1993-vanlent-21

On Sun, 29-Jul-2012   /   In vitro, Intra-articular  
van Lent PL, van den Bersselaar L, van den Hoek AE, van de Ende M, Dijkstra CD, van Rooijen N, van de Putte LB, van den Berg WB.
Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate.
Rheumatol. Int. 1993;13(1):21–30.
[toggle_content title=”Abstract”]We studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electron microscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes Control Free Clodronate
12.5 mg/ml 21.5 mg/ml EPC/Chol 77/23 MLV PBS 75 µg/6 µl
[/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
C57BL/6 mice, male, 8-12 w, 25-30 g 75 µg/6 µl intra-articular/knee MOMA-1+, MOMA-2+synovial MΦ, F4/80+NLDC-145+ phagocytes no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. No liposome prep method was cited.
  2. This concentration of encapsulated clodronate is atypically high although a typical encapsulation efficiency of 1% was reported.
  3. The authors report centrifuging at 100,000xg during the washing process so they may have collected more of the smaller liposomes which are not usually spun down during a 10,000xg centrifugation step.
[/toggle_content] [toggle_content title=”Results”]
  1. When when a single dose of clodronate liposomes, control liposomes, clodronate, or clodronate + control liposoomes was injected into normal mouse knee joints followed by histological examination on days 1, 3 and 7 revealed that
    • Free clodronate had no effect on the synovial lining cells nor did it elicit inflammatory cells into the synovium.
    • Control fluorescent liposomes and clodronate liposomes elicited some inflammatory cells into the synovial fluids which had disappeared by day 7.
    • Control fluorescent liposomes localized to the synovial lining and remained visible at least until day 7; no further time points investigated.
    • Clodronate liposomes caused enlargement and gradual depletion of synovial lining cells which reached a maximum at day 7, although the fibroblast-like cells just under the lining were unchanged.
  2. Continued observation of clodronate liposome treated animals showed
    • Synovial lining cells had begun to repopulate by day 9 on the femoral side of the synovium; complete repopulation at this site was accomplished by day 15.
    • Recovery of the dermal side of the lining began on day 20 and was only about 60% complete at day 30.
    • Only MOMA-2+ and F4/80+ cells were found in the synovial lining.
  3. Co-culturing of synovial macrophages and fibroblasts followed by treatment with clodronate liposomes confirmed that only the macrophages were affected.
  4. Proteoglycan synthesis was inhibited and degradation increased (collagen metabolism) on days 1-3 but returned to baseline by day 4.
  5. Control liposomes solicited PMN at a similar level to clodronate liposomes, but the control liposomes did not affect collagen metabolism.
  6. Immune-complex induced arthritis was initiated on day 7, but no joint swelling was measured in clodronate-liposome treated knees.
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2007-blom-147

On Sat, 28-Jul-2012   /   Intra-articular  
Blom AB, van Lent PL, Libregts S, Holthuysen AE, van der Kraan PM, van Rooijen N, van den Berg W. Crucial role of macrophages in matrix metalloproteinase–mediated cartilage destruction during experimental osteoarthritis : Involvement of matrix metalloproteinase 3. Arthritis & Rheumatism. 2007;56(1):147–57.

Abstract

Objective: To explore the involvement of synovial macrophages in early cartilage damage in osteoarthritis (OA), and to identify the role of matrix metalloproteinase 3 (MMP-3) in the pathology of early and late OA.

Methods: The role of synovial macrophages in MMP-mediated damage in OA was studied by depleting synovial macrophages prior to elicitation of a collagenase-induced instability model of OA. The expression of MMP in synovium and cartilage was monitored using TaqMan analysis. In spontaneous and induced OA, cartilage pathology was scored in MMP-3–knockout mice and control mice, by histologic assessment and VDIPEN staining.

