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Comparison of peritoneal macrophages from germfree and conventional mice
Abstract:
Morphology, lysosomal enzyme activities, and phagocytosis via immunological receptors were tested in peritoneal macrophages from germfree and conventional mice. Nonstimulated macrophages from germfree mice showed less spreading and were more easily detached when seeded on glass than conventional macrophages. The activities of the lysosomal acid phosphatase and cathepsin D were similar in the two cell groups, whereas beta-glucuronidase showed higher activity in macrophages from germfree mice. F(c) receptor-mediated phagocytosis of opsonized sheep erythrocytes was equally effective in germfree and conventional macrophages, and both cell types attached but did not internalize erythrocytes via the C(3)b receptor. Intraperitoneal injections of mineral oil caused a significantly higher influx of macrophages in conventional mice than in germfree mice, whereas the influx of polymorphonuclear cells was enhanced in both animals. Stimulation in vivo with oil or Escherichia coli endotoxin increased cell size, spreading ability, membrane ruffling, and lysosomal enzyme activities in macrophages from both conventional and germfree mice. The Fc-mediated phagocytosis was not influenced by stimulation, whereas the capacity to internalize via C(3)b receptor was triggered in macrophages from conventional mice, but not in corresponding cells from germfree mice. Similar results were obtained after stimulation with endotoxin in vitro. Culture in fetal calf serum for 72 h caused intracellular rises in all three enzyme activities tested in macrophages from conventional mice, whereas only the activity of acid phosphatase was increased in macrophages from germfree mice. Stimulation with zymosan in vitro caused selective release of lysosomal enzyme activity in macrophages from both animal groups. We conclude that peritoneal macrophages from germfree mice share several properties with cells from conventional mice, however, unstimulated beta-glucuronidase activity was increased, whereas spreading on glass, chemotactic response, in vitro induction of lysosomal enzymes, and the capacity to internalize via the C(3)b receptor after stimulation were reduced or absent.
Insights
Germfree mice macrophages show distinct differences from conventional ones, with altered enzyme activity and reduced response to certain immune stimuli, impacting their phagocytic capabilities.
Area of Science:
- Immunology
- Cell Biology
- Microbiology
Background:
- Peritoneal macrophages play a crucial role in innate immunity.
- Germ-free (GF) animals lack a microbiome, potentially altering immune cell function.
- Understanding macrophage differences between GF and conventional (CONV) mice is key to immune system research.
Purpose of the Study:
- To compare peritoneal macrophage morphology, lysosomal enzyme activity, and phagocytosis in GF and CONV mice.
- To investigate the effects of in vivo and in vitro stimulation on these macrophage functions.
Main Methods:
- Isolation and culture of peritoneal macrophages from GF and CONV mice.
- Assessment of cell morphology, spreading on glass, and detachment.
- Measurement of lysosomal enzyme activities (acid phosphatase, cathepsin D, beta-glucuronidase).
- Evaluation of phagocytosis via Fc and C3b receptors using opsonized sheep erythrocytes.
- In vivo stimulation with mineral oil or Escherichia coli endotoxin.
- In vitro stimulation with endotoxin or zymosan.
Main Results:
- Unstimulated GF macrophages exhibited reduced spreading and adherence compared to CONV.
- Beta-glucuronidase activity was higher in GF macrophages; acid phosphatase and cathepsin D were similar.
- Fc receptor-mediated phagocytosis was comparable, but C3b receptor-mediated uptake was impaired in GF macrophages, even after stimulation.
Conclusions:
- Peritoneal macrophages from GF mice share some properties with CONV mice but show reduced chemotaxis and impaired C3b receptor-mediated phagocytosis.
- GF macrophage responses to in vitro induction of lysosomal enzymes and C3b receptor stimulation are significantly diminished.
- These findings highlight specific functional deficits in GF macrophages, particularly concerning complement-mediated phagocytosis and certain inflammatory responses.