R.H.J. Beelen

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.

Monoclonal antibody EBM11 (anti-CD68) discriminates between dendritic cells and macrophages after short-term culture

Identification of dendritic cells (DC) is usually done on the basis of their strong MHC class II expression, their typical dendritic morphology and their capacity to induce a strong proliferation of allogeneic T cells. However using these criteria DC can easily be confused with MHC class II positive macrophages (M phi). In addition, the lack of an antibody directed to a specific DC marker greatly hampers the discrimination between DC and M phi. In the present study it is shown that EBM11 (anti-CD68) is a marker specific for both human M phi and DC but in a distinctive way. Human DC locate the EBM11 reactivity in a discrete juxtanuclear spot in contrast to M phi which show EBM11 reactivity throughout the cytoplasm. This greatly improves identification of DC. Light and electron microscopy showed that the CD68 epitope is associated with (phago-)lysosomes. Remarkably the EBM11 spot was only seen in DC after short-term culture, which is an essential step in all classical DC enrichment procedures. Before culture, M phi and DC were indistinguishable. These results show the close relationship between M phi and DC and suggest an important role for the structure of the lysosomal apparatus in these antigen-presenting cells.

Peritoneal dialysis induces a local sterile inflammatory state and the mesothelial cells in the effluent are related to the bacterial peritonitis incidence.

Peritoneal Dialysis Induces a Local Sterile Inflammatory State and the Mesothelial Cells in the Effluent Are Related to the Bacterial Peritonitis Incidence H.J. Bos D.G. Struijk C.W. Tuk J.C. de Veld T.J.M. Helmerhorst E.C.M. Hoefsmit L. Arisz R.H.J. Beelen Departments of Cell Biology, Obstetrics and Gynecology, Internal Medicine, Medical Faculty and University Hospital, Free University; Department of Internal Medicine, University Hospital, Academic Medical Centre, Amsterdam, The Netherlands

Enhanced killing capacity of human Kupffer cells after activation with human granulocyte/macrophage-colony-stimulating factor and interferon γ

In this study we investigated the effect of the cytokines human granulocyte/macrophage-colony-stimulating Factor (hGM-CSF) and interferon γ (IFNγ) on human Kupffer-cell-mediated cytotoxicity against the SW948 coloncarcinoma cell line. Kupffer cells were isolated from small liver wedge biopsies, taken from 14 patient who had had abdominal surgery for colon carcinoma or partial hepatectomy. The cells were incubated with hGM-CSF (100 ng/ml), or with IFNγ (100 U/ml) or with their combination and the perecentage cytotoxicity was determined using a recently described modified assay. Additional experiments were performed with tumour-necrosis-factor-α(TNFα)-sensitive U937 cells as target. The TNFα secretion of Kupffer cells was measured and we evaluated the effect of TNFα on colon tumour targets. We performed human-Kupffer cell-mediated cytotoxicity blocking experiments with anti-TNFα and used paraformaldehydefixed Kupffer cells to demonstrate lysis of TNFα-sensitive WEHI-164 cells and of SW948 cells. The overall cytotoxicity against SW948 caused by unactivated Kupffer cells (n=14), and by Kupffer cells activated with hGM-CSF (n=14), IFNγ (n=6) or their combination (n=6) was respectively: 19.5±2.6%, 25.3±2.9% 41±9.4% and 45.6±8% at E/T=1 and 28.2±2.9%, 35.6±3.2%, 55.6±9.7% and 62.8% at E/T=5. All differences were statistically significant (P<0.05). No growth-promoting activity by hGM-CSF on the SW948 tumour cells was observed. U937 cells were highly susceptible to Kupffer-cell-mediated cytotoxicity. The TNFα secretion by human Kupffer cells increased in parallel to their cytotoxicity after incubation with these cytokines. Soluble TNFα had only a slight anti-proliferative effect on SW948 cells, while specific anti-TNFα blocked Kupffer cell cytotoxicity by up to 80%. Finally, paraformaldehyde-fixed Kupffer cells were able to lyse WEHI-164 and SW948 cells. This indicates that expression of cell-associated TNFα is the main cytolytic mechanism of human-Kupffer-cell-mediated cytotoxicity. The implications for the use of hGM-CSF and IFNγ in vivo are discussed.

Ontogeny of milky spots in the human greater omentum: An immunochemical study

Milky spots in the human greater omentum are preformed specific accumulations of primarily macrophages within the stroma of the greater omentum. To obtain a better understanding of milky spots in the human greater omentum, the development and the earliest forms of milky spots in the human greater omentum were studied, with special attention to the macrophage population.