Results: On day 14 following induction of OA, MMP-mediated neoepitopes were detected in cartilage from mice with mild experimental OA (mean  SD positively stained surface area 20  3.2%). Remarkably, by depleting synovial macrophages prior to induction of OA, the generation of MMP-induced neoepitopes was largely prevented (mean  SD positively stained surface area 5  1%; P< 0.001), indicating an important role for synovial macrophages in the occurrence of MMPmediated cartilage damage. We observed a strong decrease in MMP-3 and MMP-9 expression in synovial but not cartilage tissue in macrophage-depleted joints. Among 2-year-old mice, spontaneous OA–like changes in the lining layer were significantly decreased in MMP- 3–knockout mice compared with control mice. Even more striking was the 67% reduction in the occurrence of severe cartilage damage in MMP-3–knockout mice. In addition, MMP-mediated VDIPEN expression was significantly decreased, indicating reduced MMPmediated cartilage breakdown.

Conclusion: The results of this study prove that MMP-3 is involved in the generation of severe cartilage damage in murine OA. Synovial macrophages are crucial in early MMP activity and appear to mediate MMP production in synovium rather than cartilage. [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
not specified not specified EPC/Chol not specified MLV none
[/custom_table] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice2 not specified intra-articular/knee F4/80+ synovial lining MΦ no N/A

2Base strain. Other genetically altered or knockout strains were also used.

[/custom_table]

Notes

  1. Liposome prep method referenced—van Rooijen N, Sanders A, van den Berg TK. Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine. Journal of Immunological Methods. 1996 Jun;193(1):93–9.
  2. Animals were injected 1 week prior to induction of arthritis with collagenase.
  3. No details of injection method or clodronate liposome dose were published, however referenced paper states that 6 µl (75 µg clodronate) were injected into the joint.

Results

  1. F4/80+ MΦ were reduced from about 45% to 15% by day 14 and 50% to 15% by day 21 post-injection of clodronate liposomes (7 and 14 days post-induction of OA) by immunohistological analysis.
  2. Depletion was not detected in the sublining.
  3. Although the authors state that F4/80+ MΦ had begun to repopulate at 3 w post-injection of clodronate liposomes in a cited article, the depletion level had not changed in the current experiment.
  4. F4/80+ MΦ depletion correlated with a significant down-regulation in MMP-3.
  5. The authors further pursue in the possible roles of MMP’s in MMP knockout mice without the use of clodronate liposomes.

 

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2000-barrera-1951

On Sat, 28-Jul-2012   /   Intra-articular  
Barrera P, Blom A, van Lent PLEM, van Bloois L, Beijnen JH, van Rooijen N, de Waal Malefijt M, van de Putte L, Storm G, van den Berg W.
Synovial macrophage depletion with clodronate-containing liposomes in rheumatoid arthritis.
Arthritis & Rheumatism. 2000;43(9):1951–9.
[toggle_content title=”Abstract”]Objective: To assess whether intraarticular (IA) administration of clodronate liposomes results in local macrophage depletion in patients with rheumatoid arthritis (RA). Primary goals were to address both the immunohistologic and potential toxic effects of this approach. Moreover, the correlation between immunohistologic findings and clinical assessments of disease activity and cartilage damage were assessed.

Methods: An open study was conducted in consecutive RA patients who were scheduled for knee joint replacement in our department. Synovial biopsy tissue was obtained from the knee joint at 2 weeks before and at the time of surgery. This protocol was controlled for safety and immunohistologic concordance in 6 patients. One week before surgery, 10 patients received a single IA dose of clodronate liposomes. Staining of synovial tissue for cell markers (CD68, CD14, CD3, CD38) and adhesion molecules (vascular cell adhesion molecule 1 [VCAM-1], intercellular adhesion molecule 1 [ICAM-1]) was assessed by 2 blinded observers. Local and systemic parameters of disease activity were measured before each intervention. Cartilage damage was scored using standard radiologic techniques at baseline and during surgery.

Results: A single IA dose of clodronate liposomes significantly reduced the number of CD68-positive cells (P = 0.005) and the expression of ICAM-1 and VCAM-1 in the synovial lining (P = 0.013 and P = 0.039, respectively). The intervention did not affect fibroblast-like synoviocytes, T cells, or plasma cells. No immunohistologic changes were observed in the control group. The procedure was well tolerated. The levels of ICAM-1 and VCAM-1 in the sublining layers correlated with the extent of macroscopic synovitis (P < 0.0005 and P < 0.005, respectively). The expression of ICAM-1 and CD14 in the sublining correlated with the levels of C-reactive protein (P < 0.0005 and P < 0.01, respectively). Cartilage destruction was correlated only with the expression of CD68 in the sublining (P = 0.02).

