C.D. Dijkstra

Depletion and repopulation of macrophages in spleen and liver of rat after intravenous treatment with liposome-encapsulated dichloromethylene diphosphonate

SummaryRats received a single intravenous injection with liposome-encapsulated dichloromethylene diphosphonate (Cl2MDP). This treatment resulted in the elimination of macrophages in spleen and liver within 2 days. Macrophages ingest the liposomes and are destroyed by the drug, which is released from the liposomes after disruption of the phospholipid bilayers under the influence of lysosomal phospholipases. Repopulation of macrophages in spleen and liver was studied at different time intervals after treatment. Macrophages in the liver (Kupffer cells) and red pulp macrophages in the spleen were the first cells to reappear, followed by marginal metallophilic macrophages and marginal-zone macrophages in the spleen. Different markers of the same cell did not reappear simultaneously. On the other hand, the same marker (recognized by the monoclonal antibody ED2) reappeared much more rapidly in the liver than in the spleen. The present results in the rat were different from those earlier obtained in the mouse. Red pulp macrophages were the first cells and marginal zone macrophages were the last cells to repopulate the spleen in both rodents after treatment with Cl2MDP liposomes. However, there was much more overlap in the repopulation kinetics of splenic macrophage subpopulations in the rat, when compared with the mouse.

Regulation of CD163 on human macrophages: cross-linking of CD163 induces signaling and activation

CD163 is a member of the group B scavenger receptor cysteine‐rich (SRCR) superfamily. This study describes aspects of the tissue distribution, the regulation of expression, and signal transduction after cross‐linking of this receptor at the cell surface of macrophages. CD163 showed an exclusive expression on resident macrophages (e.g., red pulp macrophages, alveolar macrophages). The expression was inducible on monocyte‐derived macrophages by glucocorticoids but not by interleukin‐4 (IL‐4), granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), and interferon‐γ. The combination of IL‐4 or GM‐CSF with glucocorticoids resulted in a further increase. Subcellular analysis of alveolar macrophages by immunoelectron microscopy showed a plasma membrane localization of the antigen. Cross‐linking of CD163 with monoclonal antibody induced a protein tyrosine kinase‐dependent signal that resulted in (1) slow‐type calcium mobilization, (2) inositol triphosphate production, and (3) secretion of IL‐6 and GM‐CSF. The data suggest a function for the SRCR‐superfamily receptor CD163 in the regulation of inflammatory processes by macrophages. J. Leukoc. Biol. 66: 858–866; 1999.

Heterogeneity of macrophages in the rat evidenced by variability in determinants: two new anti-rat macrophage antibodies against a heterodimer of 160 and 95 kd (CD11/CD18).

A set of three monoclonal antibodies (MoAbs), ED1, ED2, and ED3, has been shown to recognize in situ different subsets of macrophages in the rat. This macrophage diversity can be correlated with differences in stage of differentiation of cells belonging to one lineage. The present study quantifies this antigen distribution in the macrophage fractions of several lymphoid organs provided by Percoll centrifugation. Four new MoAbs (ED4, ED7, ED8, and ED9) raised against macrophages are included in this study. The tissue distribution of each of the four new MoAbs is determined by immuno‐ and enzyme‐histochemistry on cryostat sections. The MoAbs recognize distinct subpopulations of macrophages. The new MoAbs ED4, ED7, ED8, and ED9 recognize granulocytes and other unrelated cell types, as well as cells of the mononuclear phagocyte system. ED7 and ED8 recognize a surface heterodimer of Mr 160,000 and 95,000.

Macrophages in T cell line-mediated, demyelinating, and chronic relapsing experimental autoimmune encephalomyelitis in Lewis rats.

About 50% of the mononuclear cells in the perivascular lesions in the central nervous system (CNS) of rats suffering from experimental allergic encephalomyelitis (EAE) are blood‐borne macrophages. In this study we investigated the role of these macrophages in different variants of EAE, using a liposome‐mediated macrophage depletion technique. Intravenously injected liposomes containing dichloromethylene diphosphonate (C12MDP) are ingested by macrophages and cause temporary and selective elimination of these cells. Macrophage depletion during EAE induced by a T cell line specific for myelin basic protein (MBP; T cell‐EAE) suppresses development of neurological signs of EAE. T cell‐EAE with pronounced demyelination as induced by an additionally injected MoAb directed against myelin oligodendrocyte glycoprotein (MOG) was also significantly ameliorated after macrophage depletion. During chronic relapsing EAE (CR‐EAE) the occurrence of relapses was prevented or suppressed, provided that the liposomes were injected before the initiation of a putative relapse. A chronic progressive course of CR‐EAE was not modified by CI2MDP containing liposome treatment. Histologic examination of the CNS of liposome‐treated animals confirmed decreased infiltration of macrophages into the parenchyma in the rats with T cell and AD‐EAE, whereas T cells were still present.

Meningeal and Perivascular Macrophages of the Central Nervous System Play a Protective Role During Bacterial Meningitis

Meningeal (MM) and perivascular macrophages (PVM) constitute major populations of resident macrophages in the CNS that can be distinguished from microglial cells. So far, there is no direct evidence that demonstrates a possible role of MM and PVM in the CNS during normal or pathologic conditions. To elucidate the role of the MM and PVM during CNS inflammation, we have developed a strategy using a single intraventricular injection of mannosylated clodronate liposomes, which results in a complete and selective depletion of the PVM and MM from the CNS. Depletion of the MM and PVM during experimental pneumococcal meningitis resulted in increased illness, which correlated with higher bacteria counts in the cerebrospinal fluid and blood. This was associated with a decreased influx of leukocytes into the cerebrospinal fluid, which occurred despite an elevated production of relevant chemokines (e.g., macrophage-inflammatory protein-2) and a higher expression of vascular adhesion molecules (e.g., VCAM-1). In contrast, the higher bacterial counts correlated with elevated production of local and systemic inflammatory mediators (e.g., IL-6) indicating enhanced local leukocyte and systemic immune activation, and this may explain the worsening of the clinical signs. These findings show that the PVM and MM play a protective role during bacterial meningitis and suggest that a primary action of these macrophages is to facilitate the influx of leukocytes at the blood-brain barrier. More in general, we demonstrate for the first time that the PVM and MM play a crucial role during inflammation in the CNS.

