INFECTION AND IMMUNITY, Sept. 1975, p. 695-695 Copyright 0 1975 American Society for Microbiology
Vol. 12, No. 3 Printed in U.S.A.
NOTES Phagocytosis by Macrophages of Mycoplasma pneumoniae After Opsonization by Complement W. BREDT Institute for Medical Microbiology, Johannes Gutenberg University, D-65 Mainz, West Germany
Received for publication 14 April 1975
Mycoplasma pneumoniae cells are not phagocytized by peritoneal macrophages without opsonization. They are taken up after treatment with complement or antibody. Phagocytosis of mycoplasmas by macrophages is induced or enhanced by the addition of homologous antibodies (3-5). Recent findings indicate that the complement sequence is activated by Mycoplasma pneumoniae even in the absence of antibodies (2). In addition to damage or killing of the cells, this reaction resulted in a positive immune adherence, suggesting the presence of C3b on the mycoplasma surface. Since C3b is known to act as an opsonin, it was of interest to study the interaction of M. pneumoniae with macrophages in the presence of complement. Peritoneal macrophages were harvested from guinea pigs on day 3 after stimulation with starch gel. Cover-slip chambers with glassgrown M. pneumoniae cells (strain FH) (1) were filled with 0.2 ml of a suspension containing about 105 macrophages per ml. After settling of the cells (30 to 60 min) the chambers were rinsed once with tissue culture medium (medium 199 plus Hanks salt solution) (TCM), and one chamber at a time was refilled with TCM containing 10% of the respective serum concentration to be tested. The chamber was then closed with a cover slip and immediately placed on a microscope stage (Zeiss photomicroscope) heated to 37 C by an Air Curtain Incubator (Sage Instruments). The following sera were tested: guinea pig serum (GPS), guinea pig serum deficient in C4 (GPSC4D), rabbit serum (RS), and rabbit serum deficient in C6 (RSC6D). In addition, heat-inactivated (56 C for 30 min) guinea pig serum (GPSH56) or rabbit serum (RSH56) and a heat-inactivated rabbit anti-M. pneumoniae serum (Ab) were used as controls. The results are summarized in Table 1. Macrophages were not able to phagocytize the 694
mycoplasmas in TCM or in TCM with added heat-inactivated normal sera. Under these conditions the macrophages spread repeatedly over the mycoplasmas without any visible effect (Fig. lf to j). On the other hand mycoplasmas rounded after complement action were easily taken up as soon as they were reached by the pseudopodia of the macrophages (Fig. la to e). During the first 30 min after addition of specific antibody the cells did not alter their appearance noticeably. However, the macrophages were able to phagocytize these apparently normal cells about 10 min after addition of the antibody. I'he results suggest that macrophages are not able to efficiently phagocytize normal M. pneumoniae cells. Complement activated either via the classical (GPS, RS, RSC6D) or the alternate (GPSC4D) pathway induces phagocytosis. This is probably due to C3b bound to the surface of M. pneumoniae cells (2). The uptake is not restricted to mycoplasmas killed by the TABLE 1. Effect of various serum preparations on mycoplasma cell shape and phagocytosis by peritoneal macrophages
Beginning of phagocytosis (min)
Serum added
Mycorounding
Phagocytosis observed
None GPSH56 GPS GPSC4D RSH56 RS RSC6D Ab
+
+
Vol. 12, No. 3 Printed in U.S.A.
NOTES Phagocytosis by Macrophages of Mycoplasma pneumoniae After Opsonization by Complement W. BREDT Institute for Medical Microbiology, Johannes Gutenberg University, D-65 Mainz, West Germany
Received for publication 14 April 1975
Mycoplasma pneumoniae cells are not phagocytized by peritoneal macrophages without opsonization. They are taken up after treatment with complement or antibody. Phagocytosis of mycoplasmas by macrophages is induced or enhanced by the addition of homologous antibodies (3-5). Recent findings indicate that the complement sequence is activated by Mycoplasma pneumoniae even in the absence of antibodies (2). In addition to damage or killing of the cells, this reaction resulted in a positive immune adherence, suggesting the presence of C3b on the mycoplasma surface. Since C3b is known to act as an opsonin, it was of interest to study the interaction of M. pneumoniae with macrophages in the presence of complement. Peritoneal macrophages were harvested from guinea pigs on day 3 after stimulation with starch gel. Cover-slip chambers with glassgrown M. pneumoniae cells (strain FH) (1) were filled with 0.2 ml of a suspension containing about 105 macrophages per ml. After settling of the cells (30 to 60 min) the chambers were rinsed once with tissue culture medium (medium 199 plus Hanks salt solution) (TCM), and one chamber at a time was refilled with TCM containing 10% of the respective serum concentration to be tested. The chamber was then closed with a cover slip and immediately placed on a microscope stage (Zeiss photomicroscope) heated to 37 C by an Air Curtain Incubator (Sage Instruments). The following sera were tested: guinea pig serum (GPS), guinea pig serum deficient in C4 (GPSC4D), rabbit serum (RS), and rabbit serum deficient in C6 (RSC6D). In addition, heat-inactivated (56 C for 30 min) guinea pig serum (GPSH56) or rabbit serum (RSH56) and a heat-inactivated rabbit anti-M. pneumoniae serum (Ab) were used as controls. The results are summarized in Table 1. Macrophages were not able to phagocytize the 694
mycoplasmas in TCM or in TCM with added heat-inactivated normal sera. Under these conditions the macrophages spread repeatedly over the mycoplasmas without any visible effect (Fig. lf to j). On the other hand mycoplasmas rounded after complement action were easily taken up as soon as they were reached by the pseudopodia of the macrophages (Fig. la to e). During the first 30 min after addition of specific antibody the cells did not alter their appearance noticeably. However, the macrophages were able to phagocytize these apparently normal cells about 10 min after addition of the antibody. I'he results suggest that macrophages are not able to efficiently phagocytize normal M. pneumoniae cells. Complement activated either via the classical (GPS, RS, RSC6D) or the alternate (GPSC4D) pathway induces phagocytosis. This is probably due to C3b bound to the surface of M. pneumoniae cells (2). The uptake is not restricted to mycoplasmas killed by the TABLE 1. Effect of various serum preparations on mycoplasma cell shape and phagocytosis by peritoneal macrophages
Beginning of phagocytosis (min)
Serum added
Mycorounding
Phagocytosis observed
None GPSH56 GPS GPSC4D RSH56 RS RSC6D Ab
+
+
