Publication 1.

S. Hashimoto, J.F. Pittet, K. Hong, H. Folkesson, G. Bagby, L. Kobzik, C. Frevert, K. Watanabe, S. Tsurufuji, J. Wiener-Kronish, Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections, American Journal of Physiology – Lung Cellular and Molecular Physiology, 270 (1996) L819–L828.

Abstract

The mechanism for neutrophil (PMN) influx into infected airspaces of the lung is not known.To determine whether alveolar macrophage products are important in the initiation of chemotaxis, we depleted rats of alveolar macrophages by aerosolizing negatively charged oligolamellar liposomes complexed to clodronate disodium. Ninety-five percent of the alveolar macrophages were depleted, and lung injury and inflammation were minimized with this depletion technique. Rats depleted of alveolar macrophages were then anesthetized, and either 5 x 10 6 colony-forming units (CFU) or 5 x 10 7 CFU of Pseudomonas aeruginosa were instilled into the airspaces of these animals. When recombinant macrophage inflammatory protein (MIP-2) was intratracheally instilled into rats depleted of alveolar macrophages, PMN were recruited to their airspaces. Nonetheless, PMN numbers were decreased in the lavage fluids when moderate or large inoculums of bacteria were instilled into depleted rats, although the PMN response appeared dose dependent. Levels of bioactive tumor necrosis factor-α and immunoreactive proteins CINC/gro (cytokine-induced PMN chemoattractant) in the lavage fluids obtained from infected rats depleted of alveolar macrophages were significantly decreased compared with the levels in the lavage fluids obtained from normal infected rats. MIP-2 mRNA expression, as detected by Northern analysis, was also decreased in the infected lungs of depleted rats, and the lavage fluid from these rats had significantly decreased chemotactic activity. Therefore these results suggest that alveolar macrophage products play a direct role in the initial recruitment of PMN into infected lungs. [/toggle_content] [custom_table]

Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
7 mg/ml 1 33.4 mg/ml EPC/BPS/Chol 55/9/36 REV PBS(-)
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Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
Sprague Dawley rats, male, 300-375 g 0.05-0.25 µmoles aerosol / lungs alveolar macrophages (AM) no no change in peripheral moncytes or PMN detected
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Notes

  1. The authors state that liposomes were “extruded” through 0.2 µm syringe filters. A syringe filter is not a substitute for extrusion therefore the resulting size distribution is questionable. Liposomes >400 nm have been shown to be disrupted by aerosolization, therefore it’s likely that there was free clodronate in the aerosol.
  2. Aerotech II nebulizer at 10 L/min. MMAD = 1.6 µm; GSD = 2.5. Fluorescent liposome aerosol delivery experiments indicated that 0.1-0.5% of the dose was delivered to the lung. This value was used to estimate the clodronate dose delivered to the lungs. The authors estimate that 26X more drug is delivered by this method than by liquid installation.

Results

  1. >95% of the AM were depleted (BAL cell counts via light microscopy).
  2. The authors further state that clodronate liposome delivery by aerosol prevents neutrophilia often observed when direct instillation is used.

 

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Publication 2.

K. Kooguchi, S. Hashimoto, A. Kobayashi, Y. Kitamura, I. Kudoh, J. Wiener-Kronish, T. Sawa, Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia, Infect. Immun., 66 (1998) 3164–3169.

Abstract

To evaluate the role of alveolar macrophages (AMs) in acute Pseudomonas aeruginosa pneumonia in mice, AMs were depleted by aerosol inhalation of liposomes containing clodronate disodium. AM-depleted mice were then intratracheally infected with 5 x 10 5 CFU of P. aeruginosa . In addition to monitoring neutrophil recruitment and chemokine releases, lung injury was evaluated soon after infection (8 h) and at a later time (48 h). At 8 h, depletion of AMs reduced neutrophil recruitment, chemokine release, and lung injury. At 48 h, however, depletion of AMs decreased bacterial clearance and resulted in delayed movement of neutrophils from the site of inflammation with aggravated lung injury. With instillation of 5 x 10 7 CFU of bacteria, AM-depleted mice showed low mortality within 24 h of infection but high mortality at a later time, in contrast to non-AM-depleted mice. These results demonstrate that depletion of AMs has beneficial early effects but deleterious late effects on lung injury and survival in cases of P. aeruginosa pneumonia.

