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The development of the mononuclear phagocyte system requires macrophage colony-stimulating factor (CSF-1) signaling through the CSF-1 receptor (CSF1R, CD115). We examined the effect of an antibody against CSF1R on macrophage homeostasis and function using the MacGreen transgenic mouse (csf1r-enhanced green fluorescent protein) as a reporter. The administration of a novel CSF1R blocking antibody selectively reduced the CD115(+)Gr-1(neg) monocyte precursor of resident tissue macrophages. CD115(+)Gr-1(+) inflammatory monocytes were correspondingly increased, supporting the view that monocytes are a developmental series. Within tissue, the antibody almost completely depleted resident macrophage populations in the peritoneum, gastrointestinal tract, liver, kidney, and skin, but not in the lung or female reproductive organs. CSF1R blockade reduced the numbers of tumor-associated macrophages in syngeneic tumor models, suggesting that these cells are resident type macrophages. Conversely, it had no effect on inflammatory monocyte recruitment in models, including lipopolysaccharide-induced lung inflammation, wound healing, peritonitis, and severe acute graft-versus-host disease. Depletion of resident tissue macrophages from bone marrow transplantation recipients actually resulted in accelerated pathology and exaggerated donor T-cell activation. The data indicate that CSF1R signaling is required only for the maturation and replacement of resident-type monocytes and tissue macrophages, and is not required for monocyte production or inflammatory function.

We have developed a highly sensitive cytolysis test, the fluorolysis assay, as a simple nonradioactive and inexpensive alternative to the standard 51Cr-release assay. P815 cells were stably transfected with a plasmid expressing the enhanced green fluorescent protein (EGFP) gene. These target cells were coated with or without cognate peptide or anti-CD3 Ab and then incubated with CD8(+) T cells to allow antigen-specific or nonspecific lysis. The degree of target cell lysis was measured using flow cytometry to count the percentage of viable propidium iodide(-) EGFP(+) cells, whose numbers were standardized to a reference number of fluorochrome-linked beads. By using small numbers of target cells (200-800 per reaction) and extended incubation times (up to 2 days), the antigen-specific cytolytic activity of one to two activated CD8(+) T cells of a CTL line could be detected. The redirected fluorolysis assay also measured the activity of very few (> or =6) primary CD8(+) T cells following polyclonal activation. Importantly, antigen-specific lysis by small numbers (> or =25) of primary CD8(+) T cells could be directly measured ex vivo. This exquisite sensitivity of the fluorolysis assay, which was at least 8-33-folds higher than an optimized 51Cr-release assay, allows in vitro and ex vivo studies of immune responses that would otherwise not be possible due to low CTL numbers or frequencies.

We have studied the effects of LP-BM5 MuLV-induced murine acquired immunodeficiency syndrome (MAIDS) on concomitant murine cytomegalovirus (MCMV) infection in the livers of C57BL mice. A delayed inflammatory response in livers of mice with MAIDS (M+) on day 4 was associated with impaired clearance of MCMV-infected cells 6 days after infection. This correlated with increased levels of inflammation and serum alanine transaminase. The latter reflects enhanced hepatic necrosis, which was evident histologically. Delayed-type hypersensitivity responses to MCMV antigen were unimpaired in M+ mice and were mediated by CD8+ cells. Depletion of NK1.1+ cells from M+ mice increased MCMV replication and associated liver damage on day 6, whereas CD8+ depletion had little effect. In contrast, in the presence of CD8+ cells M- C57BL mice did not require NK1.1+ cells for control of hepatic MCMV infection, but dual NK1.1+ and CD8+ depletion dramatically potentiated hepatic MCMV replication. Our results suggest that M+ mice may acquire non-NK1.1+ and non-CD8+ cells that are able to partially control hepatic MCMV infection. These findings are discussed with reference to mortality in M+ mice after high-dose MCMV infection, as this is initially delayed but ultimately higher than in M- controls.