2008-ruiz-phd

On Sun, 01-Jul-2012   /   Intrapleural Administration  
Ruiz DF.
Filarial nematodes protect against malaria in a murine co-infection model.
[Thesis (PhD)] [Bonn]: Rheinische-Friedrich-Wilhelms University of Bonn; 2008.
[toggle_content title=”Abstract”] Helminth infections such as filariasis can be life long due to immunomodulatory mechanisms elicited by the worms that prevent their elimination by the human host. Malaria prevalence is frequently high in areas where filarial nematode infections are endemic, originating co-infections. It is still an ongoing debate the extent to which worms can alter anti-plasmodial responses in a concomitantly infected host. In order to dissect the interactions occurring between both parasites, a murine model using the filarial nematode, Litomosoides sigmodontis, and the malaria parasite, Plasmodium berghei, ANKA in BALB/c and C57BL/6 mice has been established in the present work. One third of helminth-infected BALB/c mice remained malaria negative and did not develop detectable parasitaemia after co-infection with plasmodial sporozoites. Such outcomes were not observed after co-infection with blood stage parasites, thereby circumventing the plasmodial liver stage, but were only originated after sporozoite inoculation. Protection depended on IL-10, since IL-10 deficient co-infected mice were fully susceptible to sporozoite challenge. However, systemic IL-10 levels were low, suggesting a local function of this cytokine. 44 h after P. berghei sporozoite inoculation increased CD8+ T cell numbers were found in the liver and spleen of co-infected mice. Interestingly, protection as well as numbers of CD8+ T cells correlated with the presence of microfilaraemia. Since microfilariae are potent stimulators of immune responses, their presence in the blood may enhance cross-protective responses against incoming sporozoites. Co-infected C57BL/6 mice showed significantly reduced cerebral malaria (CM) rates. CD8+ T cell recruitment to brain, a critical condition for CM development, was reduced in co-infected mice. Furthermore, in contrast to P. berghei single-infected animals, groups of double infected mice presented high levels of circulating IL-10. The absolute requirement for IL-10 in CM protection was demonstrated by the total susceptibility to the pathology in IL-10 KO co-infected mice. Hence IL-10-dependent immunoregulatory mechanisms elicited by filarial nematodes might be able to suppress the overwhelming inflammatory reaction usually triggered against malaria parasites by C57BL/6 mice, preventing full progress to CM. Taken together, this work shows that depending on the genetic background different mechanisms elicited by filarial worms improve control of both liver and blood stages of concomitant malaria parasites, and moreover ameliorate severe pathology in a murine model. The main beneficial effects driven by filariae rely on IL-10, which on the one hand prevents CM through immunomodulation but on the other enhances protective anti-malarial responses in the liver. Worm eradication programs are currently being carried out in endemic countries, but this study suggests that their implementation may cause unpredictable consequences on malaria control. [/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/ml1 23.5 mg/ml EPC/Chol 86/14 MLV none

1Author states clodronate liposome concentration as 250 mg/ml, however it is well-known that this is not achievable for clodronate liposomes. Since the liposomes were provided by the van Rooijen lab, we assume that they have the usual reported concentration of 5 mg/ml and were prepared by the usual method used by that lab.

[/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
BALB/c mice 200 µl intrapleural  Gr1highF4/80high pleural macrophages yes (i.v.) not evaluated
[/custom_table] [/toggle_content] [toggle_content title=”Notes”]
  1. No details on the intrapleural dosing technique published.
  2. Author states that 200 µl were dosed both intrapleurally and intravenously for 4 consecutive days to “L. sigmodontis co-infected mice” without specifying the time of clodronate treatment relative to infection or data collection.
[/toggle_content] [toggle_content title=”Results”]
  1. The author concluded, after FACS analysis of cells isolated from the pleural cavity, that the Gr1high+, F4/80high+ cell population which disappeared in clodronate-liposome-treated animals were macrophages.
  2. We did not find any reports of experiments investigating the effects of macrophage depletion in any models elsewhere in this thesis.
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2010-lo-6367

On Sun, 01-Jul-2012   /   Intrapharyngeal Instillation  
Lo Re S, Dumoutier L, Couillin I, van Vyve C, Yakoub Y, Uwambayinema F, Marien B, van der Brûle S, van Snick J, Uyttenhove C, Ryffel B, Renauld JC, Lison D, Huaux F.IL-17A-Producing γδ T and Th17 Lymphocytes Mediate Lung Inflammation but Not Fibrosis in Experimental Silicosis. The Journal of Immunology. 2010 Apr 26;184(11):6367–77.