Isolation of cytotoxic Kupffer cells by a modified enzymatic assay: a methodological study

Kupffer cell (KC)-mediated cytotoxicity against tumor cells is of interest, since the liver is a major site of metastatic growth of primary colorectal cancer. KC isolation methods from rat livers, to study the tumoricidal properties of these cells, are based on perfusion of the liver and are therefore not suitable for human KC isolation from liver biopsies. In view of application to isolate KC from small wedge human liver biopsies, we have developed an isolation procedure for rat KC that does not require perfusion techniques. Liver tissue fragments were incubated with pronase with continuous pH registration and neutralization. KC were subsequently separated from other non-parenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. KC and other non-parenchymal cells were identified by immunophenotyping with a cytoplasmic monoclonal antibody ED1 and by ultrastructural analysis. About 3 x 10(6) KC per gram liver were isolated with a final purity of > 95% without loss of viability. To ensure that functionally competent KC were isolated, we assayed cytotoxicity against CC531 tumor cells in a recent developed cell-mediated MTT assay. Maximum cytotoxicity of KC was approximately 40% at an effector to target ratio of 10. In conclusion our approach seems to be a useful and simple method to isolate KC with good functional properties from rat livers, without the need for perfusion techniques.

VLA molecule expression may be involved in the release of acute myeloid leukaemic cells from the bone marrow

Acute myeloid leukaemia (AML) cells have a variable capacity to egress from bone marrow into peripheral blood. This may be due to a variable lack of adhesion molecules on leukaemic cells. The expression of VLA1, 3, 4, 5, 6, beta 1-chain, LFA1, beta 2-chain, ICAM1 and NCAM appeared to be higher in bone marrow as compared to peripheral blood leukaemic cells, although this only reached significance for beta 1-chain (p less than 0.01). The number of cases with more than 20% positive cells in bone marrow leukaemic cells was lower in immature FAB-subtypes (M1, M5a) as opposed to more mature subtypes (M2, M3, M4, M5b) for the adhesion molecules tested. This reached significance for VLA5 (p less than 0.05) and beta 1-chain (p less than 0.007), while there was trend for VLA4. It is discussed that VLA4 and 5 may play a role in the release of leukaemic cells from the bone marrow.

Novel isolation and purification method permitting functional cytotoxicity studies of macrophages from milky spots in the greater omentum

Milky spots in the greater omentum are well organized perivascular infiltrates of leukocytes which are probably involved in the clearance of tumor cells from the peritoneal cavity. In milky spots, macrophages are the predominant cell type forming a distinct population of cells. To investigate whether these macrophages have a function in the control of metastatic spread in the peritoneal cavity, a novel isolation and purification method was developed in order to study the functional cytotoxicity of macrophages from milky spots in the greater omentum against tumor cells in vitro. In order to obtain a cell suspension, greater omenta of unstimulated healthy male WAG/RIJ rats were incubated in collagenase/DNase suspension and filtered. Subsequently, macrophages were isolated and purified using flow cytometry by sorting unstained cells on the basis of size and internal complexity. Macrophages and other cells were identified by routine May-Grünwald-Giemsa staining and by immunophenotyping with the specific macrophage monoclonal antibody ED 1. Furthermore, macrophage subtypes were characterized by ultrastructural analysis. Functional cytotoxicity of the isolated macrophages was assayed against the syngeneic CC 531 tumor cell line in a colorimetric MTT assay. From three greater omenta of healthy rats 1.16 +/- 0.16 x 10(6) macrophages were isolated with a purity of 83 +/- 2% and a viability of > or = 96%. The macrophages were of the exudate (monocytic), exudate-resident and resident cell type and were in equal proportions. The contaminating cells were mainly mesothelial. A maximum cytotoxicity of approximately 30% was reached with the macrophage fraction at an effector-to-target ratio of 10. Furthermore, it was established that the mesothelial cells did not exhibit cytotoxicity.

Isolation of rat and human Kupffer cells by a modified enzymatic assay.

A new rapid method is described for the isolation and purification of rat and human Kupffer cells without the need of liver perfusion techniques. Rat livers or small human liver wedge biopsies obtained peroperatively were incubated with pronase under continuous pH registration. Kupffer cells were subsequently separated from other nonparenchymal cells by Nycodenz gradient centrifugation and purified by counterflow centrifugal elutriation. Identification of Kupffer cells was achieved on the basis of ultrastructural analyses and immunophenotyping.

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

SummaryIn view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon γ (IFN) or tumour necrosis factor α (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [3H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.