N. van Rooijen

Elimination of phagocytic cells in the spleen after intravenous injection of liposome-encapsulated dichloromethylene diphosphonate

SummaryDichloromethylene diphosphonate can be used for temporary elimination of macrophages in the spleen when administered after entrapment in liposomes. No comparable effect on the macrophages of the spleen was observed with free dichloromethylene diphosphonate or in the case of empty liposomes. Marginal metallophils on the boundary between white pulp and marginal zone as well as macrophages in the marginal zone and red pulp disappeared from the spleen within one day and remained largely absent for about a week. After this time cells reappeared slowly, and at approximately four weeks after injection their presence in the spleen did not differ from that in control animals. Marginal metallophils and macrophages in the spleen were demonstrated by use of enzyme-histochemical methods and by their capacity to ingest carbon particles.

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.

Pig islet xenograft rejection is markedly delayed in macrophage-depleted mice: a study in streptozotocin diabetic animals.

Abstract: The present study aimed to evaluate the effect of depletion of macrophages and/or natural killer (NK) cells on islet xenograft rejection in the pig‐to‐mouse model. Five microliters (4000 to 5000 IEQ, islet equivalents) of adult pig islets were transplanted under the renal capsule of C57BL/6 mice with streptozotocin‐induced diabetes. Macrophages were depleted by injection of liposome‐encapsulated dichloromethylene diphosphonate (Lip‐Cl2MDP) intraperitoneally (i.p.) at a dose of 100 µl/10 g body weight (BW) 2 days before transplantation, and 50 µl/10 g BW weekly thereafter. NK cells were depleted by injection of the monoclonal antibody NK 1.1 (anti‐NK 1.1 mAb) i.p. at a dose of 100 µg/mouse 1 day before transplantation, and then 25 µg per week thereafter. Islet graft survival was monitored by daily measurements of blood glucose. Graft survival was 8 ± 1.2 days in untreated controls, 9 ± 1.0 days with anti‐NK 1.1 mAb alone, 22 ± 4.9 days with Lip‐Cl2MDP alone (P < 0.01 vs. controls), and 26 ± 3.8 days with Lip‐Cl2MDP plus anti‐NK 1.1 mAb (P < 0.01 vs. controls). In the last group, two of six animals were killed with functioning grafts 30 days after transplantation. In untreated controls, rejected xenografts were heavily infiltrated by F4/80+ macrophages and CD3+ T cells. In Lip‐Cl2MDP‐treated groups, the number of F4/80+ macrophages was markedly reduced. On the periphery of xenografts, a small number of CD3+ T cells were observed. In conclusion, our results suggest that strategies targeting macrophages may facilitate islet xenograft survival. A role for NK cells cannot be excluded, but appears to be of minor importance.

Intrabursal Injection of Clodronate Liposomes Causes Macrophage Depletion and Inhibits Ovulation in the Mouse Ovary

Abstract To investigate the role of the ovarian macrophage population in ovulation, we examined the effect of depleting this population using liposome-encapsulated clodronate. Clodronate liposomes, saline liposomes, or saline alone was injected under the ovarian bursa in gonadotropin-primed adult mice, either 84 h (Day −3) or 36 h (Day −1) before ovulation. Ovulation rates were determined by counting the number of oocytes released. The numbers of graafian follicles and corpora lutea were also counted immediately before and after ovulation. Macrophage distribution within the theca and stroma of preovulatory ovaries was examined by immunohistochemistry with specific monoclonal antibodies to the macrophage antigens macrosialin, major histocompatability complex class II (Ia), and F4/80. Injection of clodronate liposomes on Day −1 did not affect ovulation rates, whereas administration on Day −3 caused a significant reduction in ovulation rate (mean oocytes ovulated = 5.25 ± 0.6 from clodronate liposome-treated ovaries and 9.13 ± 0.9 from saline-treated ovaries, respectively, P < 0.05). The numbers of macrosialin-positive macrophages present in the theca at ovulation were reduced by treatment with clodronate liposomes on Day −1, and treatment on Day −3 reduced the numbers of Ia-positive and macrosialin-positive macrophages present in the theca. When the subsequent ovarian cycles were examined by vaginal smearing, the metestrous-2/diestrous stage was found to be extended in clodronate liposome-treated animals (7.5 ± 1.3 days vs. 3.4 ± 0.4 days for saline liposome-treated animals, P < 0.05). These results suggest that thecal macrophages may be involved in the regulation of follicular growth and rupture, as well as being important for the normal progression of the estrous cycle.

