Christine D. Dijkstra

The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3.

In the present study, a set of three monoclonal antibodies is described, each of which recognizes cells of the monocyte-macrophage lineage in the rat. The tissue distribution, in particular in lymphoid organs, of each of the three monoclonals is determined by immunoenzyme histochemistry on cryostat sections, as well as on cell suspensions. Results show that ED1 recognizes a cytoplasmic antigen in monocytes and in most macrophages, free and fixed. ED2 and ED3 recognize membrane antigens of tissue macrophages, discriminating between distinct subpopulations of macrophages, each with a characteristic localization in the compartments of lymphoid organs. No other cell types except cells of the mononuclear phagocyte system are positive for any of the three monoclonals. Possible relations between the macrophages recognized by this set of monoclonals and dendritic cells are discussed.

Suppression of experimental allergic encephalomyelitis by intraventricular administration of interferon-gamma in Lewis rats

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) which causes paralysis. Several studies have reported the involvement of Ia antigen‐expressing cells in the pathogenesis of EAE. Interferon‐gamma (IFN‐γ) can induce Ia antigen expression on a wide range of cells. We examined the effect of IFN‐γ on EAE in Lewis rats. Systemically administered IFN‐γ did not change the disease course of EAE, whereas IFN‐γ applied locally into the ventricular system of the CNS resulted in complete suppression of clinical signs. Furthermore, we found that systemic administration of anti‐IFN‐γ just prior to the onset of clinical symptoms resulted in a more severe disease course. We conclude that IFN‐γ is capable of exerting a suppressive action in EAE, possibly through induction of Ia antigen expression or through the induction of suppressive mechanisms locally in the CNS.

Immunohistological analysis of macrophages in the central nervous system of Lewis rats with acute experimental allergic encephalomyelitis

Inflammatory cells were characterized by immunohistochemistry, utilizing monoclonal antibodies specific for T cell subsets, B cells, Ia-positive cells and cells of the mononuclear phagocyte system, in cryostat sections of central nervous system of Lewis rats, sacrificed during the course of actively induced experimental allergic encephalomyelitis. The present study provides interesting information about the presence and distribution of cells of the monocyte-macrophage lineage in this immunologically mediated disease. Using these monoclonal antibodies different subpopulations of macrophages having varying distribution patterns in the central nervous system can be recognized.

Macrophages in the central nervous system of the rat

In an immunohistochemical study using monoclonal antibodies, which exclusively recognize cells of the monocyte-macrophage lineage, and monoclonal antibodies against the Ia-antigen, we describe the occurrence of macrophages in the developing and adult central nervous system (CNS). In normal adult brain, no macrophages could be detected in the CNS parenchyma; only in the meninges and the choroid plexes were a few macrophages found. During ontogeny, numerous phagocytic cells infiltrated the CNS parenchyma; these cells which did not express Ia are blood-borne. About three weeks after birth, all macrophages had disappeared from the CNS. As microglia in adult and developing brain do not stain with the anti-macrophage antibodies, we suggest that microglial cells are not related to the mononuclear phagocyte system and do not have a hematogenous origin.

Establishment of human adult astrocyte cultures derived from postmortem multiple sclerosis and control brain and spinal cord regions: immunophenotypical and functional characterization

We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3–9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte‐specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP‐positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1–3%) were present in the human adult astrocyte cultures, using a panel of macrophage‐specific markers. However, the monoclonal antibodies (mAbs KP1, EBM11, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non‐lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay). J. Neurosci. Res. 49:342–354, 1997.

