2010-winkler-4815

On Thu, 12-Jul-2012   /   Intravenous Injection, Retro-orbital  
Winkler IG, Sims NA, Pettit AR, Barbier V, Nowlan B, Helwani F, Poulton IJ, van Rooijen N, Alexander KA, Raggatt LJ, Levesque, JP. Bone marrow macrophages maintain hematopoietic stem cell (HSC) niches and their depletion mobilizes HSCs. Blood. 2010 Aug 16;116(23):4815–28.

Abstract

In the bone marrow (BM), hematopoietic stem cells (HSC) reside in specific niches near osteoblast-lineage cells at the endosteum. To investigate regulation of these endosteal niches, we studied mobilization of HSC into the bloodstream in response to granulocyte colonystimulating factor (G-CSF). We report that G-CSF mobilization rapidly depletes endosteal osteoblasts leading to suppressed endosteal bone formation and decreased expression of factors required for HSC retention and self-renewal. Importantly, G-CSF administration also depleted a population of trophic endosteal macrophages (osteomacs) that support osteoblast function. Osteomac loss, osteoblast suppression and HSC mobilization occurred concomitantly, suggesting that osteomac loss could disrupt endosteal niches. Indeed in vivo depletion of macrophages, in either macrophage Fas-induced apoptosis (Mafia) transgenic mice or by administration of clodronate-loaded liposomes to wild-type mice, recapitulated the i) loss of endosteal osteoblasts, ii) marked reduction of HSC-trophic cytokines at the endosteum, with iii) HSC mobilization into the blood as observed during G-CSF administration. Together these results establish that BM macrophages are pivotal to maintain the endosteal HSC niche and that the loss of such macrophages leads to the egress of HSC into the blood.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.4 mg/ml EPC/Chol 84/16 MLV PBS
[/custom_table]  [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Local Dosing? Local Results
C57BL/6, female mice, 8-12 w 250 µl/25 g retro-orbital F4/80+Ly-6G+CD11b+ osteomacs no N/A
[/custom_table]

Notes

  1. Liposome prep method cited — van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods. 1994 Sep 14;174(1–2):83–93.
  2. Animals were dosed with clodronate (or control) liposomes on days 2 and 4.

Results

  1. >95% of the F4/80+Ly-6G+CD11b+ osteomacs and other bone marrow macrophages were depleted by day 2; depletion continued throughout experiment (6 d).
  2. Effects on endosteal niches began within 24 h of clodronate liposome dosing.
  3. “Substantial reduction” in osteocalcin+ osteoblasts by 48 h presumably due to osteomac depletion rather than direct effect of clodronate liposomes on osteoblasts.
  4. We are curious about the mechansim of osteomac depletion by liposomal clodronate since the osteomacs are shown to exclusively associate with osteoblasts which are remotely located from the sinusoid in the bone marrow. How does the liposome enter the marrow from sinusoid and migrate through the central bone marrow to the endosteum where the osteomacs are located? Since liposomes have been shown to predominantly remain near the capillaries rather than migrating into tissue (other than those carried by macrophages), we wonder if depletion of other macrophages located perisinally elicit migration of the osteomacs toward the sinusoid.

 

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2011-chow-261

On Tue, 10-Jul-2012   /   Intravenous Injection  
Chow A, Lucas D, Hidalgo A, Méndez-Ferrer S, Hashimoto D, Scheiermann C, Battista M, Leboeuf M, Prophete C, van Rooijen N, Tanaka M, Merad M, Frenette PS. Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche. J Exp Med. 2011 Feb 14;208(2):261–71.
[toggle_content title=”Abstract”]Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.4 mg/ml EPC/Chol 84/16 MLV PBS
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Local Dosing? Local Results
C57BL/6 mice2, 8-12 w 250 µl i.v./unspecified GR-1hi/Gr-1lo monocytes, GR-1-F4/80+CD169+ macrophages no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Liposome prep method cited — van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of Immunological Methods. 1994 Sep 14;174(1–2):83–93.
  2. Animals were dosed with clodronate (or control) liposomes at 1 d (14 h), 7 d, 10 d, 16 d, or 28 d before harvest.
[/toggle_content] [toggle_content title=”Results”]
  1. 14 h post-injection of clodronate liposomes, BM MΦ (GR-1-F4/80+CD169+) were reduced by 84%; GR-1hi by 88% and GR-1lo by 74%; total BM cells were depleted by 24%.
  2. Concurrently, circulating HSC increased by 12.9X.
  3. These MΦ remained >90% depleted 7 d post-injection, had recovered to 58% by 16 d  and were fully repopulated by 28 d.
  4. GR-1+ monocytes began to return at 7 d post-injection and did not affect HSC mobilization.
  5. HSC mobilization remained elevated at least until 16 d post-injection further supporting the role of MΦ, but not monocytes, in the process.
  6. MΦ depletion also elicited HSC mobilization, albeit at a lower level, in sympathectomized animals.
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2001-emanuilidis-3919

