J. G. M. C. Damoiseaux

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.

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 (BMMPs). 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.

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.

Transcriptional organization of the DNA region controlling expression of the K99 gene cluster

SummaryThe transcriptional organization of the K99 gene cluster was investigated in two ways. First, the DNA region, containing the transcriptional signals was analyzed using a transcription vector system with Escherichia coli galactokinase (GalK) as assayable marker and second, an in vitro transcription system was employed. A detailed analysis of the transcription signals revealed that a strong promoter PA and a moderate promoter PB are located upstream of fanA and fanB, respectively. No promoter activity was detected in the intercistronic region between fanB and fanC. Factor-dependent terminators of transcription were detected and are probably located in the intercistronic region between fanA and fanB (T1), and between fanB and fanC (T2). A third terminator (T3) was observed between fanC and fanD and has an efficiency of 90%. Analysis of the regulatory region in an in vitro transcription system confirmed the location of the respective transcription signals. A model for the transcriptional organization of the K99 cluster is presented. Indications were obtained that the trans-acting regulatory polypeptides FanA and FanB both function as anti-terminators. A model for the regulation of expression of the K99 gene cluster is postulated.

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.

Three Monoclonal Antibodies to Antigen Presenting Cells in the Rat with Differential Influence on Cellular Interactions

A physical interaction between antigen presenting cells (APC) and T cells is essential for evoking an immune response1. It has been proposed that T cells first bind to APC by an antigen-independent mechanism. Antigen specific lymphocytes are then selected and activated within the APC-T cell cluster resulting in T cell proliferation2. In the first stage several accessory molecules, like LFA-1, ICAM-1, CD2, and LFA-3, are involved in the antigen-independent interaction1,2,3. Thereafter the antigen-dependent interaction, mediated by the T cell receptor (TCR), CD3, CD4, and the MHC Class II molecule, becomes more important and induces cytokine production triggering T cell proliferation1,3,4,5. In the third stage, which can occur in the absence of APC, IL-2 alone mediates proliferation of the responsive T cells.

Macrophage subpopulations and reticulum cells in rat placenta

SummaryThe placenta is a unique mixture of histoincompatible cells derived from mother and fetus. The aim of the present study was to obtain information on the development of macrophage subpopulations and reticulum cells during pregnancy in the placenta. Placentas of Wistar rats were removed at several stages of gestation, and were studied by immunohistochemical techniques applying monoclonal antibodies against macrophage subpopulations, lymphoid cells and reticulum cells. The expression of MHC class-II antigens was also studied. Throughout gestation macrophages were demonstrable in large numbers in the endometrium, in the myometrium and in the metrial gland, which is a compartment developing in the myometrium of pregnant rodents. In the labyrinth, a placenta compartment consisting of fetal cells, macrophages (probably of fetal origin) were already found on day 15. In the spongiotrophoblast and decidua basalis, which are layers of the placenta containing both maternal and fetal cells, only a few macrophages were recognized throughout gestation. The monoclonal antibody ED11, raised against reticulum cells, recognized fiber-like structures lining the blood sinuses of the spongiotrophoblast, in which only maternal blood is circulating. As the antigen recognized by ED11 is believed to play a role in the trapping of immune complexes, the spongiotrophoblast may play a role in the protection of the fetus from circulating immune complexes.

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.