Saturday, December 17, 2011

Depletion of alveolar macrophages by Clodrosome® (Clodronate encapsulated liposome)

Lung macrophages play a significant role in the biology of lung. They release over one hundred known secretory products such as cytokines, arachidonic acid metabolites and oxygen radicals. Lung macrophages are involved in diverse function such as the maintenance of lung sterility, immune modulation, identification and killing of cancer cells and prevention of allergy. Lung macrophages may also contribute to lung injury. As an example, cigarette smoking stimulates the generation of oxygen radicals by macrophages. Lung macrophages function as mobile unicellular endocrine and paracrine that are strategically located to monitor and modify the micro-environment of the lung.

Usually the term "lung macrophage" and "alveolar macrophage (AM)" are used synonymously, however this is not completely accurate. Pleural macrophages are also another sub-population of lung macrophages. Alveolar macrophages obtain energy predominantly by aerobic metabolism, since they reside in alveoli where the concentration of oxygen is high. Pleural macrophages, on the other hand, rely on glycolytic pathway to replenish their energy stores, because the concentration of oxygen in the pleural space is relatively low.

One way to investigate the in vivo function of a particular cell type is to deplete these cells in the laboratory animals such as mice and rats and to note alterations in response to experimental manipulations.

In this blog entry, we discuss the use of tracheal insufflation of liposome encapsulated clodronate to selectively deplete alveolar macrophages in laboratory animals. Clodronate liposomes target phagocytic macrophages, while minimizing exposure and possible injury to other cells that do not take up liposomes. Many papers have reported a significant depletion of alveolar macrophages in mice and rats lasting for more than 5 days after a single insufflation of clodronate liposomes. These alveolar macrophage depleted animals showed a markedly reduced ability to recruit neutrophils and to release tumor necrosis factor (TNF) into the alveolar space on endotoxin challenge. Insufflation of free and non-encapsulated clodronate drug also causes alveolar macrophage depletion. However free clodronate-induced alveolar macrophage depletion was not specific since ultra-structural studies revealed that it is also caused damage to the alveolar epithelial cells. Thus, encapsulation of clodronate into the liposomes is necessary to specifically target clodronate to macrophages.

The fact that clodronate causes lysis of alveolar macrophages buy only cytoplasmic edema of alveolar epithelial cells suggests that clodronate is preferentially more toxic to macrophages. Administration of clodronate liposomes leads to ingestion of liposomes by macrophages, which are then destroyed after phospholipase-mediated disruption of the liposomal membranes and release of encapsulated clodronate. Previous studies have shown that intravenous injection of free and non-encapsulated clodronate do not deplete hepatic and splenic macrophages. However, it has been observed that tracheal insufflation of free and non-encapsulated clodronate also depleted alveolar macrophages. The reason for this discrepancy is due to rapid clearance of clodronate which has a plasma half-life of a few minutes after intravenous injection. Because of the tight alveolar epithelial barrier the clearance of free and non-encapsulated clodronate from the alveolar space after tracheal insufflation is expected to be slow. As a result, alveolar macrophages are exposed to free and non-encapsulated clodronate for a much more longer period of time. To determine whether depletion of alveolar macrophages was depend on encapsulation of clodronate in liposomes, the effects of plain PBS liposomes and free and non-encapsulated clodronate on tracheal insufflation was studied by Breg et al (Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819). The study showed that insufflation of plain PBS liposomes (80 ul) had no effect on the number of rat alveolar macrophages, whereas insufflation of free and non-encapsulated clodronate at the dosage equivalent to the amount present in 80 ul of clodronate liposome caused a similar degree of alveolar macrophage depletion and PMN influx into the alveolar space as clodronate liposomes did.

Many studies have suggested that maximum depletion of alveolar macrophages occurred 3 days after tracheal insufflation of clodronate liposome. A single insufflation of clodronate liposome caused a dosed dependent reduction of alveolar macrophages reaching a maximal depletion of >70%at a dose of 120 ul (5 mg/ml encapsulated clodronate) of clodronate liposomes. A dose dependent increase in the number of PMNs also occurred. A time course study using 80 ul and 120 ul of clodronate liposomes shows that a significant depletion of alveolar macrophages occurred 1 day after insufflation and lasted for 7 days. However, by day 9, the number of alveolar macrophages was almost back to the baseline level. 120 ul of clodronate liposomes resulted in a higher PMN influx than did 80 ul of clodronate liposomes. PMN influx is not desirable and therefore the best dosage is the one that depletes the macrophages and minimizes the influx of PMN (See figure 1).

Figure 1- Effect of tracheal insufflation of clodronate liposomes on alveolar macrophages and neutrophils on rats (data was reproduced from the study done by Berg et al published in Journal of Applied Physiology, June 1993, vol. 74, no. 62812-2819)