On Fri, 06-Jul-2012   /   Intravenous Injection  
Emmanuilidis K, Weighardt H, Maier S, Gerauer K, Fleischmann T, Zheng XX, Hancock W, Holzmann B, Heidecke CD.
Critical Role of Kupffer Cell-Derived IL-10 for Host Defense in Septic Peritonitis.
J Immunol. 2001 Oct 1;167(7):3919–27.
[toggle_content title=”Abstract”]Intra-abdominal infection in patients following major visceral surgery is associated with high mortality. Using a macrophage depletion technique, we demonstrate that in murine septic peritonitis, Kupffer cells are a major source of systemic IL-10 levels. Kupffer cell-depleted mice were highly susceptible to the lethal effects of septic peritonitis and exhibited an increased bacterial load. Kupffer cell-depleted mice were protected by the administration of an IL-10-Fc fusion protein. Loss of Kupffer cell-derived IL-10 was associated with a weak increase in serum IL-12 levels, whereas TNF, IL-1, and IL-18 levels were not significantly elevated, suggesting that the loss of Kupffer cell-derived IL-10 did not result in a toxic cytokine release syndrome. Instead, loss of Kupffer cell-derived IL-10 was associated with a reduced splenocyte production of IFN- that is required for immune protection in murine septic peritonitis. Therefore, the results suggest that the protective function of IL-10 in septic peritonitis may not be restricted to the anti-inflammatory activities of IL-10. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.4 mg/ml EPC/Chol 84/16 MLV none
[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Local Dosing? Local Results
C57BL/6, female mice, 8-12 w 40 µl (diluted to 160 µl) i.v./not specified F4/80+ Kupffer cells no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Liposome prep method cited was a review, so standard clodronate liposome parameters assumed.
  2. Animals were dosed with clodronate liposomes 24 h prior to surgical procedure used to induce (peritonitis) sepsis.
  3. Some groups were splenectomized to determine the contribution of splenic macrophages.
[/toggle_content] [toggle_content title=”Results”]
  1. Peritoneal and aveolar macrophages were not reduced in number, but this was a 5X lower dose than often used in other papers (40 µl vs 200 µl).
  2. Authors report “complete” depletion of Kupffer cells along with splenic marginal macrophages and metallophilic macrophages, but not red pulp macrophages within 24 h by histology.
  3. Depletion lasted for at least 72 h.
  4. All other measurements on clodronate liposome-treated animals were either cytokine levels or IL-10 mRNA levels.
  5. The authors did not evaluate depletion in septic mice, therefore within the 12 h post-induction of sepsis (36 h post-clodronate liposome injection) before organs were harvested, would some migration of repopulating monocytes into tissues (including liver and spleen) occur?
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2009-zecher-7810

On Thu, 28-Jun-2012   /   Intravenous Injection  
Zecher D, van Rooijen N, Rothstein DM, Shlomchik WD, Lakkis FG.
An innate response to allogeneic nonself mediated by monocytes.
The Journal of Immunology. 2009;183(12):7810.
[toggle_content title=”Abstract”] The mammalian innate immune system has evolved diverse strategies to distinguish self from microbial nonself. How the innate immune system distinguishes self-tissues from those of other members of the same species (allogeneic nonself) is less clear. To address this question, we studied the cutaneous hypersensitivity response of lymphocyte-deficient RAG-/- mice to spleen cells transplanted from either allogeneic or syngeneic RAG-/- donors. We found that RAG-/- mice mount a specific response to allogeneic cells characterized by swelling and infiltration of the skin with host monocytes/macrophages and neutrophils. The response required prior priming with allogeneic splenocytes or skin grafts and exhibited features of memory as it could be elicited at least 4 wk after immunization. Neither depletion of host NK cells nor rechallenging immunized mice with F1 hybrid splenocytes inhibited the response, indicating that the response is not mediated by NK cells. Depletion of host monocytes/macrophages or neutrophils at the time of rechallenge significantly diminished the response and, importantly, the adoptive transfer of monocytes from alloimmunized RAG-/- mice conferred alloimmunity to naive RAG-/- hosts. Unlike NK- and T cell-dependent alloresponses, monocyte-mediated alloimmunity could be elicited only when donor and responder mice differed at non-MHC loci. These observations indicate that monocytes mount a response to allogeneic nonself, a function not previously attributed to them, and suggest the existence of mammalian innate allorecognition strategies distinct from detection of missing self-MHC molecules by NK cells. [/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 PBS

1Clodronate and lipid concentrations assumed based on information in paper (dosed 2 mg in 0.2 ml).

