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.
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  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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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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