Phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus by mouse fibroblasts expressing human Fcgamma receptor type IIa (CD32).
Phagocytes bear more than one class of receptors for the Fc domain of IgG (FcgammaR). In addition the same ligand can interact with different classes of FcgammaR. This complexity makes it difficult to study the contribution of the various classes of FcgammaR to antimicrobial functions. To circumvent this difficulty, in the present study mouse 3T6 fibroblasts transfected with cDNA encoding for human FcgammaR type IIa (FcgammaRIIa-expressing cells) were used to determine the role of this receptor in phagocytosis and intracellular killing of serum-opsonized Staphylococcus aureus. Experiments using microbiological and fluorescent techniques to discriminate between cell-adherent and intracellular bacteria revealed that serum-opsonized bacteria are phagocytized by FcgammaRIIa-expressing cells, but not by parental fibroblasts. Non-opsonized bacteria were poorly internalized by FcgammaRIIa-expressing as well as parental fibroblasts. Furthermore, incubation of FcgammaRIIa-expressing cells with opsonized bacteria at 4oC and incubation of FcgammaRIIa-expressing cells with cytochalasin E prior to addition of opsonized bacteria inhibited the phagocytosis of these bacteria almost completely. Phagocytosis of opsonized bacteria by FcgammaRIIa-expressing cells was partly inhibited by selective inhibition of protein tyrosine kinases (PTK). FcgammaRIIa cross-linking initiated transient tyrosine phosphorylation of various proteins in FcgammaRIIa-expressing cells. These data indicate that activation of PTK is involved in the FcgammaRIIa-mediated phagocytosis of opsonized S. aureus by transfected fibroblasts. Human serum from normal individuals and agammaglobulinemic patients triggered the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts. Surprisingly, heat-inactivated human serum, IgG and incubation with anti-FcgammaRII antibodies followed by a bridging secondary antibody did not stimulate the killing process. The possibility that these ligands did not interact with FcgammaRIIa on the cells can be excluded since they induced tyrosine phosphorylation of cellular proteins. The serum factor that stimulates the intracellular killing of bacteria by FcgammaRIIa-expressing cells is not yet identified. Oxygen-independent mechanisms are thought to be responsible for the killing of intracellular bacteria by these cells since the NADPH oxidase inhibitor diphenylene iodonium did not affect the serum-stimulated intracellular killing of S. aureus and no reactive oxygen and nitrogen intermediates were produced by FcgammaRIIa-expressing cells after appropiate stimulation. Taken together, these data show that phagocytosis but not intracellular killing of S. aureus is mediated via FcgammaRIIa on cells expressing this receptor.
Nibbering
P H
PH
Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands. nibbering@rullf2.medfac.leidenuniv.nl
Broug-Holub
E
E
Bezemer
A C
AC
Jansen
R
R
van de Winkel
J G
JG
Geertsma
M F
MF
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*; D001422:Bacterial Adhesion; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism* / Q000382:microbiology; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D042541:Intracellular Space / Q000382:microbiology; D051379:Mice; D008856:Microscopy, Fluorescence; D009573:Nitrites / Q000378:metabolism; D009861:Onium Compounds / Q000494:pharmacology; D009895:Opsonin Proteins / Q000276:immunology; D010587:Phagocytosis / Q000276:immunology / Q000502:physiology*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D017452:Receptors, IgG / Q000096:biosynthesis / Q000276:immunology / Q000502:physiology*; D044967:Serum / Q000276:immunology; D013211:Staphylococcus aureus / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D013696:Temperature; D014443:Tyrosine / Q000378:metabolism; D020032:Tyrphostins / Q000494:pharmacology
D015703:Antigens, CD; D004791:Enzyme Inhibitors; C080568:Fc gamma receptor IIA; D009573:Nitrites; D009861:Onium Compounds; D009895:Opsonin Proteins; D017452:Receptors, IgG; D020032:Tyrphostins; C060152:tyrphostin 47; D014443:Tyrosine; C007517:diphenyleneiodonium; D006861:Hydrogen Peroxide; D011505:Protein-Tyrosine Kinases; D016650:Fluorescein-5-isothiocyanate