Lymphatic uptake and biodistribution of liposomes after subcutaneous injection - IV. Fate of liposomes in regional lymph nodes

The ability of clodronate-containing liposomes to deplete lymph nodes of macrophages was used as a tool to investigate the fate of liposomes in regional lymph nodes after subcutaneous (s.c.) administration. Reduced lymph node localization of liposomes in macrophage-depleted lymph nodes confirmed that phagocytosis by macrophages plays an important role in lymph node retention of liposomes. Depletion of macrophages had less effect on lymph node localization of small liposomes than on the lymph node localization of large liposomes. Inclusion of distearoylphosphatidylethanolamine (DSPE)-poly(ethyleneglycol) (PEG-PE) into the liposomes, which is known to oppose macrophage uptake, did not affect lymph node localization in macrophage-depleted or control lymph nodes. We conclude that PEG-liposomes retained by lymph nodes are also taken up by lymph node macrophages. Morphological observations visualizing the uptake of PEG-liposomes by lymph node macrophages support this conclusion.

Reversible depletion of synovial lining cells after intra-articular treatment with liposome-encapsulated dichloromethylene diphosphonate

SummaryWe studied the depletion and repopulation of synovial lining cells in mice. A single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL2MDP) in the mouse knee joint caused selective elimination of synovial lining cells. Depletion of cells occurred within a few days as evidenced by light microscopic, electronmicroscopic and immunohistochemical studies. Maximal depletion was seen on day 7. Repopulation was observed in the following weeks, starting at the bone side of the joint. Until day 30, full recovery (60% recovery) was not observed in the lining lying adjacent to the dermis. Side effects on cartilage metabolism, such as inhibition of proteoglycan synthesis or degradation of proteoglycans from the matrix was minor but significant, 1 and 2 days after liposome treatment but thereafter full recovery was observed. Selective elimination of lining cells from the joint enabled us to study the in vivo role of these cells in the onset and subsequent pathology of experimental arthritis. An immune-complex-mediated experimental arthritis elicited in lining cell depleted joints that had received CL2MDP-liposomes 7 days earlier prevented inflammation as compared to controls.

Lymphocyte capping and lymphocyte migration as associated events in the in vivo antigen trapping process. an electron-microscopic autoradiographic study in the spleen of mice

SummaryThe fate of 125I labeled antigen-antibody complexes in the first two hours after intravenous injection was followed in the spleen of mice by light and electron microscopic autoradiography. It appeared that AAC were phagocytized by granulocytes and by macrophages in the marginal zone and were present over the surface of lymphocytes in that area. By rinsing the spleen before fixation it could be shown that AAC were indeed bound to the lymphocyte membrane and not merely present in the blood plasma between the cells. The label was present in a spotty fashion or over a so-called uropod. The majority of labeled uropods (13 out of 17) pointed away from the follicle. From this it was inferred that these lymphocytes moved to and most probably into, the follicles. Inside the follicles, at 1 hour post injectionem, most of the label was associated with interfaces between lymphocytes. At 2 hours post injectionem there was a preferential localization over interfaces between lymphocytes and dendritic reticulum cells. It is conceivable that antigen that is introduced into the circulation is ultimately presented to dendritic reticulum cells in a complexed form with antibody, probably with complement, and with the B-cell receptor, since receptor shedding is a normal event following capping.

