Ed A. Döpp

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.

Interferon-β directly influences monocyte infiltration into the central nervous system

Interferon-beta (IFN-beta) has beneficial effects on the clinical symptoms of multiple sclerosis (MS) patients, but its exact mechanism of action is yet unknown. We here suggest that IFN-beta directly modulates inflammatory events at the level of cerebral endothelium. IFN-beta treatment resulted in a marked reduction of perivascular infiltrates in acute experimental allergic encephalomyelitis (EAE), the rat model for MS, which was coupled to a major decrease in the expression of the adhesion molecules ICAM-1 and VCAM-1 on brain capillaries. In vitro, IFN-beta reduced the mRNA levels and protein expression of adhesion molecules of brain endothelial cell cultures and diminished monocyte transendothelial migration. Monocyte adhesion and subsequent migration was found to be predominantly regulated by VCAM-1. These data indicate that IFN-beta exerts direct antiinflammatory effects on brain endothelial cells thereby contributing to reduced lesion formation as observed in MS patients.

Macrophage phagocytosis of myelin in vitro determined by flow cytometry: phagocytosis is mediated by CR3 and induces production of tumor necrosis factor-α and nitric oxide

Demyelination of axons in the central nervous system (CNS) during multiple sclerosis (MS) and its animal model experimental allergic encephalomyelitis (EAE) is a result of phagocytosis and digestion by macrophages (M phi) and the local release of inflammatory mediators like tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO). We have investigated the process of myelin phagocytosis by M phi in vitro using flow cytometric analysis. The binding and uptake of CNS-derived myelin was dose dependent, was abolished in the presence of EDTA and was enhanced after opsonization with complement. The phagocytosis of opsonized myelin could be inhibited by antibodies directed against complement receptor type 3 (CR3). Furthermore, CR3 also contributes to phagocytosis of non-opsonized myelin, e.g. under serum-free conditions. The phagocytosis of CNS-derived myelin induced the production of substantial amounts of TNF-alpha and NO by the M phi. Our results indicate an important role for CR3 in myelin phagocytosis. The induction of TNF-alpha and NO which accompanies this phagocytosis may further contribute to the overall process of demyelination during MS or EAE.

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.

Characterization and Expression of the Antigen Present on Resident Rat Macrophages Recognized by Monoclonal Antibody ED2

Because of the absence of a specific marker for labeling resident macrophages in the rat, there is almost no information available regarding the properties of individual resident macrophages in different organs. The recently described and in our laboratory developed mAb ED2, has been shown to exclusively recognize resident macrophages. The present study examines expression, function and structure of the ED2 antigen to obtain more information about the marker and therefore, more information about resident macrophages. In earlier studies, the expression of ED2 could not be induced by a range of macrophage stimulating factors under non-adherent culture conditions. We show a highly inducible expression of the ED2 antigen under adhering, non proliferating conditions as well as in long-term bone marrow cultures. ED2 appears to recognize a surface protein on resident macrophages consisting of three protein chains of 175, 160, and 95 kDa.

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.

Macrophage scavenger receptor MARCO: in vitro and in vivo regulation and involvement in the anti-bacterial host defense.

Abstract Recently, a novel murine member of the scavenger receptor class A family, designated MARCO, has been cloned [4] . Scavenger receptors have a characteristic broad ligand specificity and are able to bind various substances, including bacteria cell surface components. The receptor MARCO is expressed by a distinct subset of macrophages in spleen and lymph nodes. Using a panel of monoclonal antibodies (mAbs) specifically directed against MARCO, we investigated the regulation of this receptor in vitro and in vivo. Stimulation of the mouse macrophage cell line J774.2 in vitro with bacterial lipopolysaccharides (LPS) induces upregulation of MARCO surface expression. In accordance with this observation, in mice suffering from endotoxin shock caused by bacterial infection, expression of MARCO is induced on macrophages in the liver, lung and spleen, which normally do not express this receptor. We found that two of the anti-MARCO mAbs, ED29 and ED31, were able to block ligand binding. They inhibited the uptake of heat-killed Escherichia coli by MARCO expressing CHO cells. Further experiments will be needed to confirm the importance of MARCO in the anti-bacterial host defense.

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.

Complementary Adhesion Molecules Promote Neutrophil- Kupffer Cell Interaction and the Elimination of Bacteria Taken Up by the Liver

Most bacteria that enter the bloodstream are taken up by the liver. Previously, we reported that such organisms are initially bound extracellularly and subsequently killed by immigrating neutrophils, not Kupffer cells as widely presumed in the literature. Rather, the principal functions of Kupffer cells demonstrated herein are to clear bacteria from the peripheral blood and to promote accumulation of bactericidal neutrophils at the principal site of microbial deposition in the liver, i.e., the Kupffer cell surface. In a mouse model of listeriosis, uptake of bacteria by the liver at 10 min postinfection i.v. was reduced from approximately 60% of the inoculum in normal mice to ∼15% in mice rendered Kupffer cell deficient. Immunocytochemical analysis of liver sections derived from normal animals at 2 h postinfection revealed the massive immigration of neutrophils and their colocalization with Kupffer cells. Photomicrographs of the purified nonparenchymal liver cell population derived from these infected mice demonstrated listeriae inside neutrophils and neutrophils within Kupffer cells. Complementary adhesion molecules promoted the interaction between these two cell populations. Pretreatment of mice with mAbs specific for CD11b/CD18 (type 3 complement receptor) or its counter-receptor, CD54, inhibited the accumulation of neutrophils in the liver and the elimination of listeriae. Complement was not a factor; complement depletion affected neither the clearance of listeriae by Kupffer cells nor the antimicrobial activity expressed by infiltrating neutrophils.

Ontogenetic development of T- and B-lymphocytes and non-lymphoid cells in the white pulp of the rat spleen

SummaryThe location and temporal appearance of the different classes of lymphocytes were investigated in the developing white pulp of the rat spleen. Additionally, indications were sought for the involvement of non-lymphoid cells in the localization of lymphocytes. The lymphocytes were demonstrated by their surface determinants (W3/13, IgM, IgG, IgA, Ia) in a two-step immunoperoxidase method; the non-lymphoid cells were characterized by immuno-enzyme-histochemical techniques. The results showed that 1) already at birth strongly Ia-positive cells are present in the T-cell area; 2) the marginal zone develops as a distinct compartment, independent of the PALS and follicles; 3) follicles are recognizable at day 14, the capacity to trap immune complexes on follicular dendritic cells occurring one week later.A possible relation between the development of the follicles and the differentiation of the follicular dendritic cell is discussed.