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Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
not stated 11 mg/ml EPC/Chol 57/43 REV none
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Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
CD-1, male,  35-37 g not stated aerosol / lungs alveolar macrophages (AM) no not evaluated
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Notes

  1. Authors state that liposomes were prepared as in Publication 1, however lipid composition is different as is final volume. Encapsulated clodronate is not published. Lipid concentration calculated assuming 100% recovery.
  2. As discussed for publication 1., passing liposomes through a syringe filter is not equivalent to extrusion.
  3. Aerotech II nebulizer at 12 L/min. No further details published.

 

Results

  1. >95% of the AM were depleted (BAL cell counts via light microscopy).
  2. No neutrophils at t=0 post-innoculation, therefore neutrophilia is not observed in this model.
  3. No control liposomes dosed; could chemokine levels be altered by liposomes alone?

 

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Publication 3.

A.C.P. Elder, R. Gelein, G. Oberdörster, J. Finkelstein, R. Notter, Z. Wang, Efficient Depletion of Alveolar Macrophages Using Intratracheally Inhaled Aerosols of Liposome-Encapsulated Clodronate, Experimental Lung Research, 30 (2004) 105–120.

Abstract

Rat alveolar macrophages (AMs) were depleted via intratracheal inhalation (ITIH) of clodronate- containing liposomes. AM depletion following ITIH delivery of clodronate liposomes was 33.2±14.2 on day 1, 88.1±6.2 on day 3, and 91.4±1.8 on day 4 relative to control rats given saline-containing liposomes. Almost all (~99%) of the AMs remaining at the 3-day time point were peroxidase negative, suggesting that immature macrophages were not recruited from the circulation to replace those undergoing cell death on that day. Only 0.5%±0.5% of bronchoalveolar lavage (BAL) cells were neutrophils at this time (normalized to controls). Whole-body inhalation did not induce as much AM depletion at 3 days (37.6%±10.1%) and required larger amounts of liposome- encapsulated clodronate compared to ITIH. Intratracheal instillation (as opposed to inhalation) of clodronate liposomes produced a significant inflammatory response characterized by the influx of both polymorphonuclear neutrophils (PMNs) and macrophages. In subsequent pilot studies, the response to intratracheally instilled crystalline silica (75 mg) was found to be markedly reduced in rats depleted of AMs by the ITIH method. We conclude that ITIH of clodronate liposomes in rats is both efficient and useful for examining the role of AMs in pulmonary toxicology.
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Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5.6 mg/ml 1 28.4 mg/ml EPC/BPS/Chol 55/9/36 REV PBS
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Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Systemic Dosing? Systemic Results
Fischer 344 rats, 8 wks, 240-260 g 186 µg intratracheal aerosol / lungs alveolar macrophages (AM) no not evaluated
3.8 µg whole-body aerosol inhalation / lungs alveolar macrophages (AM) no not evaluated
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Notes

  1. As discussed for publication 1., passing liposomes through a syringe filter is not equivalent to extrusion.
  2. Animals were dosed either by intratracheal inhalation (aerosol), whole-body inhalation (aerosol), or liquid bolus instilled via teflon catheter inserted transorally into the trachea.
  3. Intratracheal inhalation (ITIH)
    • Lovelace Respiratory Research Inst. low-flow jet nebulizer at 1.67 L/min (50 psi). MMAD = 1.1 µm; GSD = 1.9 µm. Liposomes >400 nm have been shown to leak during nebulization.
    • Animals connected to a constant flow respirator system via transoral catheter; inspiratory pressure = 20 cm H 2 O; inspir./expir. = 1.5/0.5 sec.
    • 0.8-0.9 ml clodronate liposomes mixed with 3.1-3.2 ml saline for nebulization. Other clodronate liposome formulations have been shown to leak when diluted in PBS.
    • 1% Halothane mixed inline with liposomes to maintain anesthesia. Halothane is lipid soluble and can induce leakage in liposomes.
  4. Whole-Body Inhalation
    • Aerotech II nebulizer at 15 L/min (40 psi) in 31 L exposure chamber containing 2.5 ml liposomes.
    • Exposure time: ~13 min
    • Estimated dose: 3.8 µg clodronate
  5. Intratracheal Instillation
    • ~180 µl (1 mg clod) liposomal clodronate diluted to 250 µl in saline.
    • Delivered through transoral intratracheal catheter during inspiratory phase.
    • 1 animal dosed with 0.5 mg clod/250 µl X 2 days.
  6. Silica dosed via intratracheal instillation 3 days post-ITIH of clodronate liposomes as in Point 3.
  7. Cells from BAL stained or treated and counted by hemocytometer.

