Selectivity of the approach with respect to macrophages

Since the liposome-mediated macrophage suicide technique is based on a rapid internalization and consecutive intracellular degradation of liposomes, it is not surprising that macrophages are the only cells to be affected. Indeed the approach allows the selective removal of mononuclear phagocytes from heterogeneous spleen cell populations in vitro. No effect was found on non-phagocytic spleen cells as measured by growth, protein production, antigen presentation, and antigen-specific T cell proliferation (22). An important question concerns the possible depletion of neutrophil granulocytes as a consequence of treatment with clodronate-liposomes. These play a crucial role in the clearance of many micro-organisms. Neutrophils, however, appeared neither morphologically nor functionally to be affected by clodronate-liposomes administered in vivo (23). Dendritic cells (DC), which are mainly responsible for processing and presentation of antigens to T lymphocytes form another important cell population that might be affected. However, DC isolated from animals treated with clodronate-liposomes were not impaired in their ability to induce primary CTL responses (24). Recently it has been shown that two distinct populations of DC are present in the spleen. In addition to the classical population of DC in the T cell areas of the white pulp, a new subpopulation of DC has been described at the border between the marginal zone and the red pulp of the spleen. These so-called marginal DC (25) are able to phagocytose particulates in vivo, express markers characteristic of both DC and macrophages, and have a high turnover rate. In

Clodronate Administration Routes

Figure 2 The four most frequently applied administration routes of clodronate-liposomes are shown, (a) Intravenous (IV) administration of clodronate-liposomes may induce depletion of macrophages in spleen (SP), liver (LI), and bone marrow (BM), since liposomes may unhindered leave the blood vessels (BV) here, (b) Intraperitoneal (IP) administration of clodronate-liposomes may at first induce depletion of macrophages in the peritoneal cavity (PE). From there liposomes are drained towards the parathymal lymph nodes (LN2) where macrophages are also depleted. Via the ductus thoracicus, lymph is carried into the circulation. As a consequence macrophages in the organs mentioned in Figure 2a may also be depleted if the injected dose is high enough.

(c) Intratracheal (IT) Instillation of clodronate-liposomes may lead to a depletion of alveolar macrophages (AL) in the lung (LU), since these have direct access to the alveolar lumen.

(d) Subcutaneous administration of clodronate-liposomes in the draining area (DA) of a lymph node (LN1) may induce depletion of macrophages in that lymph node. If there are enough of the liposomes left after lymph node passage, the remaining liposomes, which are earned to a next lymph node station with the efferent lymph, may also lead to macrophage depletion in that lymph node (LN2). N.B. Macrophages in the testis (TE) and phagocytic synovial lining cells in knee joints (SY) may be depleted by direct local injections, but macrophage depletion in many other organs such as in the thymus (TH), eyes (EY), kidneys (Kl), gut (GU), or brain (BR) is difficult and at best it is incomplete.

contrast, DC in the T cell areas are not phagocytic in vivo and have a low turnover rate. Not surprisingly, the marginal DC were completely depleted by a single intravenous injection with clodronate-liposomes, whereas DC in the T cell areas were not affected (25). However given the finding that the antigen presenting capabilities of DC, isolated from spleen of animals that were previously treated with clodronate-liposomes, were not affected, the question remains whether the new DC have to be considered macrophages or DC.

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