Abstract

IL-17-producing T lymphocytes play a crucial role in inflammation, but their possible implication in fibrosis remains to be explored. In this study, we examined the involvement of these cells in a  mouse model of lung inflammation and fibrosis induced by silica particles. Upregulation of IL-17A was associated with the development of experimental silicosis, but this response was markedly reduced in athymic, γd T cell-deficient or CD4+ T cell-depleted mice. In addition, γd T lymphocytes and CD4+ T cells, but not macrophages, neutrophils, NK cells or CD8 T cells, purified from the lungs of silicotic mice markedly expressed IL-17A. Depletion of alveolar macrophages or neutralization of IL-23 reduced upregulation of IL-17A in the lung of silicotic mice. IL-17R–deficient animals (IL-17R-/- ) or IL-17A Ab neutralization, but not IL-22 -/- mice, developed reduced neutrophil influx and injury during the early lung response to silica. However, chronic inflammation, fibrosis, and TGF-β expression induced by silica were not attenuated in the absence of IL-17R or -22 or after IL-17A Ab blockade. In conclusion, a rapid lung recruitment of IL-17A–producing T cells, mediated by macrophage-derived IL-23, is associated with experimental silicosis in mice. Although the acute alveolitis induced by silica is IL-17A dependent, this cytokine appears dispensable for the development of the late inflammatory and fibrotic lung responses to silica. [/toggle_content] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 86/14 MLV PBS
[/custom_table] [custom_table]
Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6 mice2 100 µl pharyngeal aspiration / lungs alveolar macrophages (AM) no not evaluated

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

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Notes

  1. No details on pharyngeal dosing technique published.
  2. Citations related to macrophage depletion compare intratracheal and aerosol administration methods, not pharyngeal administration.

Results

  1. >80% of the AM were depleted (BAL cell counts via light microscopy) 3 days post-treatment (data not shown).
  2. Complete inhibition of IL-7A production is only other depletion-related data published.

 

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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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2012-ivanov-413

On Wed, 27-Jun-2012   /   Intranasal Instillation  
Ivanov S, Fontaine J, Paget C, MachoFernandez E, Maele LV, Renneson J, Malliet I, Wolf NM, Rial A, Léger H, Ryffel B, Frisch B, Chabalgoity J, Sirard JC, Benecke A, Faveeuw C, Trottein F.
Key role for respiratory CD103+ dendritic cells, IFN-γ and IL-17 in protection against Streptococcus pneumoniae infection in response to α-galactosylceramide.
J Infect Dis. [Internet]. 2012 Jun 21 [cited 2012 Jun 27]; Available from: http://jid.oxfordjournals.org/content/early/2012/06/21/infdis.jis413
[toggle_content title=”Abstract”] Background. Exogenous activation of pulmonary invariant Natural Killer T (iNKT) cells, a population of lipid-reactive αβ T lymphocytes, by means of mucosal α-galactosylceramide (α-GalCer) administration, is a promising approach to control respiratory bacterial infections. We undertook the present study to characterize mechanisms leading to α-GalCer-mediated protection against lethal infection with S. pneumoniae serotype 1, a major respiratory pathogen in humans.
Methods and Results. α-GalCer was administered by the intranasal route before infection with S. pneumoniae. We showed that respiratory dendritic cells (DCs), most likely the CD103+ subset, play a major role in the activation (IFN-γ and IL-17 release) of pulmonary iNKT cells, whilst alveolar and interstitial macrophages are minor players. After challenge, S. pneumoniae was rapidly (4h) eliminated in the alveolar spaces, a phenomenon that depended on respiratory DCs and neutrophils, but not macrophages, and on the early production of both IFN-γ and IL-17. Protection was also associated with the synthesis of various interferon-dependent and IL-17-associated genes as revealed by transcriptomic analysis.
Conclusions. These data imply a new function for pulmonary CD103+ DCs in mucosal activation of iNKT cells and establish a critical role for both IFN-γ and IL-17 signalling pathways in mediating the innate immune response to S. pneumoniae.[/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
C57BL/6, CD11c mice2, male, 8 w 20 µg intranasal instillation alveolar MΦ no NA

2Base strains; mutants or transgenic animals also used.

[/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, van Nieuwmegen R. Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate. An enzyme-histochemical study. Cell Tissue Res. 1984;238(2):355–8.
[/toggle_content][toggle_content title=”Results”]
  1. ~74% of the alveolar macrophages collected by lavage were depleted 48 h post-administration of clodronate liposomes; counts at other time points were not taken.
  2. Alveolar macrophage depletion did not affect pulmonary CFU at 4 h post-infection or mortality rate.
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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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