 

Results

  1. AM depletion measured 3 days post-treatment
    • ITIH: 86 % depletion; PMN: 0.9 % inc.; 99 % remaining; peroxidase negative (not recruited monocytes).
    • Whole-body inhalation: 38 % depletion; PMN: 0.6 % inc.
    • Intratracheal instillation: 7 % increase ; PMN: 70.3 % inc.
    • Intratracheal instillation X2: 89 % depletion; PMN: 1.7 % inc.
  2. ITIH clodronate liposome treatment -3 days prior to silica exposure reduced PMN by 58 % at 24 h post-silica.
  3. Although intratracheal instillation results appear atypical in terms of depletion, neutrophilia appears to be related to clodronate liposome concentration and possibly method of administration.

 

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Publication 4.

M.M. Kelly, K. McNagny, D.L. Williams, N. van Rooijen, L. Maxwell, C. Gwozd, C.H. Mody, P. Kubes, The Lung Responds to Zymosan in a Unique Manner Independent of Toll-Like Receptors, Complement, and Dectin-1, American Journal of Respiratory Cell and Molecular Biology, 38 (2007) 227–238.

Abstract

In vitro studies indicate that the inflammatory response to zymosan, a fungal wall preparation, is dependent on Toll-like receptor (TLR) 2, and that this response is enhanced by the dectin-1 receptor. Complement may also play an important role in this inflammatory response. However, the relevance of these molecules within the in vivo pulmonary environment remains unknown. To examine pulmonary in vivo inflammatory responses of the lung to zymosan, zymosan was administered by intratracheal aerosolization to C57BL/6, TLR2- TLR4-, MyD88-, and complement-deficient mice. Outcomes included bronchoalveolar fluid cell counts. We next examined effects of dectin-1 inhibition on response to zymosan in alveolar macrophages in vitro and in lungs of C57BL/6, TLR2-, and complement-deficient mice. Finally, the effect of alveolar macrophage depletion on in vivo pulmonary responses was assessed. Marked zymosan-induced neutrophil responses were unaltered in TLR2-deficient mice despite a TLR2-dependent response seen with synthetic TLR2 agonists. TLR4, MyD88, and complement activation were not required for the inflammatory response to zymosan. Although dectin-1 receptor inhibition blocked the inflammatory response of alveolar macrophages to zymosan in vitro, in vivo pulmonary leukocyte recruitment was not altered even in the absence of TLR2 or complement. Depletion of alveolar macrophages did not affect the response to zymosan. Neither complement,macrophages, nor TLR2, TLR4, MyD88, and/or dectin-1 receptors were involved in the pulmonary in vivo inflammatory response to zymosan.

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Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
5 mg/ml 23.5 mg/ml EPC/Chol 84/16 MLV none
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Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6, 25-30 g, 6-10 wks 25 µg in 100 µl aerosol / lungs alveolar macrophages (AM) no not evaluated
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Notes

      1. Aerosolization was accomplished via the Penn-Century Microsprayer (Model IA-1C)

Results

      1. ~78% of the AM were depleted (BAL cell counts via light microscopy).
      2. Slight, but statistically insignificant increase in PMN noted.

 

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Publication 5.

K.M. Roth, Immune response to primary aerosol infection with Francisella novicida, Thesis (PhD), Ohio State University, 2008.