[/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/6, C57BL/10 mice 2 , 8-20 w 250 µl intravenous/tail vein CD115+ F4/80int monocytes; F4/80+
Gr-1int splenic monocytes/MΦ; CD115+ F4/80+ bone marrow MΦ		 no NA

2 Base or background strain. Variants, mutants or otherwise genetically altered strains were also used in this study.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper, although references provide little information as to final clodronate concentrations.
  2. Reference for clodronate liposome preparation – van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of immunological methods. 1994;174(1-2):83–93.
[/toggle_content][toggle_content title=”Results”]
  1. % Control Cells Remaining Post-Clodronate Liposome Treatment [custom_table]
    Tissue 16 h 24 h 48 h
    Bone Marrow 40 75 130
    Blood Monocytes 5 10 110
    Spleen 1 1 1

    Numbers extrapolated from supplemental figure 1 in paper.[/custom_table]

  2. MΦ/monocytes or neutrophil depletion reduces the allogenic non-self response (pinnae swelling or DTH) by 50%.
  3. PBS control liposome administration does not affect the response.
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1990-vanrooijen-215

On Wed, 27-Jun-2012   /   Intravenous Injection  
van Rooijen N, Kors N, vd Ende M, Dijkstra CD.
Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate.
Cell Tissue Res. 1990 May;260(2):215–22.
[toggle_content title=”Abstract”] Rats received a single intravenous injection with liposome-encapsulated dichloromethylene diphosphonate (C12MDP). This treatment resulted in the elimination of macrophages in spleen and liver within 2 days. Macrophages ingest the liposomes and are destroyed by the drug, which is released from the liposomes after disruption of the phospholipid bilayers under the influence of lysosomal phospholipases. Repopulation of macrophages in spleen and liver was studied at different time intervals after treatment. Macrophages in the liver (Kupffer cells) and red pulp macrophages in the spleen were the first cells to reappear, followed by marginal metallophilic macrophages and marginal-zone macrophages in the spleen. Different markers of the same cell did not reappear simultaneously. On the other hand, the same marker (recognized by the monoclonal antibody ED2) reappeared much more rapidly in the liver than in the spleen. The present results in the rat were different from those earlier obtained in the mouse. Red pulp macrophages were the first cells and marginal zone macrophages were the last cells to repopulate the spleen in both rodents after treatment with C12MDP liposomes. However, there was much more overlap in the repopulation kinetics of splenic macrophage subpopulations in the rat, when compared with the mouse.[/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

