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Macrophages (M phi s) undergo a physiological response known as the macrophage disappearance reaction (MDR) in response to certain stimuli in the peritoneal compartment. The types of stimuli that can cause the MDR, the relationship of the MDR to the host immunological response, and the possible role of the MDR in M phi activation are reviewed. The data indicate that the MDR occurs in response to both acute nonspecific inflammatory and specific immune delayed hypersensitivity processes and that the MDR may play an important role in M phi activation.

Resident peritoneal macrophages (M phi) were labeled in situ by intraperitoneal (i.p.) injection of the green fluorescent cell tracking dye PKH-1. After immunofluorescence staining with M phi specific monoclonal antibodies (Mabs) and phycoerythrin (PE) second antibody, the resident M phi were labeled with both the green dye and red Mab label, while recruited M phi were labeled only with the red Mab tag. These populations were distinguished by two-color flow cytometry. PKH-1 labeled resident peritoneal M phi were followed for 1-49 days in mice that received no further treatment (steady state). Dye labeled M phi were still detectable after 49 days in vivo, although their green fluorescence intensity had decreased steadily over time. The decrease in dye intensity was limited to M phi, as the fluorescence intensity of PKH-1 labeled peritoneal lymphocytes did not change. Resident M phi populations were clearly separated from recruited M phi by the intensity of their staining with PKH-1 for up to 28 days. No decrease in the number of resident (dye labeled) peritoneal M phi was observed over 1-28 days. These data indicate that resident peritoneal M phi were not replaced by recruited blood monocytes in the steady state.

A novel method for labeling resident peritoneal macrophages (M phi) by injection of a dye into the peritoneal cavity is described. The dye, which fluoresces green, is selectively taken up by the resident M phi. Dye labeled cells can be further characterized by labeling of cell surface antigens with monoclonal antibodies (Mabs) and phycoerythrin conjugated second antibody. After such labeling with the Mabs F4/80 or Mac 1 the resident M phi were labeled by both the green dye and the red Mab markers, while recruited M phi or neutrophils were labeled with just the red Mab; the two populations of cells were readily distinguished by two-color flow cytometry. This technique enabled identification of resident and recruited M phi in each animal without the use of radioisotopes, irradiation, or bone marrow ablation. Sufficient numbers of cells can be analyzed from each animal so that individual animals could be evaluated. We found no adverse effects of this labeling technique on expression of cell surface antigens or M phi mediated cytotoxicity. We did find evidence that the i.p. injection induced a mild inflammation in the peritoneal cavities of animals injected with either the dye or the balanced salt solution vehicle. Examination of the intracellular staining pattern indicated that the label rapidly sequestered in the cytoplasm of the M phi, possibly in the lysosomes. Dye solubility studies showed that the dye was partially soluble at the concentration used for in vivo labeling. We hypothesize that the M phi labeling occurred by a combination of phagocytosis of dye aggregates and endocytosis of labeled plasma membrane.