Conclusion: A single IA administration of clodronate liposomes leads to macrophage depletion and decreased expression of adhesion molecules in the synovial lining in patients with longstanding RA. The procedure is well tolerated, and its therapeutic potential is currently under investigation. The expression of adhesion molecules in the sublining layers reflects ongoing inflammation.[/toggle_content] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
4-10 mg/ml not specified EPC/Chol not specified LUV, 120-160 nm none
[/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
Humans, >18 yr, scheduled for knee joint prosthesis 160 ± 35 mg intra-articular/knee CD68+ synovial lining/sublining MΦ/synoviocytes no N/A
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Liposome prep method did not specify amount of EPC used, but appeared to be a printing error. Maintaining the 6:1 molar ratio typically employed by van Rooijen would have called for 1.1 g EPC per 98 mg chol which was specified in the text. If we assume final prep volume was 10 ml, then the total lipid concentration would have been ~115 mg/ml. EPC/chol MLV were extruded through 0.2, 0.1 and 0.05 µm filters. Only 60 mg/ml clodronate used for resuspension (van Rooijen method uses 250 mg/ml) and encapsulation efficiency reported at 10-14%. Using the 10 ml total prep volume assumption and 10% encapsulation efficiency, patients received ~27 ml injections. We have not previously seen this prep method and the assumptions may not have provided the correct injection volumes.
  2. The authors stated that the dose was extrapolated from animal studies, but no further details were given.
  3. Patients were treated 2 weeks prior to knee joint prosthetic surgery during which histolgical samples were collected and other observations recorded.
  4. Study goal was to evaluate safety and tolerance.
[/toggle_content] [toggle_content title=”Results”]
  1. Median CD68+ cells in synovial lining reduced from 4 to 0.45 (immunohistological analysis).
  2. Median CD68+ cells in synovial sublining reduced from 2.65 to 1.65, although authors speculate that the reduction was not due to liposomes entering the sublining.
  3. VCAM-1 and ICAM-1 were significantly reduced in the synovial lining in treated patients which correlated with a reduction in synovitis and macroscopic swelling at the time of surgery.
  4. CD14 and ICAM-1 correlated with CRP.
  5. CD68 staining in the sublining correlated with cartilage damage.
  6. In similarly designed animal studies, depleted macrophages were completely repopulated in 2-4 weeks post-treatment.
  7. Patients reported no discomfort or pain upon clodronate liposome injection and no adverse reactions were noted.
  8. Hisological analysis revealed substantial changes in the appearance of the synovial linings.
[/toggle_content]
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2006-qualls-802