1 Clodronate and lipid concentrations assumed based on information in paper (dosed 2 mg in 0.2 ml).

[/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
Wistar rats, female,  adult 20 mg in 2 ml intravenous ED1+, ED2+, ED3+, ED7+ MΦ no NA
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper, although references provide little information as to final clodronate concentrations.
  2. Reference for clodronate liposome preparation — van Rooijen N, Kors N, ter Hart H, Claassen E. In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate. Virchows Arch., B, Cell Pathol. 1988;54(4):241–5.
[/toggle_content][toggle_content title=”Results”]
  1. The repopulation kinetics of spleen and liver macrophage subpopulations differ considerably between rats (data from this paper) and mice (historical data).
  2. In rat spleens, only cellular remnants of ED1+/ED2+/ED3+ macrophages remain on days 2 and 4 post-injection. ED1+ cells began to reappear in the red pulp on day 8. ED2+ and ED3+ cells were detected on day 16 in selective regions. ED1+ cells returned to baseline values by day 32. ED2+/ED3+ cells were back to normal levels by day 65.
  3. Interdigitating dendritic cells were not depleted.
  4. Other macrophages in the white pulp appeared rounded and swollen on day 8, but did not die.
  5. Rat liver ED1+/ED2+ macrophages were depleted by day 2 and ED2+ cells did not reappear until day 8. A few small, round ED1+ cells were found on day 4, but had regained their normal morphology by day 8. Both populations had returned to baseline levels on day 16.
  6. Bone marrow, peritoneal and other lymphoid tissues were examined, however only some showed a slight decrease in ED1+ and ED3+ cells. Alveolar macrophages were not collected.
  7. After day 16, ED3+ cells appeared in lymphoid tissues where they are not normally found; the authors speculated that autoimmune mechanisms were at play.
  8. The presence of swollen high endothelial venules were observed in bronchial and tracheal lymph nodes which the authors also proposed to be due to autoimmune responses.
  9. The fact that various subpopulations return to tissues at various rates should be considered when interpreting results from depletion experiments. Cell-surface receptor variability and functional differences between the subpopulations may result in abnormal macrophage responses when experimental data is collected during macrophage repopulation after depletion.
  10. Repopulation rates must be carefully considered when experimental protocols are dependent on the absence of phagocytic activity in all or select tissues. For example, monocytes may begin to repopulate before maximal depletion is reached in the spleen and liver.
  11. To reiterate, depletion and repopulation rates will also differ between species including within rodent species. Therefore, historical data from mice may not be applicable when running depletion experiments in rats or guinea pigs, for example.
  12. We also wonder if the various immunocompromised mutants could demonstrate atypical depletion and repopulation kinetics within a species.
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1998-leenen-2166

On Tue, 26-Jun-2012   /   Intravenous Injection  
Leenen PJM, Radosevic K, Voerman JSA, Salomon B, van Rooijen N, Klatzmann D, van Ewijk W.
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 title=”Abstract”] In the normal mouse spleen, two distinct populations of dendritic cells (DC) are present that differ in microanatomical location. The major population of marginal DC is found in the “marginal zone bridging channels” and extends into the red pulp. The interdigitating cells (IDC) are localized in the T cell areas in the white pulp. The aim of the present study was to characterize these two splenic DC populations with regard to their phenotype, in vivo phagocytic function, and turnover. Both marginal DC and IDC are CD11c+ and CD13+, but only IDC are NLDC-145+ and CD8α+. Notably, both populations, when freshly isolated, express the macrophage markers F4/80, BM8, and Mac-1. To study the phagocytic capacity of these cells, we employed the macrophage “suicide” technique by injecting liposomes loaded with clodronate i.v. Marginal DC, but not IDC, were eliminated by this treatment. Phagocytosis of DiI-labeled liposomes by DC confirmed this finding. The two DC populations differed significantly with regard to their turnover rates, as studied in a transgenic mouse model of conditional depletion of DC populations with high turnover. In these mice, marginal DC were completely eliminated, but the IDC population remained virtually intact. From these data we conclude that the marginal DC population has a high turnover, in contrast to the IDC population. Taken together, the present results indicate that marginal DC and IDC represent two essentially distinct populations of DC in the mouse spleen. They differ not only in location, but also in phenotype, phagocytic ability, and turnover. [/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 information in paper (dosed 2 mg in 0.2 ml).

[/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/6, C57BL/10 mice 2, 8-20 w 200 µl intravenous CD11c+, CD13+, F4/80+, etc. splenic DC / IDC no NA

2Base or background strain. Variants, mutants or otherwise genetically altered strains were also used in this study.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper, although references provide little information as to final clodronate concentrations.
  2. Reference for clodronate liposome preparation – van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of immunological methods. 1994;174(1-2):83–93.
[/toggle_content][toggle_content title=”Results”]
  1. Splenic marginal, but not interdigitated, dendritic cells were completely depleted at 48 h post-liposomal clodronate treatment as judged by histological analysis.
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2003-vanrijn-2522

