Sunderkötter C, Nikolic T, Dillon MJ, van Rooijen N, Stehling M, Drevets DA, Leenen PJ.
Subpopulations of Mouse Blood Monocytes Differ in Maturation Stage and Inflammatory Response.
J Immunol. 2004 Apr 1;172(7):4410–7.
[toggle_content title=”Abstract”]

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6Chigh subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6Chigh monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6Chigh monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6Cmed/high monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.
[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
10 mg/ml 1 46 mg/ml EPC/Chol 86/14 MLV none

1Clodronate and lipid concentrations assumed based on referenced paper.

[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Adjunct Dosing? Adjunct Dosing Route
C57BL/6J female mice. 8-16 w 200 µl intravenous CD11b+, Mac-1+, Ly-6C+, etc. monocytes no NA
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper.
  2. Reference for liposome preparation — Leenen PJM, Radosevic K, Voerman JSA, Salomon B, van Rooijen N, Klatzmann D, et al. Heterogeneity of Mouse Spleen Dendritic Cells: In Vivo Phagocytic Activity, Expression of Macrophage Markers, and Subpopulation Turnover. The Journal of Immunology. 1998;160(5):2166–73.
[/toggle_content][toggle_content title=”Results”]
  1. >90% monocytes depleted in blood 18 h post-injection of liposomal clodronate and begin to reappear 30 h later (Ly-C6hi); returned to baseline values within 4 d as Ly-C6hi while Ly-C6lo did not reappear until >7 d.
  2. DiD lipos (no clodronate) removed monocytes from circulation by 30 m, unlabelled Ly-6Chi reappeared at 2 hr and labeled Ly-6Clo (marginated) began to reappear at 16 h, therefore early (<2 h) monocyte “depletion” is actually margination.
  3. Ly-6Cmed/hi monocyte recruitment into the peritoneal cavity by sterile FCS was inhibited at 24 h by concomitant intravenous injection of clodronate liposomes.