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Updated: Jul 5, 2026
Isolation and In Vitro Culture of Murine and Human Alveolar Macrophages
Published on: April 20, 2018
Characterization of immortalized MARCO and SR-AI/II-deficient murine alveolar macrophage cell lines
Hongwei Zhou1, Amy Imrich, Lester Kobzik
1Department of Environmental Health, Harvard School of Public Health, Boston, MA, 02115, USA. lkobzik@hsph.harvard.edu.
Background:
Alveolar macrophages (AM) avidly bind and ingest unopsonized inhaled particles and bacteria through class A scavenger receptors (SRAs) MARCO and SR-AI/II. Studies to characterize the function of these SRAs have used AMs from MARCO or SR-AI/II null mice, but this approach is limited by the relatively low yield of AMs. Moreover, studies using both MARCO and SR-AI/II-deficient (MS-/-) mice have not been reported yet. Hence, we sought to develop continuous cell lines from primary alveolar macrophages from MS-/- mice.
Results:
We used in vitro infection of the primary AMs with the J2 retrovirus carrying the v-raf and v-myc oncogenes. Following initial isolation in media supplemented with murine macrophage colony-stimulating factor (M-CSF), we subcloned three AM cell lines, designated ZK-1, ZK-2 and ZK-6. These cell lines grow well in RPMI-1640-10% FBS in the absence of M-CSF. These adherent but trypsin-sensitive cell lines have a doubling time of approximately 14 hours, exhibit typical macrophage morphology, and express macrophage-associated cell surface Mac-1 (CD11b) and F4/80 antigens. The cell lines show robust Fc-receptor dependent phagocytosis of opsonized red blood cells. Similar to freshly isolated AMs from MS-/- mice, the cell lines exhibit decreased phagocytosis of unopsonized titanium dioxide (TiO2), fluorescent latex beads and bacteria (Staphylococcus aureus) compared with the primary AMs from wild type (WT) C57BL/6 mice.
Conclusion:
Our results indicated that three contiguous murine alveolar macrophage cell lines with MS-/- (ZK1, ZK2 and ZK6) were established successfully. These cell lines demonstrated macrophage morphology and functional activity. Interestingly, similar to freshly isolated AMs from MS-/- mice, the cell lines have a reduced, but not absent, ability to bind and ingest particles, with an altered pattern of blockade by scavenger receptor inhibitors. These cell lines will facilitate in vitro studies to further define MARCO and SR-AI/II function, and may also be useful to identify other novel scavenger-type macrophage receptors and for additional studies of particle toxicology.
Insights
Established murine alveolar macrophage cell lines lacking MARCO and SR-AI/II (MS-/-) exhibit reduced particle uptake. These cell lines aid in studying scavenger receptor function and particle toxicology.
Area of Science:
- Immunology
- Cell Biology
Background:
- Alveolar macrophages (AMs) utilize scavenger receptors (SRs), including MARCO and SR-AI/II, for binding and ingesting unopsonized particles.
- Previous studies on SR function were limited by low AM yields and lack of studies on combined MARCO and SR-AI/II deficiency (MS-/-).
Purpose of the Study:
- To develop continuous cell lines from primary alveolar macrophages derived from MS-/- mice.
- To establish a model for studying the function of MARCO and SR-AI/II in particle uptake.
Main Methods:
- Primary AMs from MS-/- mice were infected with a J2 retrovirus encoding v-raf and v-myc oncogenes.
- Three stable cell lines (ZK-1, ZK-2, ZK-6) were established and characterized for morphology, growth, and surface markers.
- Phagocytic activity of the cell lines was assessed using opsonized and unopsonized particles and bacteria.
Main Results:
- Three continuous murine alveolar macrophage cell lines (ZK-1, ZK-2, ZK-6) were successfully established from MS-/- mice.
- These cell lines exhibit typical macrophage morphology, express macrophage markers (Mac-1, F4/80), and demonstrate Fc-receptor dependent phagocytosis.
- The MS-/- cell lines showed reduced, but not absent, phagocytosis of unopsonized particles (TiO2, beads, S. aureus) compared to wild-type AMs.
Conclusions:
- The established MS-/- alveolar macrophage cell lines provide a valuable tool for in vitro research.
- These cell lines mimic the reduced particle uptake of primary MS-/- AMs, facilitating studies on MARCO and SR-AI/II function.
- The cell lines may aid in identifying novel scavenger receptors and in particle toxicology research.