On Tue, 26-Jun-2012   /   Intravenous Injection  
van Rijn RS, Simonetti ER, Hagenbeek A, Hogenes MCH, de Weger RA, Canninga-van Dijk MR, Weijer K, Spits H, Storm G, van Bloois L, Rijkers G, Martens AC, Ebeling SB. 
A New Xenograft Model for Graft-Versus-Host Disease by Intravenous Transfer of Human Peripheral Blood Mononuclear Cells in RAG2-/- ɣc-/- Double-Mutant Mice.
Blood. 2003 Oct 1;102(7):2522–31.
[toggle_content title=”Abstract”] The safe application of new strategies for the treatment of graft-versus-host disease (GVHD) is hampered by the lack of a clinically relevant model for preclinical testing. Current models are based on intraperitoneal transfer of human peripheral blood mononuclear cells (huPBMCs) into NOD-SCID (nonobese diabetic-severe combined immunodeficient)/SCID mice. Intravenous transfer would be preferred but this has always been ineffective. We developed a new model for xenogeneic GVHD (X-GVHD) by intravenous transfer of huPBMCs into RAG2-/- γc-/- mice. Our results show a high human T-cell chimerism of more than 20% (up to 98%) in more than 90% of mice, associated with a consistent development of XGVHD within 14 to 28 days and a total mortality rate of 85% shorter than 2 months. After murine macrophage depletion, engraftment was earlier and equally high with lower doses of huPBMCs. Human macrophages were also absent in these mice. Purified huCD3+ cells showed a similar X-GVH effect with contribution of both CD4 and CD8 phenotypes. Human immunoglobulins and cytokines were produced in diseased mice. One of 30 mice developed chronic X-GVHD with skin histology similar to human GVHD. In conclusion, we present a new model for X-GVHD by intravenous transfer of huPBMCs in RAG2-/- γc-/- mice. Murine and human macrophages do not seem to be necessary for acute X-GVHD in this model. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
2-2.5 mg/ml 69. 4 mg/ml 1 EPC/Chol/EPG 80/12/8 MLV PBS

1 Phospholipid concentration; cholesterol not assayed.

[/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
RAG2 -/- γc -/- mice, 8-34 w 200 µl intravenous systemic MΦ no NA
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. We commend the authors for assaying both clodronate and lipid concentrations in their preparation, however they selected an atypical formulation of clodronate liposomes for which they cite nor acknowledge any depletion studies performed with this particular formulation. Confirmed depletion of any population of macrophages at any time point was not reported in this paper. Depletion was assumed.
  2. The study cited for macrophage depletion was one in which rat spleen and liver macrophage depletion and repopulation was compared to those previously reported for mice. The earliest time point assessed was 2 days, while this van Rijn, et. al. study assumed depletion after one day at which time the huPBMC were injected.
  3. The clodronate-to-lipid wt ratio was significantly smaller 2.5/69 than that of other commonly used formulations (5/23 range), although the volume dosed was the same as that used for more common formulations. These animals received about half the usual clodronate dose and 3X more lipid than the usual dose.
[/toggle_content] [toggle_content title=”Results”]
  1. The goal of the authors was to develop a model of GVHD post-intravenous, rather than intraperitoneal, injection of huPBMC to more accurately mimic human GVHD caused by the intravenous introduction of donor PBMC. The engraftment rate leading to the development of GVHD was enhanced in the clodronate-liposome treated groups vs PBS-liposome treated groups suggesting that some level depletion of some population of phagocytes was critical to the development of this model. However, the authors failed to establish the extent, time of maximal depletion and repopulation rate of any phagocyte population. We believe that characterizing the phagocyte depletion parameters is absolutely necessary in establishing and optimizing this model. Additionally, it seems to us that determining which phagocyte populations are or are not involved in enhancing the engraftment rate is an important piece of information in characterizing GVHD.
  2. The authors speculate that the clodronate liposomes deplete the phagocytes in the injected huPBMC, however this is extremely unlikely, if not impossible, since the huPBMC were injected 1 day post-injection of the clodronate liposomes. These clodronate liposomes would not remain in the circulation for more than a few hours.
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2006-tacke-583

On Fri, 22-Jun-2012   /   Intravenous Injection  
Tacke F, Ginhoux F, Jakubzick C, van Rooijen NV, Merad M, Randolph GJ.
Immature Monocytes Acquire Antigens from Other Cells in the Bone Marrow and Present Them to T Cells After Maturing in the Periphery.
 J Exp Med. 2006 Mar 20;203(3):583–97.
[toggle_content title=”Abstract”] Monocytes are circulating precursors for tissue macrophages and dendritic cells (DCs) but are not recognized to directly participate in antigen presentation. We developed techniques to label mouse monocyte subsets with particulate tracers in vivo. Gr-1 lo but not Gr-1 hi monocytes were stably labeled by intravenous injection of 0.5-μm microspheres. Gr-1 hi monocytes could be labeled when the microspheres were injected after systemic depletion of blood monocytes and spleen macrophages. In this condition, the phagocytic tracer was transferred to immature bone marrow monocytes by neutrophils and B cells that first carried the particles to the bone marrow. Moreover, antigens from B cells or proteins conjugated to the tracer particles were processed for presentation by monocytes and could induce T cell responses in the periphery. Cell-associated antigen taken up by bone marrow monocytes was retained intracellularly for presentation of the antigen days later when monocyte-derived DCs migrated to lymph nodes or in vitro after differentiation with granulocyte/macrophage colony-stimulating factor. These data reveal that immature monocytes unexpectedly sample antigen from the bone marrow environment and that they can present these antigens after they leave the bone marrow. [/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