Abstract

Francisella tularensis causes tularemia and death in humans without treatment. It has been classified as a group A bioterror agent due to its infectivity, extreme virulence and rapid dissemination. Aerosolized F. tularensis infection poses the greatest threat because pneumonic tularemia results in high mortality and would likely be the route used in a bioterrorist attack. Despite this, little is known about the host immune response to this pathogen, particularly after primary aerosol infection. Therefore, we have developed a novel aerosol model in mice using Francisella novicida, a subspecies of F. tularensis, to elucidate host immune responses to primary, pulmonary tularemia. Here we show that a F. novicida aerosol model in mice provides a useful tool for investigating immune responses that can be applied to human tularemia. We also report for the first time that primary F. novicida infection causes dystrophic cardiac calcinosis (DCC) in BALB/c mice and demonstrate that F. novicida interferes with antigen-specific CD4+ T cell responses by disrupting IFN-γ signaling in macrophages. In addition, we have shown that STAT1-/- mice are more susceptible to tularemia.
Characterization of this murine aerosol model has shown that F. novicida causes severe lung pathology and disseminates to the liver and the heart, where it causes pericardial calcification. We have also confirmed that macrophages are the prominent cell population in the lung following aerosol infection. Using this model, we show development of DCC in BALB/c mice is associated with significant induction of RANKL but not osteopontin (OPN) mRNA in their organs. Depletion of lung macrophages prior to infection markedly reduces pericarditis and calcification in BALB/c mice but does not affect their survival.
We have also demonstrated the effect of F. novicida infection on antigen-specific CD4+ T cell responses. We have shown that F. novicida disrupts STAT1-dependent IFN-γ signaling in macrophages by down-regulation of IFN-γRα expression. This disruption decreases MHC class II surface expression on macrophages and ultimately decreases antigen-specific CD4+ T cell responses. This is the first report of F. novicida interference in antigen-specific CD4+ T cell responses following primary infection. [/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 84/16 MLV PBS
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Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6, DBA/2, BALB/c, 8-10 wks, sex-matched 100 µl aerosol / lungs alveolar macrophages (AM) 200 µl i.v.,400 µl i.p. see results
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Notes

      1. Liposomes are not described in the thesis, however van Rooijen is a co-author on the first part of the thesis. Therefore his typical preparation is assumed to be that used in the models.
      2. Aerosolization was accomplished via the Penn-Century Microsprayer (Model A-1C)

Results

      1. >95% depletion by FACS of BAL stated but no data or further details presented.
      2. Pericardial calcification was significantly reduced in animals treated with aerosol clodronate liposomes compared to animals treated with aerosol PBS liposomes, systemically treated or control animals.

 

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Publication 6.

K.M. Roth, S. Oghumu, A.A. Satoskar, J.S. Gunn, N. van Rooijen, A.R. Satoskar, Respiratory infection with Francisella novicida induces rapid dystrophic cardiac calcinosis (DCC), FEMS Immunology & Medical Microbiology, 53 (2008) 72–78.

Abstract

Francisella tularensis causes pulmonary tularemia and death in humans when left untreated. Here, using a novel aerosol infection model, we show that acute pulmonary Francisella novicida infection not only causes pneumonia and liver damage, but also induces dystrophic cardiac calcinosis (DCC) in BALB/c mice. C57BL/6 mice also develop pneumonia and hepatic damage, but fail to develop DCC. Development of DCC in BALB/c mice is associated with significant induction of RANKL but not osteopontin in their organs. Depletion of lung macrophages prior to infection markedly reduces pericarditis and calcification in BALB/c mice but does not increase their susceptibility to infection. [/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 84/16 MLV PBS
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Animal Description Clodronate Dose Dosing Site/Method Target Phagocytes Systemic Dosing? Systemic Results
C57BL/6, BALB/c, 8-10 wks, sex-matched 100 µl aerosol / lungs alveolar macrophages (AM) 200 µl i.v.,400 µl i.p. see results
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Notes

      1. This paper is the published version of the study from the author’s thesis (Publication 4.)
      2. Liposomes are not described in the thesis, however van Rooijen is a co-author on the first part of the thesis. Therefore his typical preparation is assumed to be that used in the models.
      3. Aerosolization was accomplished via the Penn-Century Microsprayer (Model A-1C)

Results

      1. >95% depletion by FACS of BAL stated but no data or further details presented.
      2. Pericardial calcification was significantly reduced in animals treated with aerosol clodronate liposomes compared to animals treated with aerosol PBS liposomes, systemically treated or control animals.

 

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1 Clodronate concentration usually assumed. This value is the result of a clodronate assay.

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