Abstract
The opsonization and phagocytosis of group B streptococci of types Ia, Ib, and Ic were studied in vitro by measuring the uptake of radioactivity by coverslip cultures of rabbit alevolar macrophages during incubation with radiolabeled, nonviable bacteria which had been exposed to rabbit serum. The uptake of counts per minute was quantitative, reproducible, and reversibly inhibited by cold, indicating that it was largely a measurement of phagocytic ingestion rather than of attachment of bacteria-immunoglobulin complexes to macrophage membranes. Moreover, suspended macrophages killed approximately 90% of viable streptococci in the presence of specific antiserum. The opsonic activity of immune serum was heat stable, and phagocytosis of streptococci was insignificant after incubation with normal serum and antiserum to some heterologous group B streptococci. By absorption studies, it was possible to identify the effect of antibodies to specific bacterial antigens. Phagocytosis of streptococci containing the corresponding antigens was maximal after opsonization with homologous or heterologous sera containing antibody to IaCHO, IbCHO, or Ibc protein. Phagocytosis of all three serotypes was intermediate when opsonization could be attributed to anti-IabcCHO. The opsonization of a specific group B streptococcus is complex and may involve two or more antigen-antibody systems.
Highlights
This paper summarizes a series of experiments that were designed to develop a quantitative assay of phagocytosis and to identify the critical microbial antigens and serum requirements for the opsonization of group B streptococci
Coverslips without macrophages did not accumulate radioactivity when incubated with labeled bacteria exposed to homologous antiserum; this last control was regularly included in all experiments but does not appear in subsequent figures
When stained coverslips were examined under light microscopy, macrophages that had been incubated with streptococci exposed to specific antiserum appeared to contain numerous cocci (Fig. 2 A), but cultures incubated with bacteria treated with normal rabbit serum (NRS) did not (Fig. 2 B)
Summary
For each set of macrophage cultures from a single animal, the number of viable, adherent cells per tube was estimated by treating one or two coverslips with 1 ml of 0.25% trypsin in phosphate-bufferedsaline, pH 7.4, diluting the trypsin-cell suspension with 0.2% trypan blue in a leukocyte pipette and counting in a hemocytometer. 0.1 ml of the bacteria-serum mixtures and 0.9 ml of McCoy's medium with 10% FCS were added to each coverslip for a bacterial CFU:macrophage ratio of 100:1, and the Leighton tubes were agitated gently at 37°C on a rocking platform At 0, 30, 60, and 120 min, anumber of coverslips were washed three times with McCoy's medium and 10% FCS to remove free bacteria, air dried, and immersed in scintillation vials to determine radioactive uptake by macrophages. The macrophages were free of contamination by B. bronchiseptica
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