Timo K. van den Berg

Apoptosis of macrophages induced by liposome-mediated intracellular delivery of clodronate and propamidine

Liposomes can be used as vehicles for intracellular delivery of drugs into phagocytic cells. Clodronate and propamidine, delivered into macrophages in this way, will kill these cells as a result of intracellular accumulation and irreversible metabolic damage. The so-called liposome-mediated macrophage suicide approach, which is based on this principle, is now frequently applied in studies aimed at unravelling macrophage function. In the present study, the mechanism of phagocytic cell death induced by liposome encapsulated drugs was investigated in vitro. Peritoneal macrophages and macrophages of the RAW 264 cell line were cultured in the presence of the liposome encapsulated drugs clodronate, propamidine and several forms of ethylenediaminetetraacetic acid (EDTA). The results obtained suggest that apoptotic death is induced in phagocytic cells both by liposomally delivered clodronate and by liposomally delivered propamidine. Although intracellular EDTA did induce apoptosis in a minority of the experiments, the results support earlier findings that EDTA does not deplete macrophages as effectively as clodronate and propamidine.

Reactive oxygen species are required for the phagocytosis of myelin by macrophages.

Reactive oxygen species (ROS) are thought to be involved in the pathogenesis of multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE). In this study we showed that the phagocytosis of myelin by macrophages triggers the production of ROS. We also demonstrated that ROS play a crucial role in the myelin phagocytosis. Blocking the ROS production with NADPH oxidase inhibitors (100 microM DPI or 10 mM Apocynin) essentially prevented the phagocytosis of myelin. Furthermore, scavenging of ROS with catalase (H2O2) or mannitol (OH-) decreased the phagocytosis of myelin by macrophages, whereas superoxide dismutase (O2-) did not show this effect. In addition, Lipoic acid (LA), a non-specific scavenger of ROS, also decreased the phagocytosis of myelin by macrophages. In our results, we demonstrate for the first time that ROS appear to play a regulatory role in the phagocytosis of myelin.

Core α1→3-fucose is a common modification of N-glycans in parasitic helminths and constitutes an important epitope for IgE from Haemonchus contortus infected sheep

Synthesis of parasite specific IgE plays a critical role in the defence against helminth infections. We report here that IgE from serum from Schistosoma mansoni infected mice and Haemonchus contortus infected sheep recognizes complex‐type N‐glycans from Arabidopsis thaliana, which contain R‐GlcNAcβ1→4(Fucα1→3)GlcNAcβ1‐Asn (core α1→3‐Fuc) and Xylβ1→2Manβ1→4GlcNAcβ1‐R (core β1→2‐Xyl) modifications, and honeybee phospholipase A2, which carries N‐glycans that contain the core α1→3‐Fuc epitope. Evidence is presented that core α1→3‐fucosylated N‐glycans bind a substantial part of the parasite specific IgE in serum of H. contortus infected sheep. These results suggest that the core α1→3‐Fuc antigen may contribute to induction of a Th2 response leading to the production of IgE. In addition we show here that N‐glycans carrying core α1→3‐Fuc and β1→2‐Xyl antigens are synthesized by many parasitic helminths and also by the free living nematode Caenorhabditis elegans. Since N‐glycans containing the core α1→3‐Fuc have also been implicated in honeybee and plant induced allergies, this conserved glycan might represent an important common IgE epitope.

The role of perivascular and meningeal macrophages in experimental allergic encephalomyelitis

The perivascular (PVM) and meningeal (MM) macrophages constitute a major population of resident macrophages in the central nervous system (CNS). To investigate a possible role of PVM and MM during CNS inflammation, we have analysed PVM and MM during experimental allergic encephalomyelitis (EAE), an experimental model for MS, in the rat. Our results demonstrate a remarkable increase in the expression of the ED2 antigen on PVM and MM (already at day 9 post-EAE induction), which precedes the onset of clinical symptoms and infiltration of leukocytes into the CNS (at day 13). Therefore, the onset of EAE is accompanied by alterations of PVM and MM, and the ED2 antigen provides an early marker of pathology during CNS inflammation. Moreover, selective depletion of the ED2-positive macrophages in the CNS using clodronate liposomes resulted in a suppression of the clinical symptoms. These observations indicate that PVM and MM play a role during the early stages of EAE development.

A method for the selective depletion of perivascular and meningeal macrophages in the central nervous system.

The perivascular (PVM) and meningeal (MM) macrophages form a distinct population of resident CNS cells, selectively expressing the mature macrophage marker ED2 in the rat. In order to elucidate the role of the PVM and MM in rats during normal functioning of the brain and pathology, we have developed a strategy employing a single intraventricular injection of clodronate liposomes. This resulted in a complete depletion of the PVM and MM. Clodronate liposomes did not deplete the microglial cells. In other parts of the body, a temporal and mild depletion effect was observed, which was restored within 1 week. Detailed analysis of the elimination and repopulation kinetics of the PVM and MM revealed a slow repopulation of the CNS, starting at 14 days post depletion. This selective depletion method of the PVM and MM will enable us to get direct insight in their functions during normal and pathologic conditions of the CNS.

