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
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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
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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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2009-erler-35

On Tue, 10-Jul-2012   /   Unspecified  
Erler JT, Bennewith KL, Cox TR, Lang G, Bird D, Koong A, Le QT, Giaccia AJ.  Hypoxia-Induced Lysyl Oxidase Is a Critical Mediator of Bone Marrow Cell Recruitment to Form the Premetastatic Niche. Cancer Cell. 2009 Jan;15(1):35–44.

Abstract

Tumor cell metastasis is facilitated by ‘‘premetastatic niches’’ formed in destination organs by invading bone marrow-derived cells (BMDCs). Lysyl oxidase (LOX) is critical for premetastatic niche formation. LOX secreted by hypoxic breast tumor cells accumulates at premetastatic sites, crosslinks collagen IV in the basement membrane, and is essential for CD11b+ myeloid cell recruitment. CD11b+ cells adhere to crosslinked collagen IV and produce matrix metalloproteinase-2, which cleaves collagen, enhancing the invasion and recruitment of BMDCs and metastasizing tumor cells. LOX inhibition prevents CD11b+ cell recruitment andmetastatic growth. CD11b+ cells and LOX also colocalize in biopsies of human metastases. Our findings demonstrate a critical role for LOX in premetastatic niche formation and support targeting LOX for the treatment and prevention of metastatic disease.[/toggle_content]

 

[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] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Local Dosing? Local Results
Nude mice not specified not specified CD11b+ bone marrow derived cells no N/A
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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; van Rooijen N, van Kesteren-Hendrikx E. In vivo “depletion of macrophages by liposome-mediated”suicide. Methods in Enzymology [Internet]. 2003 [cited 2012 Feb 25]. p. 3–16.
  2. No details provided on route of administration, volume or amount dosed or time post-treatment at which depletion was evaluated.

Results

 

  1. The authors report that depletion of CD11b+ bone marrow-derived cells reduce those cells present in two mouse tumor models as well as the number and size of metastatic foci found in these models.
  2. The authors further acknowledge that clodronate liposome depletion is not specific to CD11b+ cells and that the effects of depletion may reduce metastatic tumor size and number by mechanisms not related to CD11b+ cell depletion.

 

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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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2011-alexander-1511

On Mon, 02-Jul-2012   /   Intradefect (Bone)  
Alexander KA, Chang MK, Maylin ER, Kohler T, Müller R, Wu AC, van Rooijen N, Sweet MJ, Hume DA, Raggatt LJ, Pettit AR.
Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model.
Journal of Bone and Mineral Research. 2011 Jul;26(7):1517–32.
[toggle_content title=”Abstract”] Bone-lining tissues contain a population of resident macrophages termed osteomacs that interact with osteoblasts in vivo and control mineralization in vitro . The role of osteomacs in bone repair was investigated using a mouse tibial bone injury model that heals primarily through intramembranous ossification and progresses through all major phases of stabilized fracture repair. Immunohistochemical studies revealed that at least two macrophage populations, F4/80 + Mac-2 -/low TRACP – osteomacs and F4/80+Mac-2 hi TRACP – inflammatory macrophages, were present within the bone injury site and persisted throughout the healing time course. In vivo depletion of osteomacs/macrophages (either using the Mafia transgenic mouse model or clodronate liposome delivery) or osteoclasts (recombinant osteoprotegerin treatment) established that osteomacs were required for deposition of collagen type 1 + (CT1 + ) matrix and bone mineralization in the tibial injury model, as assessed by quantitative immunohistology and micro–computed tomography. Conversely, administration of the macrophage growth factor colony-stimulating factor 1 (CSF-1) increased the number of osteomacs/macrophages at the injury site significantly with a concurrent increase in new CT1 + matrix deposition and enhanced mineralization. This study establishes osteomacs as participants in intramembranous bone healing and as targets for primary anabolic bone therapies. [/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
7 mg/ml 1 23.5 mg/ml EPC/Chol 86/14 MLV PBS

1 Based on typical encapsulation results reported by van Rooijen and Sanders (1994).

[/custom_table] [/toggle_content] [toggle_content title=”Animals and Dosing”] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
Mafia 2 mice, 11-12 w 100 µl intradefect/bone injury site F4/80+ osteomacs yes (i.p. 10 µl/g) not quantitated

2 Transgenic mice; C57BL/6 mice were used as controls.

[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Mice were dosed by injecting 100 µl clodronate liposomes into the tibial bone injury site at the time of surgical injury followed by 10 µl/g clodronate liposomes (200-250 µl) dosed i.p. daily X 9 d.
[/toggle_content] [toggle_content title=”Results”]
  1. FACS analysis of cells isolated from the control bone (contralateral uninjured tibia) showed an average 25.5% depletion in F4/80+ macrophages as a result of the i.p. injections.
  2. Immunohistochemistry of the injured bone confirmed depletion of F4/80+ macrophages.
  3. µCT confirmed a statistically significant reduction in bone density in mice treated with clodronate liposomes vs. PBS liposomes.
  4. Authors did not directly compare osteomac counts in control vs. injured bone, therefore we cannot determine the contribution of the intradefect injection of clodronate liposomes.
  5. Could pre-treatment of mice 18-24 hours before bone injury have effected the bone healing? Or, could withholding clodronate liposome treatment until 3 days post-surgery after the inflammatory phase have made a difference? Although the study focused on days 4 through 9 after surgery, we wonder how, or if, macrophage depletion during (vs before or after) the inflammatory phase changes the timing or extent of anabolic bone modeling (days 4-7) and catabolic modeling/remodeling (days 8-9).
  6. Direct delivery of a significant volume (100 µl) of clodronate liposomes to the site of the injury most likely resulted in some free clodronate being released in the region where osteoclasts reside. As free clodronate is known to kill osteoclasts, we believe that a free clodronate control group was critical to this study.
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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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