2000-cheng-1402

On Fri, 22-Jun-2012   /   Subconjunctival Administration  
Cheng H, Tumpey TM, Staats HF, van Rooijen N, Oakes JE, Lausch RN.Role of macrophages in restricting herpes simplex virus type 1 growth after ocular infection.Invest. Ophthalmol. Vis. Sci. 2000 May;41(6):1402–9.

 

PURPOSE
To investigate the role macrophages play in controlling herpes simplex virus (HSV)-1 replication after infection of the murine cornea.

METHODS
Macrophage depletion in selected tissues before or after virus infection was achieved by repeated subconjunctival (SCJ) and/or intravenous (IV) injection of liposomes containing dichloromethylene diphosphonate (L-Cl2MDP). Controls received liposomes containing phosphate-buffered saline (L-PBS). The efficiency of depletion was evaluated by histologic examination. Virus content in infected tissues was determined by standard plaque assay. Delayed-type hypersensitivity (DTH) responsiveness was assessed using the ear-swelling assay. Antibody isotype responses to virus antigens and cytokine production were monitored by enzyme-linked immunosorbent assay.

RESULTS
Balb/c mice given SCJ injection of L-Cl2MDP 4 and 2 days before HSV-1 corneal infection were found to have ocular virus titers as much as 105-fold higher than that seen in the L-PBS-treated controls 8 days after infection. When L-Cl2MDP treatment was delayed until 2 and 4 days after infection, virus titers in the eye were analogous to those in the control animals. Subconjunctival and submandibular lymph node macrophages in mice given local (SCJ) L-Cl2MDP pretreatment were profoundly reduced, whereas the number of corneal Langerhans’ cells and lymph node dendritic cells remained unchanged. Local L-Cl2MDP pretreatment was associated with significantly reduced DTH responsiveness to HSV-1 antigen, and an alteration in selected antibody isotype production. Depletion of macrophages in the subconjunctival tissue before corneal infection was not accompanied by enhanced virus growth at early times (2 or 4 days) after infection.

CONCLUSIONS
Macrophages play an important role in restricting HSV-1 growth after corneal infection. These cells appear to be required for the development of an acquired immune response, presumably by functioning in antigen processing and presentation. The hypothesis that macrophages are major participants in innate immunity to HSV-1 corneal infection was not supported.

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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 PBS

1Clodronate and lipid concentrations assumed based on referenced paper.

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Animal Description Clodronate Dose Dosing Method/Site Target Phagocytes Adjunct Dosing? Adjunct Dosing Results
BALB/c female mice, 12-14 w, 15-17 g 10 µl subconjunctivally subconjunctival MΦ 200 µl i.v. subconjunctival MΦ not depleted
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  1. Clodronate or lipid concentrations are stated in the paper nor does the referenced paper clearly state concentrations.
  2. Reference for liposome preparation — 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.

 

 

  1. Subconjunctival administration of liposomal clodronate resulted in depletion of subconjunctival and submandibular macrophages (presumably due to drainage of ocular lymph into submandibular lymph nodes) but not dendritic nor Langerhans cells as judged by histological assessment of acid phosphatase+ cells.
  2. Subconjunctival administration of liposomal clodronate pre-infection reduced the acquired immune response (DTH) but had no effect when dosed either post-infection or intravenously.
  3. Subconjunctival administration of liposomal clodronate pre-infection increased the titers of anti-HSV IgG2a (2.8X) and IgM (43X).
  4. Clodronate liposome treatment by either route before or after infection did not affect viral production.
  5. Neither liver nor splenic macrophage depletion was assessed when clodronate liposomes were administered intravenously, but subconjuntival macrophages were not depleted post-i.v. treatment with clodronate liposomes.

 

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1998-kooguchi-3164

On Fri, 23-Mar-2012   /   Nasal Inhalation  
Kooguchi K, Hashimoto S, Kobayashi A, Kitamura Y, Kudoh I, Wiener-Kronish J, Sawa,T.
Role of alveolar macrophages in initiation and regulation of inflammation in Pseudomonas aeruginosa pneumonia.
Infect. Immun. 1998 Jul;66(7):3164–9.
[toggle_content title=”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 105 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 107 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
[/custom_table] [/toggle_content] [toggle_content title=”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.
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  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? Not likely but important control.
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1996-hashimoto-L819

On Thu, 22-Mar-2012   /   Nasal Inhalation  
Hashimoto S, Pittet JF, Hong K, Folkesson H, Bagby G, Kobzik L, Frevert C, Wanatabe K, Tsurufuji S, Weiner-Kronish, J.
Depletion of alveolar macrophages decreases neutrophil chemotaxis to Pseudomonas airspace infections.
American Journal of Physiology – Lung Cellular and Molecular Physiology. 1996 May 1;270(5):L819 –L828.
[toggle_content title=”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 106 colony-forming units (CFU) or 5 x 107 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] [toggle_content title=”Clodronate Liposome Parameters”] [custom_table]
Clodronate Concentration Total Lipid Concentration Lipid Composition Lipid Mole % Liposome Type Control Liposomes
7 mg/ml 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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  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.
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  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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