Identification of Common Molecular Pathways in Granulopoiesis Associated with C/EBP Epsilon and Gfi-1-Deficient Neutrophil Specific Granule Deficiency (SGD).

Abstract

Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype to SGD patients, and functional mutations in the C/EBPε gene have been identified in two patients with SGD. However, other patients with a similar disease phenotype do not have functional C/EBPε mutations, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a functional C/EBPε mutation demonstrated elevated protein levels of C/EBPε and significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1) in peripheral blood neutrophils from this SGD patient. However, no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. SGD can therefore be classified as C/EBPε negative or C/EBPε positive. We hypothesize that during granulopoiesis defects in a common molecular pathway occur in both C/EBPε- positive and -negative SGD resulting in the SGD phenotype. In order to gain insight into the molecular features of the two SGD phenotypes in the face of severe limitations on primary material from SGD patients, we have generated and characterized two cell line models. Using bone marrow from both C/EBPε−/− and Gfi-1+/− mice and their corresponding wildtype (WT) littermates, we generated EML (erythroid, myeloid, lymphoid) cell lines by transducing bone marrow with a retroviral vector expressing the dominant negative RARα and selecting for immortalized cells. Upon induction of the knock out and WT EML cell lines with all-trans retinoic acid (ATRA) to the neutrophil stage, we confirmed that both C/EBPε−/− and Gfi-1+/− cells share morphologic and functional abnormalities corresponding to their respective knockout mice, and hence to SGD. To further characterize the molecular defects in SGD, we have initiated microarray and proteomics analyses using RNA and protein prepared from both knockout cell lines. Using this approach, we may delineate defective pathways in both knock out cell types and correlate these observations to the specific SGD type. Our study should shed light on the relationship between Gfi-1 and C/EBPε during neutrophil development and should address the issue as to whether common or divergent molecular pathways are responsible for C/EBPε-negative and -positive SGD.