PH Nibbering¹, E Broug-Holub², AC Bezemer¹, R Jansen¹, JGJ van de Winkel³, MF Geertsma¹.

1 Dept. of Infectious Diseases, University Hospital, Leiden, The Netherlands.
2 Dept. of Cell Biology and Immunology, Free University, Amsterdam, The Netherlands.
3 Dept. of Experimental Immunology, University Hospital, Utrecht, The Netherlands.

Received 12/7/95; Accepted 12/29/95; On-line 1/1/96

The present results indicate that phagocytosis but not intracellular killing of serum-opsonized S. aureus is mediated by FcgammaRIIa fibroblasts expressing this receptor. The conclusion that human FcgammaRIIa mediates phagocytosis of heat-inactivated serum-opsonized, S. aureus by mouse 3T6 fibroblasts stably transfected with this receptor is based on the following lines of evidence. First, by means of two different techniques to discriminate between cell-adherent and intracellular bacteria, we have demonstrated that bacteria are truly phagocytized by FcgammaRIIa-expressing fibroblasts. Second, incubation of FcgammaRIIa-expressing fibroblasts and opsonized bacteria at 4°C or pre-incubation of the fibroblasts with cytochalasin E inhibited phagocytosis of opsonized S. aureus, as has been found for professional phagocytes (16). Third, FcgammaRIIa-expressing, but not parental, fibroblasts efficiently phagocytized opsonized S. aureus. Fourth, non-opsonized bacteria were poorly phagocytized by these cells. These observations are in agreement with reports that human FcgammaRIIa introduced into murine 3T6 fibroblasts (13), II A 1.6 B cells (14-15) and P388D1 macrophage-like cells (26) mediated phagocytosis of opsonized particles. Interestingly, transfection of the same receptor into CHO cells resulted in enhanced binding but not internalization of opsonized erythrocytes (12). Clearly, many cell types (but not all) are equipped to phagocytize particles including opsonized bacteria. Another important finding is that selective inhibition of PTK reduced the phagocytosis of opsonized S. aureus by FcgammaRIIa-expressing fibroblasts, as reported earlier for professional phagocytes (9, 25-27). Reduction of the phagocytosis of opsonized bacteria by the PTK inhibitor tyrphostin-47 was not due to a cytotoxic effect of the inhibitor or suppression of expression of human FcgammaRIIa by the compound (unpublished observations), as has been reported earlier for human monocytes (8).

The second conclusion pertains to the ability of FcgammaRIIa-expressing fibroblasts to intracellularly kill bacteria. Our observation that serum, both from healthy controls and from patients suffering from agammaglobulinemia, induced intracellular killing of opsonized S. aureus indicates that 3T6 fibroblasts exhibit antimicrobial activity. The identity of the serum factor that stimulates the killing process is not clear, since FcgammaR-specific stimuli, such as heat-inactivated serum, IgG and cross-linking FcgammaRIIa with a specific mAb and bridging second antibody, did not trigger the killing process. The possibility that these FcgammaR-specific stimuli do not result in intracellular signaling can be excluded since FcgammaRIIa cross-linking caused rapid tyrosine phosphorylation of cellular proteins in fibroblasts expressing this receptor. Of course, it could be that signaling down-stream of tyrosine phosphorylation of proteins after FcgammaRIIa cross-linking on the cells is impaired. In this connection, it can be speculated that cross-linking of FcgammaRIIa on fibroblasts does not lead to the formation of complexes of FcgammaRIIa and g-chains. These complexes may be essential to the stimulation of the killing process, as has recently been reported for interleukin-2 release, immune complex internalization and antigen presentation by FcgammaRIIa-expressing murine B cells IIA.1.6. (14-15). The possible explanation that the antimicrobial effector mechanisms in fibroblasts are rather limited is very attractive. We have found that the NADPH oxidase-inhibitor DPI did not affect the killing process in FcgammaRIIa-expressing fibroblasts. Upon stimulation with opsonized bacteria and phorbol ester reactive oxygen and nitrogen intermediates were not produced by FcgammaRIIa-expressing fibroblasts. In contrast to human monocytes (8,28) the mechanism(s) underlying the intracellular killing of S. aureus by FcgammaRIIa-expressing fibroblasts is most probably oxygen-independent. It has been reported that in these fibroblasts, opsonized erythrocytes end up in phagolysosomes where they are believed to be degraded by the contents of lysosomes (13). It could be that phagocytized S. aureus are degraded by the action of lysosomal proteins in FcgammaRIIa-expressing fibroblasts as well.