Sialoadhesin on macrophages: its identification as a lymphocyte adhesion molecule.

In this study we present evidence that the mouse and rat sialoadhesin (originally named sheep erythrocyte receptor) on macrophages can function as a lymphocyte adhesion molecule. Lymphocytes were shown to bind to the splenic marginal zone, and lymph node subcapsular sinus and medulla in a frozen section assay. Selective depletion experiments showed that binding was mediated by macrophages. Adhesion was blocked by preincubation of the sections with monoclonal antibodies against mouse or rat sialoadhesin. Binding was temperature dependent, divalent cation independent, and involved sialic acid residues on the lymphocyte, as it could be inhibited by prior neuraminidase treatment or addition of the ganglioside GD1a. Binding to sialoadhesin was confirmed using the purified receptor and was observed among T cells, T blasts, B cells, and B blasts. Isolated macrophages or dendritic cells showed little binding. Sialoadhesin provides the first example of a macrophage-restricted lymphocyte adhesion molecule.

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.

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 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.

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.

Regulation of CD 163 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-gamma. 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.

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

Dendritic immune complex trapping cells in the spleen of the snake, Python reticulatus.

The spleen of the snake Python reticulatus, was investigated as to its general histology as well as the presence of immune complex trapping cells both at the light and electron microscopical level. Histological examination revealed that the spleen of this reptile was encapsulated and contained some trabeculae. In the splenic parenchyma two different regions could be distinguished: viz. red and white pulp. The white pulp appeared to be arranged around "central arterioles" and their smaller branches extending towards the periphery of the white pulp. The red pulp was composed of blood sinusoids and cell cords. Electron microscopy revealed at least three types of non-lymphoid cells in the white pulp of the spleen of python: reticulum cells, forming the framework; some macrophages and dendritic cells predominantly located in the periphery of the white pulp. Of these types of non-lymphoid cells, only dendritic cells were able to trap and to retain intravenously injected horseradish peroxidase (HRP)-rabbit-anti-HRP immune complexes on their cell surface as determined by enzymehistochemistry at the light and electron microscopical level. These dendritic cells were frequently found in association with collagen fibres and did not engulf large quantities of carbon particles. These data suggest that dendritic cells in the spleen of the python might be the phylogenetic precursors of the mammalian follicular dendritic cells.

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.

Expression of the ED3 antigen on rat macrophages in relation to experimental autoimmune diseases.

Susceptibility to experimental autoimmune diseases (EAD) is rat strain dependent. Susceptible animals are reported to have a defective glucocorticoid response. Although many EAD are regarded as preferentially T cell-mediated, macrophages (M phi) play an important role in several different stages of these diseases. In this study we have investigated the possible effect of the disturbed hypothalamic-pituitary (HPA) axis on M phi phenotype. Therefore we studied M phi differentiation in several different rat strains, especially with regard to the M phi specific differentiation antigen as recognized by monoclonal antibody (mAb) ED3. This mAb is, in normal healthy rats, reactive with very restricted M phi subpopulations present in the lymphoid organs only. However, in autoimmune diseased tissues many of the infiltrated M phi are also ED3-positive. It appeared that M phi, in vitro derived from monocytes out of susceptible rat strains, showed a high ED3 expression in contrast to monocyte-derived M phi out of resistant rat strains. This difference in ED3 expression appeared to be T cell-mediated. Our results are suggestive for the fact that the impaired HPA-axis in EAD susceptible rat strains affects M phi differentiation. The relevance of the observed differences with respect to disease induction, maintenance, or suppression is discussed and obviously needs further investigation.

Transport of immune complexes from the subcapsular sinus into the lymph node follicles of the rat.

To study the mode of transport of immune complexes from the subcapsular sinus into the follicles of draining popliteal lymph nodes, horse radish peroxidase (HRP)-anti HRP was injected in rat footpads. Within six minutes, complexes were already present in the subcapsular sinus freely or attached to the plasma membrane of different types of cell including cells forming the stroma. A few minutes later, complexes were also seen in the deeper part of the outer cortex, and after two hours they had reached the periphery of the follicles. They were always seen scattered between lymphoid and non-lymphoid cells. After one day, complexes were present on well-developed follicular dendritic cells. After injection of HRP, no localization of this antigen was observed in the deeper part of the outer cortex including the follicles. These results strongly suggest that HRP-anti HRP complexes are passively transported through the outer cortex into the follicles where they are trapped and retained by follicular dendritic cells.

Liposome mediated affection of monocytes.

Dichloromethylene diphosphonate (Cl2MDP) enclosed in liposomes, when injected intravenously, selectively eliminates all phagocytizing macrophages that are in direct contact with the blood circulation of the spleen and the liver. We examined whether Cl2MDP containing liposomes affect, in addition, rat monocytes and bone marrow macrophage precursors (BMMP's). In addition to Phosphatidylcholine-Cholesterol liposomes (PC liposomes), we also used mannosylated PC liposomes (PCMAN liposomes), which are reported to bind more efficiently to macrophages. Monocytes appeared to be affected 24 h after injections of 2 ml of Cl2MDP containing PC as well as PCMAN liposomes. In addition, almost no macrophages could be detected in one week cultures of blood leukocytes isolated from these animals; cultures from control animals contained +/- 50% macrophages. Bone marrow macrophage precursors did not appear to be affected.

Macrophages in experimental autoimmune diseases in the rat: a review.

In the pathogenesis of most experimental autoimmune diseases T lymphocytes play a crucial role in the initiation, whereas macrophages are essential in the effector phase. This review deals with several methods to elucidate the exact role macrophages play in different stages of autoimmune models in the rat. By using monoclonal antibodies an inventory has been made on the different macrophage subsets that are present in the infiltrates of the affected tissues. That macrophages play a decisive role in provoking the clinical signs has been shown by several macrophage elimination studies. The severe tissue damage caused by macrophages is brought about by the release of inflammatory mediators. Especially interference with the production or action of these products could provide new therapeutical means.