A critical role for alveolar macrophages in elicitation of pulmonary immune fibrosis

Hapten immune pulmonary interstitial fibrosis (HIPIF) is induced by a recall cell‐mediated immune response against the hapten 2,4,6‐trinitrobenzene sulphonic acid (TNBS) in the lung. Studies here dissect the role of the cellular components of the bronchoalveolar lavage (BAL) cells (alveolar macrophages [AMs] versus monocytes and immature dendritic cells) in the fibrogenic inflammatory response. BAL cells from HIPIF mice were generally more activated and produced a greater amount of tumour necrosis factor‐α (TNF‐α) than controls. Liposome‐encapsulated dichloromethylene diphosphonate (Cl2MDP) that was inoculated intranasally (i.n.) into mice selectively depleted AMs. Following AM depletion, the number of TNF‐α‐containing cells was reduced, and both the number of immune inflammatory cells recruited into the alveolar space and the subsequent collagen deposition (hydroxyproline) were decreased in the sensitized and intratracheally (i.t.) challenged mice. In conclusion, AMs are required, in part, for the development of pulmonary fibrosis in HIPIF because AM‐derived factors such as TNF‐α are needed for initiation of chemokine and cytokine pathways and accumulation of immune inflammatory cells.

Cell type-specific type I interferon antagonism influences organ tropism of murine coronavirus

ABSTRACT Previous studies have demonstrated that mouse hepatitis virus (MHV) hepatotropism is determined largely by postentry events rather than by availability of the viral receptor. In addition, mutation of MHV nonstructural protein 2 (ns2) abrogates the ability of the virus to replicate in the liver and induce hepatitis but does not affect replication in the central nervous system (CNS). Here we show that replication of ns2 mutant viruses is attenuated in bone marrow-derived macrophages (BMM) generated from wild-type (wt) mice but not in L2 fibroblasts, primary astrocytes, or BMM generated from type I interferon receptor-deficient (IFNAR−/−) mice. In addition, ns2 mutants are more sensitive than wt virus to pretreatment of BMM, but not L2 fibroblasts or primary astrocytes, with alpha/beta interferon (IFN-α/β). The ns2 mutants induced similar levels of IFN-α/β in wt and IFNAR−/− BMM, indicating that ns2 expression has no effect on the induction of IFN but rather that it antagonizes a later step in IFN signaling. Consistent with these in vitro data, the virulence of ns2 mutants increased to near that of wt virus after depletion of macrophages in vivo. These data imply that the ability of MHV to replicate in macrophages is a prerequisite for replication in the liver and induction of hepatitis but not for replication or disease in the CNS, underscoring the importance of IFN signaling in macrophages in vivo for protection of the host from hepatitis. Our results further support the notion that viral tissue tropism is determined in part by postentry events, including the early type I interferon response.

MUCOSAL IMMUNOADJUVANT ACTIVITY OF LIPOSOMES: ROLE OF ALVEOLAR MACROPHAGES

Previously, we have reported on a liposomal adjuvant system for stimulation of both systemic IgG and mucosal s‐IgA responses against viral antigens (influenza virus subunit antigen or whole inactivated measles virus) administered intranasally to mice. Immune stimulation is observed with negatively charged, but not with zwitterionic, liposomes and is independent of a physical association of the antigen with the liposomes. Furthermore, liposome‐mediated immune stimulation requires deposition of the liposomes and the antigen in the lower respiratory tract. In the present study, it is shown that alveolar macrophages (AM) are the main target cells for negatively charged liposomes administered to the lungs of mice. AM isolated from animals, to which negatively charged liposomes were administered beforehand, showed large intracellular vacuoles, suggestive of massive liposome uptake. Under ex vivo conditions, both AM and RAW 264 cells exhibited a high capacity to take up negatively charged liposomes. The deposition of negatively charged liposomes, but not zwitterionic, liposomes in the lung reduced the phagocytic and migratory behaviour of AM, as assessed on the basis of transport of carbon particles to the draining lymph nodes of the lungs. Depletion of AM in vivo with dichloromethylene diphosphonate, facilitated an enhanced systemic and local antibody response against influenza subunit antigen deposited subsequently to the lower respiratory tract. In conclusion, these data provide support for the hypothesis that uptake of negatively charged liposomes blocks the immunosuppressive activity of AM, thereby facilitating local and systemic antibody responses.