title: Abnormal regulation in the signal transduction in neutrophils from patients with severe congenital neutropenia: relation of impaired mobilization of cytosolic free calcium to altered chemotaxis, superoxide anion generation and F-actin content.
abstract: Severe congenital neutropenia (SCN) can be corrected in vivo by treatment with pharmacological dosages of recombinant human granulocyte colony-stimulating factor (rhG-CSF). In order to analyze the decreased chemotaxis of neutrophils from SCN patients receiving rhG-CSF, neutrophil functions essential for chemotaxis were investigated. The mobilization of cytosolic calcium ([Ca2+]i) and the functional state of cytoskeletal proteins in neutrophils from SCN patients were compared with either neutrophils from healthy donors (or, in selected experiments, from patients with cyclic neutropenia) and neutrophils from patients with chemotherapy-induced neutropenia also receiving rhG-CSF. Using flow cytometric analysis, two neutrophil subpopulations were detected in SCN patients in response to N-formylmethionine leucyl-phenylalanine (FMLP) (10(-9) M to 10(-7) M), one of which was unable to respond to this stimulus with an increase in [Ca2+]i. Whereas a homogeneous increase in [Ca2+]i in normal neutrophils occurred at 10(-9) M FMLP, neutrophils from SCN patients required 10(-6) M FMLP to respond homogeneously with an increase in [Ca2+]i. In contrast, G-CSF induced neutrophils from patients with cyclic neutropenia and from patients with chemotherapy-induced neutropenia showed a normal increase in [Ca2+]i after stimulation. The [Ca2+]i-dependent superoxide anion (O2-) generation in response to FMLP was also significantly diminished in neutrophils from SCN patients compared to normal neutrophils. However, O2- generation elicited by phorbolester (PMA), which directly activates protein kinase C (PKC), was not affected in SCN neutrophils. The total immunoreactive actin content and basal F-actin content in neutrophils from SCN patients were elevated as compared to normal neutrophils and neutrophils from patients with chemotherapy-induced neutropenia. The increase in F-actin content following FMLP activation was much lower in neutrophils from SCN patients as compared with normal neutrophils. These data suggest a defect in the signal transduction pathway in neutrophils from SCN patients between FMLP ligand-receptor interaction and Ca2+ mobilization, whereas upstream of PKC, triggered events seem to be unaffected. Therefore, [Ca2+]i-dependent neutrophil function in response to FMLP, such as actin disassembly, chemotaxis and O2- generation are diminished in SCN neutrophils. The pathomechanism responsible for the defective [Ca2+]i increase might be an initial step in understanding the underlying pathophysiology of SCN.
authors.affiliation: Fraunhofer Institute ITA, Department of Immunobiology, Hannover, Germany.
authors.forename: J
authors.initials: J
authors.lastname: Elsner
authors.forename: J
authors.initials: J
authors.lastname: Roesler
authors.forename: A
authors.initials: A
authors.lastname: Emmendörffer
authors.forename: M L
authors.initials: ML
authors.lastname: Lohmann-Matthes
authors.forename: K
authors.initials: K
authors.lastname: Welte
mesh_terms: D000199:Actins / Q000097:blood; D000838:Anions; D002118:Calcium / Q000097:blood*; D002634:Chemotaxis, Leukocyte; D003600:Cytosol / Q000378:metabolism; D016179:Granulocyte Colony-Stimulating Factor / Q000494:pharmacology; D006801:Humans; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009503:Neutropenia / Q000097:blood / Q000139:chemically induced / Q000151:congenital*; D009504:Neutrophils / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*; D013481:Superoxides / Q000097:blood; D014404:Tubulin / Q000097:blood
chemical_list: D000199:Actins; D000838:Anions; D014404:Tubulin; D013481:Superoxides; D016179:Granulocyte Colony-Stimulating Factor; D009240:N-Formylmethionine Leucyl-Phenylalanine; D002118:Calcium