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Isolation and Characterization of Neutrophils with Anti-Tumor Properties
Published on: June 19, 2015
Changes in the localization of catalase during differentiation of neutrophilic granulocytes
C A Ballinger1, C Mendis-Handagama, J R Kalmar
1Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
Insights
Catalase compartmentalization shifts from granules in immature myeloid cells to the cytosol in mature neutrophils. This differentiation-linked change in catalase localization may impact acute leukemia classification and drug resistance.
Area of Science:
- Cell Biology
- Hematology
- Biochemistry
Background:
- The intracellular localization of catalase in human myeloid cells is not fully understood.
- Investigating catalase compartmentalization is crucial for understanding myeloid cell function and differentiation.
Purpose of the Study:
- To elucidate the subcellular localization of catalase in human myeloid cells, specifically HL-60 cells and mature neutrophils.
- To determine how catalase localization changes during myeloid cell differentiation.
Main Methods:
- Indirect immunofluorescence and ultrastructural immunocytochemistry using a specific anti-catalase antibody.
- Differential centrifugation and Percoll-sucrose density gradient centrifugation of cell lysates.
- Enzyme activity assays for catalase and other myeloid markers (MPO, beta-hexosaminidase, beta-glucuronidase, lysosomal alpha-mannosidase, LDH).
- Proteolysis protection assays and digitonin titration experiments.
Main Results:
- Immature HL-60 cells displayed punctate cytoplasmic staining for catalase, indicating compartmentalization.
- Mature neutrophils showed diffuse cytoplasmic staining, suggesting cytosolic localization.
- Differential centrifugation revealed a fragile catalase compartment in HL-60 cells compared to other granule markers.
- Density gradient centrifugation showed catalase localized in a less dense compartment than MPO in HL-60 cells, and comigrated with LDH in neutrophils.
- Ultrastructural studies confirmed multiple catalase compartments in HL-60 cells and primarily cytosolic localization in neutrophils.
Conclusions:
- Catalase compartmentalization undergoes a significant shift from a granular/compartmentalized state in immature myeloid cells to a predominantly cytosolic state in mature neutrophils.
- This dynamic change in catalase localization during myeloid differentiation may be relevant for classifying acute leukemias and understanding drug resistance mechanisms.
Abstract:
The nature of the compartmentalization of catalase in human myeloid cells is an unresolved issue. Using a rabbit polyclonal antibody specific for catalase, indirect immunocytofluorescence of immature leukemic promyelocytes (HL-60 cells) showed a pattern of small, sharp, punctate staining in the cytoplasm of all cells, while mature neutrophils showed a larger diffuse, flocculent pattern of cytoplasmic staining. Differential centrifugation of nitrogen cavitates of HL-60 cells indicated that the putative catalase-containing compartment was relatively fragile compared with the compartment(s) that contained myeloperoxidase (MPO), beta-hexosaminidase, beta-glucuronidase, and lysosomal alpha-mannosidase activities. Parallel studies using dimethylsulfoxide (DMSO)-induced HL-60 cells and mature neutrophils showed that, in the course of differentiation, there was an apparent shift in the localization of catalase from the granule fraction to the cytosolic fraction. Percoll-sucrose density gradient centrifugation of HL-60 cell cavitates showed a catalase-containing compartment with a mean peak density (1.05 g/mL) significantly lower than that of the major myeloperoxidase-containing compartment (1.08 g/mL); in mature neutrophils, catalase activity comigrated with lactate dehydrogenase (LDH) activity. Catalase in isolated fractions was protected from proteolysis in the absence, but not in the presence, of 0.1% Triton X-100. Digitonin titration experiments confirmed the compartmentalized nature of catalase in immature HL-60 cells and were consistent with a cytosolic localization in mature neutrophils. Ultrastructural localization of catalase by Protein A-gold immunocytochemistry demonstrated four to six catalase-containing compartments in all HL-60 cell profiles. In mature neutrophils, catalase was localized primarily in the cytoplasmic matrix, although in fewer than 2% of the cell profiles, one to two catalase-containing compartments were observed. The changes in catalase localization that occur during myeloid differentiation appear to be similar to the changes that occur during erythroid and megakaryocytic differentiation, and may have potential clinical significance in the classification of acute leukemia and in the development of drug resistance.