On Wed, 25-Jul-2012   /   Intrarectal  
Qualls JE, Kaplan AM, van Rooijen N, Cohen DA.
Suppression of experimental colitis by intestinal mononuclear phagocytes.
Journal of leukocyte biology. 2006;80(4):802–15.
[toggle_content title=”Abstract”]The contribution of innate immunity to inflammatory bowel disease (IBD) remains an area of intense interest. Macrophages (MØ) and dendritic cells (DC) are considered important factors in regulating the onset of IBD. The goal of this study was to determine if intestinal mononuclear phagocytes (iMNP) serve a pathological or protective role in dextran sulfate sodium (DSS)-induced colitis in mice. Using a conditional MØ/DC depletion transgenic mouse line—MØ Fas-induced apoptosis— to systemically deplete iMNP, DSS colitis histopathology was shown to be more severe in MØ/DC-depleted compared with MØ/DC-intact mice. Similarly, localized iMNP depletion by clodronate- encapsulated liposomes into C57BL/6, BALB/c, and CB.17/SCID mice also increased DSS colitis severity, as indicated by increased histopathology, weight loss, rectal bleeding, decreased stool consistency, and colon length compared with MØ/DC-intact, DSS-treated mice. Histology revealed that iMNP depletion during DSS treatment led to increased neutrophilic inflammation, increased epithelial injury, and enhanced mucin depletion from Goblet cells. iMNP depletion did not further elevate DSS-induced expression of TNF- and IFN- mRNA but significantly increased expression of CXCL1 chemokine mRNA. Myeloperoxidase activity was increased in colons of MØ/DCdepleted, DSS-treated mice, compared with DSS alone, coincident with increased neutrophil infiltration in diseased colons. Neutrophil depletion combined with MØ/DC depletion prevented the increase in DSS colitis severity compared with MØ/DC depletion alone. This study demonstrates that iMNP can serve a protective role during development of acute colitis and that protection is associated with MØ/DC-mediated down-regulation of neutrophil infiltration.[/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 Systemic Dosing? Sytemic Results
C57BL/6, BALB/c, CB.17/SCID, MaFIA female mice, 5-8 w 100 µl intrarectal CD45+Ly-6G-CD11b+ MΦ 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. Mice were anesthesized with an i.p. injection of 200 µl 2.5% Avertin. 100 µl clodronate liposomes were injected intrarectally using a micropipette. No other details were provided; it is not clear whether a Pipetman-type polycarbonate tip or a glass capilllary type micropipette was used.
  3. Authors note that depletion was minimal in the more proximal regions of the colon.
  4. Authors further state that intrarectal injection of control liposomes resulted in a partial reduction of total colonic MΦ and could effect the physiology of the remaining colonic MΦ (data not shown). Therefore, they chose not to include empty liposome controls.
[/toggle_content] [toggle_content title=”Results”]
  1. A single intrarectal treatment (MaFIA mice) with clodronate liposomes resulted in a 92% of the MΦ in the descending colon at 24 h post-treatment.
  2. Lesser effects were observed in the transverse and ascending colon, however no depletion was observed in the bone marrow and peritoneum.
  3. C57BL/6, BALB/c and CB.17/SCID female mice were injected intrarectally with 100 µl clodronate liposomes on days -1, +1, +3 and +5 days while dextran sodium sulfate (DSS) was added to their drinking water beginning at day 0. On day 7 (day 5 for CB.17/SCID) the % of CD45+Ly-6G-CD11b+ MΦ was about half the control values indicating a relatively rapid repopulation of colonic MΦ.
  4. Although the different mouse strains demonstrated different levels of disease responses, all strains had significantly more severe disease activity overall when MΦ were depleted.
  5. Spleens and mestenteric lymph nodes were cultured to determine if MΦ depletion resulted in a release of bacteria from colon, but little or no bacteria were found.
  6. MΦ depletion correlated to an increase in chemokine CXCL1 and neutrophils, but not other cytokines.
  7. Concurrent neutrophil depletion prevented the increase in disease severity resulting from MΦ depletion.
  8. Given that control liposomes had a significant effect on colonic MΦ and that the decrease in these MΦ was not likely due to MΦ killing by the liposomes, we wonder what results control liposomes would have produced. Could the increase in neutrophil activity be the result of MΦ killing and not exclusively due to the absence of macrophages?
[/toggle_content]
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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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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
[/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; 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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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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2011-drabek-517