Enhanced tumour growth in the rat liver after selective elimination of Kupffer cells

The evidence that Kupffer cells are capable of controlling metastatic growth in the liver in vivo is largely circumstantial. The best approach when studying natural cytotoxicity activities of Kupffer cells is to investigate the effect of Kupffer cell elimination on tumour growth. Until now it has not been possible to eliminate Kupffer cells without affecting other cell populations. We have recently developed a new method to eliminate Kupffer cells selectively: intravenous injection of liposome-encapsulated (dichloromethylene)bisphosphonate (Cl2MDP-liposomes) leads to effective elimination of all Kypffer cells, without affecting non-phagocytic cells. Wag/Rij rats were injected with Cl2MDP-liposomes. After 48 h, rats were inoculated with syngeneic CC531 colon carcinoma cells by injection in the portal system. The results show a strongly enhanced tumour growth in the liver of the Cl2MDP-liposometreated rats. In these animals, livers were almost completely replaced by tumour and had increased in weight, whereas in the control groups only a few (four to eight) small (1-mm) tumour nodules were found. These data show that selective elimination of Kupffer cells results in enhanced tumour growth in the liver, implying that Kupffer cells play a crucial role in controlling tumour growth in the liver.

The origin of osteoclasts: An immunohistochemical study on macrophages and osteoclasts in embryonic rat bone

SummaryThe origin of osteoclasts was studied in embryonic rat bone primordia using a set of monoclonal antibodies (ED1, ED2, and ED3) that exclusively recognize monocytes and macrophages. ED1 recognizes monocytes and macrophages. Mononuclear phagocytes which were ED1 positive were found in the perichondrium/periosteum of developing bone. These cells started to infiltrate the primordia when the cartilage became hypertrophic. During bone formation, multinucleated ED1-positive cells with the morphological characteristics of osteoclasts were found in the developing bone marrow cavity and against the bone collar. The present findings support the notion that osteoclasts arise by fusion of mononuclear phagocytes derived from blood monocytes.

Low-dose cyclosporin A induces relapsing remitting experimental allergic encephalomyelitis in the Lewis rat

Experimental allergic encephalomyelitis (EAE) in Lewis rats is an acute monophasic autoimmune disease. It can be treated prophylactically and therapeutically with high doses of cyclosporin A (CsA). Here we demonstrate that low-dose CsA does not prevent a first attack of EAE, but, on the contrary, induces a chronic relapsing form of the disease in 100% of Lewis rats examined. Possible explanations for the high relapse rate after low-dose CsA treatment are discussed. Further studies will be needed to evaluate the immunological mechanisms responsible for these results.

Liposome-mediated monocyte depletion during wallerian degeneration defines the role of hematogenous phagocytes in myelin removal.

Newly recruited hematogenous mononuclear cells of the monocyte/macrophage system are suggested to be important effector cells in myelin removal during Wallerian degeneration. Their role has extensively been studied in various in vitro and in vivo models. However, there has been much controversy concerning the role of hematogenous vs. resident cells of the peripheral nervous system in Wallerian degeneration. The present study used a recently established technique to deplete the hematogenous monocyte population by application of dichloromethylene diphosphonate‐containing liposomes. Intravenously injected liposomes containing dichloromethylene diphosphonate (Cl2MDP) are ingested by macrophages and monocytes and cause temporary and selective depletion of these cells. The number of LFA‐1‐and Mac‐l‐ positive macrophages within the nerves was significantly reduced when liposomes were injected shortly after nerve transsection. In these nerves, myelin degradation was significantly less, indicating an essential role of newly recruited phagocytes in this process. Macrophage invasion of degenerating nerves occurred within the first 2 days after transsection. Resident cells of the peripheral nerve participate in myelin removal since macrophage depletion did not completely abolish myelin degradation. These results confirm the important role of hematogenous phagocytes in myelin removal during Wallerian degeneration.

The role of macrophages in demyelination

Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS). In particular in the CsA-induced chronic relapsing form (CREAE), pronounced demyelination occurs, in temporal association with relapses. It is still a matter of discussion which cell type ultimately is responsible for the actual process of demyelination. Macrophages, cytotoxic T lymphocytes and also astrocytes are possible candidates. In this short overview, the role of macrophages in the pathogenesis of EAE is discussed. It is shown that in particular, newly recruited macrophages play a crucial role in the generation of clinical signs. Possible mechanisms by which macrophages are involved in the pathogenesis of demyelinating diseases are presented.