CXC-chemokines KC and macrophage inflammatory protein-2 (MIP-2) synergistically induce leukocyte recruitment to the central nervous system in rats.

Intracisternal injection of the CXC-chemokines KC or macrophage inflammatory protein (MIP)-2 induced a pleocytosis in the cerebrospinal fluid (CSF) of rats in a dose dependent way. MIP-2 was much more potent than KC. The concurrent injection of both chemokines revealed a profound synergistic effect on leukocyte recruitment into CSF.

Dexamethasone promotes phagocytosis and bacterial killing by human monocytes/macrophages in vitro

One of the actions of glucocorticoids (GC) in multiple sclerosis (MS) is an inhibitory effect on demyelination. This can be caused by a reduction in the number of infiltrating macrophages and/or by an effect on the phagocytosis of myelin. Here we investigate the effect of GC on the phagocytosis of myelin. Contrary to what was expected, we found that incubation of human monocytes with dexamethasone (DEX) for 48 h augmented (approximately threefold) the phagocytosis of myelin. This enhancement of phagocytosis by human monocytes was not restricted to myelin. Phagocytosis of various particles mediated by different macrophage receptors was increased by DEX. We found that not only the phagocytosis of Staphylococcus aureus bacteria was augmented, but also the killing of these bacteria was at least twice as effective after culture with DEX. Tumor necrosis factor α production of human monocyte‐derived macrophages induced by lipopolysaccharide and S. aureus was suppressed by DEX. Together our results show that DEX promotes the phagocytosis of particles by human monocytes and thereby may contribute to tissue repair after immune‐mediated tissue damage or infection. These data shed a new light on the clinical application of GC. J. Leukoc. Biol. 67: 801–807; 2000.

Innate immune ‘self’ recognition: a role for CD47–SIRPα interactions in hematopoietic stem cell transplantation

Self-nonself discrimination is a central property of the immune system. This paradigm was originally established in the context of tissue transplantation, leading to the discovery of major histocompatibility complex molecules as signals of self. However, accumulating evidence has shown that innate immune cells are regulated in a similar fashion. Recent evidence has suggested that interactions between the self molecule CD47 and the innate inhibitory receptor signal regulatory protein-alpha expressed on macrophages may be a critical determinant of transplant engraftment, supporting the concept that self-awareness is a general property of all immune cells.

Ultrastructural Localisation of Sialoadhesin (Siglec- 1) on Macrophages in Rodent Lymphoid Tissues

In previous studies it has been demonstrated that sialoadhesin is a macrophage-restricted adhesion receptor for lymphocytes and myeloid cells. It is under normal circumstances expressed by subpopulations of macrophages in lymphoid and haemopoietic tissues. In this study different immunoelectronmicroscopical techniques are used to investigate the ultrastructural localisation of sialoadhesin within the lymph node and spleen of rodents. The results show that sialoadhesin is selectively expressed by a subset of macrophages in peripheral lymphoid tissues. Sialoadhesin was localised predominantly on the plasma membrane and in particular in areas of intimate contact with lymphocytes, thereby visualizing putative local interaction between these cells. Interestingly, sialoadhesin was also detected in intracellular vesicles that were apparently taken up by macrophages. These findings are consistent with the putative role of sialoadhesin in local cell-cell interactions in lymphoid tissues. Surprisingly, sialoadhesin was also found at contact points of macrophages with other macrophages, sinus-lining cells and reticulum cells, suggesting that sialoadhesin also mediates interactions with these cell types.

C1 Inhibitor Treatment Improves Host Defense in Pneumococcal Meningitis in Rats and Mice

In spite of antibiotic treatment, pneumococcal meningitis continues to be associated with significant morbidity and mortality. The complement system is a key component of innate immunity against invading pathogens. However, activation of complement is also involved in tissue damage, and complement inhibition by C1 inhibitor (C1-inh) is beneficial in animal models of endotoxemia and sepsis. In the present study, we demonstrate classical pathway complement activation during pneumococcal meningitis in rats. We also evaluate the effect of C1-inh treatment on clinical illness, bacterial clearance, and inflammatory responses in rats and mice with pneumococcal meningitis. C1-inh treatment was associated with reduced clinical illness, a less-pronounced inflammatory infiltrate around the meninges, and lower brain levels of proinflammatory cytokines and chemokines. C1-inh treatment increased bacterial clearance, possibly through an up-regulation of CR3. Hence, C1-inh may be a useful agent in the treatment of pneumococcal meningitis.