Therapeutic effect of the D2-dopamine agonist bromocriptine on acute and relapsing experimental allergic encephalomyelitis.

We examined the effect of bromocriptine (BCR) treatment on the duration and severity of neurological symptoms of acute experimental allergic encephalomyelitis (EAE), an animal model for demyelinating diseases, particularly multiple sclerosis. To mimic the clinical situation, BCR treatment was started after the onset of clinical signs. Furthermore, the effect of BCR treatment on the course of a chronic relapsing form of EAE was studied. BCR was injected at daily intervals in a dose that resulted in sustained suppression of plasma concentrations of prolactin, a pituitary hormone that plays a role in immunoregulation. In acute EAE, BCR therapy reduced both severity and duration of the clinical signs. In chronic relapsing EAE, BCR treatment did not affect the severity and duration of the first attack, but reduced the duration of the subsequent, second attack. Thus, BCR treatment improves the clinical course in animals with ongoing disease. These findings may have implications for the search for new therapeutic approaches in multiple sclerosis.

Rat bone marrow and monocyte cultures: influence of culture time and lymphokines on the expression of macrophage differentiation antigens.

A set of seven monoclonal antibodies (moabs) has been shown to discriminate in situ between distinct subpopulations of macrophages in the rat. It is still controversial if this heterogeneity is caused by the existence of different lineages or by differentiation of a common precursor. In both cases, the differentiation process might be regulated by microenvironmental factors. The present study examines the expression of the macrophage markers recognized by the seven ED‐moabs in bone marrow and monocyte cultures. Furthermore, the impact of culture time and stimulating factors on the antigen expression in these cultures was tested. The expression of the ED3 antigen is highly inducible in bone marrow cultures. Factors that might be responsible for the increased ED3 expression are investigated. This strong ED3 expression by bone marrow‐derived macrophages is nearly absent by monocyte‐derived macrophages. This implies that the ability to express ED3 is blocked before the macrophage precursor cells enter the circulation to become monocytes. The ED2 expression cannot be induced under the tested circumstances during culture. The expression of ED7 and ED8 is also highly inducible because bone marrow macrophages in vivo do not express these antigens. In culture, these macrophages stain positive for these markers already after the first day of culturing. The other three antigens are expressed on all macrophages under all tested circumstances.

Cellular binding mechanism on rat macrophages for sialylated glycoconjugates, inhibited by the monoclonal antibody ED3.

The mAb ED3 recognizes a subpopulation of rat macrophages, with a highly restricted tissue distribution. The tissue distribution as well as the in vitro expression of the ED3 antigen and of the sheep erythrocyte receptor (SER), binding unopsonized erythrocytes in the mouse, are very similar. This receptor has almost the same binding characteristics, although a different tissue distribution, as the sialic acid binding receptor (SAR), binding ganglioside‐coated erythrocytes in the rat In this study we summarize the available literature concerning these sialic acid binding receptors (SER and SAR). Furthermore we have identified ED3 as SER by inhibition studies of erythrocyte binding with mAb ED3, as well as by the newly developed equivalents ED16 and ED17. We also show that light trypsin treatment of alveolar macrophages, expressing SAR, results in SER‐like activity. This obtained SER‐like activity could not be blocked by the mAb ED3, indicating that SER and SAR are different receptors. It appears that rat macrophages can express two receptors for sialylated glycoconjugates, a high‐affinity receptor SER, recognized by mAb ED3, and a low‐affinity receptor SAR, not recognized by mAb ED3.

Mechanism of immune complex trapping by follicular dendritic cells

The follicular dendritic cell (FDC) is a major constituent of the microenvironment of the lymphoid follicle. FDC have the characteristic and unique property of binding antigens in the form of antigen-antibody complexes and retaining these complexes, without ingestion, for long periods of time. The presence of antigen-antibody complexes, also called immune complexes, on FDC is believed to play a crucial role in the development of B cell memory and the affinity maturation of the antibody response against T cell-dependent antigens.

Stromal components in rat bone marrow frozen sections compared to long-term rat bone marrow cultures

The haematopoietic microenvironment is believed to play an important role in controlling the haematopoietic process. Ultrastructural studies have shown that the haematopoietic stroma is composed of cellular as well as extracellular components. Relatively little is known about the distribution of the different stromal components in the bone marrow. The knowledge on the bone marrow microenvironment is mainly based on studies in which in vitro long-term bone marrow cultures have been used. Although this culture system offers a unique possibility to study haematopoietic in vitro, it does not fully represent the complexity of intact bone marrow.In the present study we describe the immunohistochemical distribution of different cellular and extracellular stromal components in frozen sections of rat bone marrow as well as in long-term bone marrow cultures, in order to compare the haematopoietic microenvironment used in in vitro studies in the in situ situation. We found that in situ a specific compartmentalization of stromal components exists in the bone marrow. Under culture conditions however, most stromal components are indeed present but the architecture present in the in situ situation had almost completely disappeared. The interaction between the different stromal elements was studied in the long-term bone marrow cultures. It appeared that under the chosen conditions, nodules were formed with a core of reticular cells and extracellular matrix. In close contact with this core immature macrophages appeared to proliferate and differentiate into mature, non-dividing macrophages.