1 Clodronate 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/6 & BALB/c mice 2 250 µl intravenous CD115+, F4/80+ monocytes no NA

2 Base or background strain. Variants, mutants or otherwise genetically altered strains were also used in this study.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Clodronate and lipid concentrations assumed based on referenced paper, although references provide conflicting information as to final clodronate concentrations.
  2. Reference for clodronate liposome preparation – van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of immunological methods. 1994;174(1-2):83–93.
[/toggle_content] [toggle_content title=”Results”]
  1. >90% of monocytes (based on estimate from figure) depleted in blood 18 h post-injection of liposomal clodronate and returned to baseline at 48 h.
  2. Depletion corresponded to reduced pulmonary permeability (vascular leakage) post-LPS challenge.
  3. Very large numbers of monocytes marginated in organs during low dose LPS challenge, therefore total monocyte count in blood grossly underestimates total number of monocytes in the vascular system (also true for neutrophils). This could result in larger-than-expected levels of inflammatory cytokines in the circulation during some inflammatory events.
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2009-o’dea-1155

On Fri, 22-Jun-2012   /   Intravenous Injection  
O’Dea KP, Wilson MR, Dokpesi JO, Wakabayashi K, Tatton L, van Rooijen N, Takada M.
Mobilization and margination of bone marrow Gr-1high monocytes during subclinical endotoxemia predisposes the lungs toward acute injury.
The Journal of Immunology. 2009;182(2):1155.
[toggle_content title=”Abstract”]The specialized role of mouse Gr-1high monocytes in local inflammatory reactions has been well documented, but the trafficking and responsiveness of this subset during systemic inflammation and their contribution to sepsis-related organ injury has not been investigated. Using flow cytometry, we studied monocyte subset margination to the pulmonary microcirculation during subclinical endotoxemia in mice and investigated whether marginated monocytes contribute to lung injury in response to further septic stimuli. Subclinical low-dose i.v. LPS induced a rapid (within 2 h), large-scale mobilization of bone marrow Gr-1high monocytes and their prolonged margination to the lungs. With secondary LPS challenge, membrane TNF expression on these premarginated monocytes substantially increased, indicating their functional priming in vivo. Zymosan challenge produced small increases in pulmonary vascular permeability, which were markedly enhanced by the preadministration of low-dose LPS. The LPS-zymosan-induced permeability increases were effectively abrogated by pretreatment (30 min before zymosan challenge) with the platelet-activating factor antagonist WEB 2086 in combination with the phosphatidylcholine-phospholipase C inhibitor D609, suggesting the involvement of platelet-activating factor/ceramide-mediated pathways in this model. Depletion of monocytes (at 18 h after clodronate-liposome treatment) significantly attenuated the LPS-zymosan-induced permeability increase. However, restoration of normal LPS-induced Gr-1high monocyte margination to the lungs (at 48 h after clodronate-liposome treatment) resulted in the loss of this protective effect. These results demonstrate that mobilization and margination of Gr-1high monocytes during subclinical endotoxemia primes the lungs toward further septic stimuli and suggest a central role for this monocyte subset in the development of sepsis-related acute lung injury. [/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/ml1 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 mice. 8-12 w 200 µl intravenous CD11b+, Gr-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 — Reference for clodronate liposome preparation – van Rooijen N, Sanders A. Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. Journal of immunological methods. 1994;174(1-2):83–93.
[/toggle_content][toggle_content title=”Results”]
  1. Monocyte depletion at 18 h post-clodronate-liposome treatment prevented increase in pulmonary vascular permeability induced by LPS; LPS effect returned at 48 h post-clodronate-liposome treatment when monocyte levels returned to near baseline.
  2. In the absence of depletion, the large numbers of marginated monocytes in organs are not detectable in blood, although marginated monocytes may be producing unexpectedly high levels of cytokines in the bloodstream.
  3. Monocyte margination should be taken into account when interpreting monocyte depletion studies as well as monocyte behavior in pathogen-induced inflammation models in general.
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2004-sunderkötter-4410

On Fri, 22-Jun-2012   /   Intravenous Injection  
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.
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