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Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils
Abstract:
The contributions of complement and antibody to phagocytosis and, as a separate process, intracellular killing of Proteus mirabilis, were investigated using mouse peritoneal exudate neutrophils. Phagocytosis of P. mirabilis was promoted by both immune mouse (IMS) and normal mouse (NMS) sera. Opsonization by IMS promoted significantly greater phagocytosis than did NMS, as did NMS compared with heated IMS (HIMS). The ability of NMS to opsonize P. mirabilis for both phagocytosis and phagocytic killing was diminished by chelation with EGTA and abolished by chelation with EDTA. This suggested that fixation of complement by both alternative and classical pathways provided optimal opsonization of this organism in NMS. In order to study intracellular killing as a process separate from phagocytosis, peritoneal exudate cell suspensions were exposed to P. mirabilis, previously incubated with 1% NMS, 1% IMS, 10% HNMS (heated normal mouse serum) or 10% HIMS, followed by centrifugation of the phagocyte-bacteria mixtures on Percoll density gradients. Populations of neutrophils containing viable intracellular bacteria, and relatively free of extracellular bacteria (less than 7% of total) were recovered in washed suspensions of cells fractionated at densities greater than 1.069 g/ml. For P. mirabilis that had been opsonized with 1% NMS before phagocytosis, the continued presence of extracellular serum was necessary for intracellular killing. NMS stimulated significantly greater intracellular killing than did HNMS, which stimulated some intracellular killing compared with the absence of serum, in which no killing occurred. IMS was similar to NMS in its ability to stimulate intracellular killing. EGTA partially blocked the stimulation of intracellular killing by NMS, and EDTA abolished it. These findings suggested that (as for optimal opsonization) complement activated via both alternative and classical pathways was responsible for optimal stimulation of intracellular killing.
Insights
Complement and antibodies enhance neutrophil phagocytosis and killing of Proteus mirabilis. Complement activation via both classical and alternative pathways is crucial for optimal opsonization and intracellular bacterial killing.
Area of Science:
- Immunology
- Microbiology
- Cell Biology
Background:
- Neutrophils are key immune cells involved in host defense against bacterial infections.
- Phagocytosis and intracellular killing are critical mechanisms employed by neutrophils to eliminate pathogens.
- The roles of complement and antibodies in these processes are well-established but require specific investigation for different bacterial species.
Purpose of the Study:
- To investigate the distinct contributions of complement and antibody to phagocytosis and intracellular killing of Proteus mirabilis.
- To elucidate the specific complement pathways involved in opsonization and intracellular killing of P. mirabilis.
- To differentiate the processes of phagocytosis and intracellular killing using purified neutrophil populations.
Main Methods:
- Utilized mouse peritoneal exudate neutrophils for in vitro experiments.
- Assessed phagocytosis of P. mirabilis opsonized with normal mouse serum (NMS), immune mouse serum (IMS), and heated sera.
- Employed Percoll density gradient centrifugation to isolate neutrophils with intracellular bacteria, separating phagocytosis from intracellular killing.
Main Results:
- Both NMS and IMS significantly promoted P. mirabilis phagocytosis, with IMS providing superior opsonization.
- Complement fixation via both alternative and classical pathways, as indicated by EGTA and EDTA chelation studies, was essential for optimal opsonization.
- Intracellular killing of P. mirabilis by neutrophils required the presence of extracellular complement, with NMS and IMS demonstrating significant killing activity.
Conclusions:
- Complement activation through both alternative and classical pathways is critical for effective opsonization of P. mirabilis.
- Complement plays a vital role in stimulating intracellular killing of P. mirabilis by neutrophils, in addition to promoting phagocytosis.
- Antibodies, in conjunction with complement, enhance both the uptake and destruction of P. mirabilis by neutrophils.