Peritoneal neutrophils and macrophages, bone marrow cells and neutrophils isolated from bone marrow of mice homozygous for Ncf1m1J fail to produce superoxide upon stimulation in vitro with N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate 13-acetate (PMA), as determined by kinetic spectrophotometric analysis of cytochrome c reduction. Western blot analysis detected no full-length NCF1/p47phox protein in cells from these mice; a faint band of slightly smaller molecular size than the wild type NCF1 protein was observed on probing with antibodies to NCF1. To exclude the possibility that the NCF1 protein is produced in cells of mutant mice but is degraded rapidly by endogenous proteases, bone marrow cells were isolated and samples prepared for western blot analysis in the presence of diisopropyl fluorophosphate (DFP); no difference was observed upon analysis of freshly prepared cell lysates made with and without DFP, indicating that NCF1 protein is not expressed in mutant cells (Huang et al. 2000).
Analysis for other NADPH oxidases involved in neutrophil superoxide production revealed that NCF2/p67phox was present at wild type levels and CYBB/gp91phox and CYBA/p22phox were expressed at higher than wild type levels. Other neutrophil products were assayed and found not to differ in bone marrow cells of C57BL/6J vs. mutant mice: GR-1, a granulocyte marker present on approximately 40% of marrow cells by fluorescence activated cell sorting (FACS) analysis; NCF4/p40phox and RAC1/RAC2 proteins, analyzed by western blot analysis; and expression of mRNAs encoding the granule proteins myeloperoxidase, elastase, cathepsin, lysozyme, lactoferrin and gelatinase as detected by reverse transcriptase - polymerase chain reaction (RT-PCR) (Huang et al. 2000).
The response of Ncf1m1J/Ncf1m1J mice to infectious or irritant (inflammatory stimulus) challenge has not been investigated (Huang et al. 2000). However, mice homozygous for the targeted mutation Ncf1tm1Shl (Stock No. 027331) - which disrupts the gene near the point of the Ncf1m1J mutation and, like the latter, results in failure of phagocytes to generate a superoxide burst following PMA stimulation in vitro - developed spontaneous infections with a variety of microorganisms including Staphylococcus xylosus, Lactobacillus, a hyaline septate mold and the fungus Paecilomyces, despite being maintained under specific pathogen free conditions. Ten-fold more Staphyloccoccus aureus bacteria survived intracellularly following in vitro uptake by phagocytes inblood from these mice than from control littermates, demonstrating a defect in the intracellular bactericidal activity of the mutant mice. Following intraperitoneal injection of the sterile irritant thioglycolate, twice as many leukocytes were recruited to the peritoneal cavities of Ncf1tm1Shl homozygous mice than of littermate controls; proportions of neutrophils in mutant and control exudates were similar (Jackson et al., J Exp Med 182:751-758 1995).
