Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils

P. H. Hart, L. K. Spencer, N. L. Hill, P. J. McDonald, J. J. Finlay-Jones

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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 (<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.

Original languageEnglish
Pages (from-to)89-100
Number of pages12
JournalBritish Journal of Experimental Pathology
Volume68
Issue number1
Publication statusPublished - 5 May 1987
Externally publishedYes

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Exudates and Transudates
Neutrophils
Antibodies
Proteus mirabilis
Phagocytosis
Serum
Egtazic Acid
Bacteria
Edetic Acid
Suspensions
Classical Complement Pathway
Alternative Complement Pathway
Phagocytes
Centrifugation

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Hart, P. H. ; Spencer, L. K. ; Hill, N. L. ; McDonald, P. J. ; Finlay-Jones, J. J. / Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils. In: British Journal of Experimental Pathology. 1987 ; Vol. 68, No. 1. pp. 89-100.
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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 (<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.",
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Roles of antibody and complement in the bactericidal activity of mouse peritoneal exudate neutrophils. / Hart, P. H.; Spencer, L. K.; Hill, N. L.; McDonald, P. J.; Finlay-Jones, J. J.

In: British Journal of Experimental Pathology, Vol. 68, No. 1, 05.05.1987, p. 89-100.

Research output: Contribution to journalArticle

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