On Mon, 02-Jul-2012   /   Intrahippocampal Injection  
Drabek T, Janata A, Jackson EK, End B, Stezoski J, Vagni VA, Janesko-Feldman K, Wilson CD, van Rooijen N, Tisherman SA, Kochanek PM.
Microglial depletion using intrahippocampal injection of liposome-encapsulated clodronate in prolonged hypothermic cardiac arrest in rats.
Resuscitation. 2012 Apr;83(4):517–26.
[toggle_content title=”Abstract”] Trauma patients who suffer cardiac arrest (CA) from exsanguination rarely survive. Emergency preservation and resuscitation using hypothermia was developed to buy time for resuscitative surgery and delayed resuscitation with cardiopulmonary bypass (CPB), but intact survival is limited by neuronal death associated with microglial proliferation and activation. Pharmacological modulation of microglia may improve outcome following CA. Systemic injection of liposome-encapsulated clodronate (LEC) depletes macrophages. To test the hypothesis that intrahippocampal injection of LEC would attenuate local microglial proliferation after CA in rats, we administered LEC or PBS into the right or left hippocampus, respectively. After rapid exsanguination and 6 min no-flow, hypothermia was induced by ice-cold (IC) or room-temperature (RT) flush. Total duration of CA was 20 min. Pre-treatment (IC, RTpre) and post-treatment (RTpost) groups were studied, along with shams (cannulation only) and CPB controls. On day 7, shams and CPB groups showed neither neuronal death nor microglial activation. In contrast, the number of microglia in hippocampus in each individual group (IC, RTpre, RTpost) was decreased with LEC vs. PBS by ∼34–46% (P < 0.05). Microglial proliferation was attenuated in the IC vs. RT groups (P < 0.05). Neuronal death did not differ between hemispheres or IC vs. RT groups. Thus, intrahippocampal injection of LEC attenuated microglial proliferation by ∼40%, but did not alter neuronal death. This suggests that microglia may not play a pivotal role in mediating neuronal death in prolonged hypothermic CA. This novel strategy provides us with a tool to study the specific effects of microglia in hypothermic CA. [/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 20.4 mg/ml EPC/Chol/Aminophenyl mannose 69/21/10 MLV PBS
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Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
Sprague Dawley rats, male, 300-350 g 5 µl stereotaxic inj/hippocampus Iba-1+ microglia no no change in peripheral moncytes or PMN detected
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. Detailed description of stereotaxic injection of 5 µl of liposomal clodronate (R hemisphere) and 5 µl control liposomes (L hemisphere) over 10 min.
  2. Needle was left in place for 3 min. post-injection and withdrawn at 1 mm/min.
  3. To assess potential increase in intracranial pressure (ICP) due to injection, 10 µl liposomal clodronate was injected in control animals and ICP monitored.
  4. Liposome treatment occured either 24 hr prior to or 24 hr after exsanguination and resusitation experimental protocol; animals sacrificed 7 days post-exsanguination protocol.
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  1. ICP did not change significantly during injection of 10 µl of liposomal clodronate.
  2. Iba-1 detects activated microglia which were reduced overall by 37-42% (immunohistology) by liposomal clodronate.
  3. The post-trauma treatment appeared to reduce the microglia by 50% compared to the pre-trauma treatment in both liposome treatment groups.
  4. While neuronal death overall was not affected, again the neuronal death was lower in both liposome treatment groups when comparing pre- and post-trauma treatment groups.
  5. The authors attribute these results (points 3 and 4) to the effect of the pre- and post-trauma treatment regimen which would be supported by the data if the liposomal PBS control group values did not change with treatment regimen. However, since the effect is observed in both liposomal PBS and liposomal clodronate groups, it seems that the effect is a result of the presence of liposomes (with or without clodronate). Therefore, it is critical to compare buffer-injected controls to both liposomal clodronate and liposomal PBS treated animals in determining the effect of treatment regimen on microglial depletion and neuronal death. If we speculatively assume that buffer treatment will have no effect, then liposomes (with or without clodronate) themselves appear to reduce neuronal death depending on treatment regimen. There is even a slight suggestion that liposomal PBS is more effective than liposomal clodronate (31% vs 19% reduction in neuronal death).
  6. These observations also hold true for the microglial depletion data except that liposomal clodronate does reduce the number of activated microglia as the authors conclude. However, the presence of liposomes, even without clodronate, reduces the number of active microglia by at least half when dosed 24 hr post-trauma rather than 24 hr pre-trauma. Although the specific reduction in activated microglia by liposomal clodronate does not translate to neuronal sparing, the overall reduction in activated microglia when either liposomal PBS or liposomal clodronate is dosed 24 hr post-trauma does correlate to neuronal sparing.
  7. The authors viewed this study as a proof-of-concept trial and acknowledged several other parameters, including determining the optimal liposomal clodronate dose and treatment regimen, and evaluating effects on other inflammatory cell types (i.e. PMN). However, the observation that liposomes, even without clodronate, attenuates microglial activation/proliferation and neuronal death by a mechanism apparently unrelated to macrophage depletion points in a different direction for neuroprotection post severe CV trauma— can liposomes, perhaps incombination with other anti-inflammatory agents, ameliorate (or prevent) neuronal loss post-CV-trauma?
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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.
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  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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