on the function of leukemic and normal granulocytes. To further investigate the pathophysiology of the retinoic acid syndrome, future studies may need to use endothelial cells, cytokines, or granulocytes obtained from APL patients.