Nel Kors

Macrophage subset repopulation in the spleen: differential kinetics after liposome-mediated elimination.

Different macrophage subsets can be discriminated in the well defined compartments of the mouse spleen by specialized functions and the presence of specific surface determinants. Red pulp macrophages, marginal zone macrophages, and marginal metallophilic macrophages are eliminated simultaneously within 24 hr by a single injection with liposome‐entrapped dichloromethylene diphosphonate (DMDP). After such elimination, these subsets show a striking difference in their kinetics of reappearance: Red pulp macrophages are back in normal numbers after 1 week, the marginal metallophilic macrophages take 2 weeks to regain fully their position at the border of the marginal zone and periarteriolar lymphocyte sheath, but it takes over 1 month for complete reappearance of the marginal zone macrophages. Marginal zone lymphocytes, also affected by treatment with the liposome‐entrapped drug, reappeared in the marginal zone within 2 weeks, indicating that marginal zone macrophages are not required for their localization and/or retention there. Approximately 2 weeks after treatment, all cells in the spleen have returned to normal numbers with the exception of marginal zone macrophages, which can be found only sporadically at that time. The results indicate that these macrophage subpopulations must have different precursor requirements. The differential reappearance of the macrophages creates the possibility of studying lineage analysis and will help to unravel the precise function of the marginal zone macrophages and marginal metallophilic macrophages in particular.

Effects of intracellular diphosphonates on cells of the mononuclear phagocyte system:In vivo effects of liposome-encapsulated diphosphonates on different macrophage subpopulations in the spleen

SummaryThe intracellular effects of different diphosphonates on splenic macrophages were compared after intravenous injection of liposomes with encapsulated dichloromethylene diphosphonate (C12MDP), 3-amino-1-hydroxypropane-1,1-diphosphonate (APD), or 1 hydroxyethane-1,1-diphosphonate (EHDP) in similar concentrations. Intracelular C12MDP was by far the most toxic compound for macrophages in the spleen. APD encapsulated in liposomes in the highest possible concentration eliminated marginal zone macrophages only and EHDP did not affect any of the macrophage subpopulations in the spleen.

In vitro and in vivo elimination of macrophage tumor cells using liposome-encapsulated dichloromethylene diphosphonate

SummaryIt is shown in the present study that RAW 264 tumor cells can be killed by liposomeentrapped dichloromethylene diphosphonate (DMDP), both in vitro and in vivo. DMDP is ingested by phagocytic cells when entrapped in liposomes. Once phagocytized the liposomal membranes are digested and the drug is released into the cell and is ready for action. In vitro, even low doses of liposome-entrapped DMDP caused an significant reduction in cell numbers. In vivo, liposome-encapsulated DMDP markedly reduced tumor formation in the liver, when given 1 day after injection of 1 × 106 RAW 264 tumor cells. Liposome-encapsulated DMDP, given 4 or more days after injection of the tumor cells had no significant effect. We concluded that tumor formation by RAW 264 cells is only susceptible to in vivo treatment with liposome-entrapped DMDP during a short period of time after injection of the cells. Obviously, phagocytosis of the tumor cells is reduced after this period making the cells less susceptible to treatment with the liposome-entrapped drug.

Morphology, Kinetics and Secretory Activity of Antibody-forming Cells

Specific antibody-forming cells from spleen, bone marrow and popliteal lymph nodes were studied in mice after subcutaneous priming and intravenous boosting with horseradish peroxidase (HRP). Functional antibody-secreting capacity of these cells was correlated with their morphology at the cell population level. For this purpose, cells synthesizing anti-HRP antibody from the same cell suspensions were studied simultaneously by light and electron microscopy and by a plaque assay. It appeared that the population of cells responsible for antibody synthesis as well as antibody secretion was morphologically heterogeneous: besides plasma cells, considerable numbers of antibody-forming lymphocytes, antibody-forming plasmablasts and antibody-forming immature plasma cells were observed. Immature plasma cells constituted the majority of antibody-forming as well as antibody-secreting cells. Among the immature plasma cells in the popliteal lymph nodes proliferation occurred. Evidence is presented that the light-microscopically identified mature plasma cell is not the main antibody-forming cell. It does not show 3H-Thymidine incorporation and should be considered as a non-dividing end-cell.

The in situ immune response in popliteal lymph nodes of mice after macrophage depletion. Differential effects of macrophages on thymus-dependent and thymus-independent immune responses.

Mice were subcutaneously (SC) injected in the left hind footpad with dichloromethylene diphosphonate (Cl2MDP)-containing liposomes to eliminate macrophages lining the subcapsular sinus (SCS) and those in the medulla of draining popliteal lymph nodes (PLN). In order to study the effect of depletion of these macrophages on the in situ immune response in the PLN, liposome-treated mice were SC injected in the same footpad with thymus-independent (TI) type 1 antigen trinitrophenylated lipopolysaccharide (TNP-LPS), TI-type 2 antigen TNP-Ficoll or thymus-dependent (TD) antigen TNP-keyhole limpet haemocyanin (TNP-KLH). No major differences were observed in antibody-serum titers of liposome-treated and control animals. After primary as well as secondary immunization with the TD-antigen TNP-KLH, an increase in the number of antibody-forming cells (AFC) was found and the peak of response was delayed in the PLN of liposome-treated animals. Such differences were not observed with the TI-antigens. These results indicate that macrophages lining the SCS and those in the medulla of the PLN are not essential for the induction of an immune response. The positive effect of macrophage-depletion on the number of AFC may be explained by competition for the antigen by macrophages and other antigen-presenting cells.

The influence of the route of antigen administration on the development of specific antibody-producing cells in the follicles of the popliteal lymph nodes of rabbits

SummaryRabbits received an injection of liposome-associated human serum albumin (HSA) and six weeks later a booster injection of HSA free in solution. Anti-HSA antibody-forming cells were demonstrated in the popliteal lymph nodes by use of a HSA-horseradish peroxidase (HRP) conjugate for their detection. After a single subcutaneous injection into the footpads, specific antibody-forming cells were found, but after a single intravenous injection no anti-HSA-forming cells were observed in the lymph nodes. During the secondary immune response specific anti-HSA antibody-producing cells were found in the lymph nodes of all animals. The localization pattern of anti-HSA antibody-forming cells within the lymph nodes depended on the route of antigen administration used for the primary and booster injection. Anti-HSA-forming cells were mainly found in the cords of the medulla and in the outer cortex. During the secondary immune response, however, they were also detected in the follicles but only when at least one of the two antigen injections was given subcutaneously.

THE SECONDARY IMMUNE RESPONSE AGAINST LIPOSOME ASSOCIATED ANTIGENS

In the present experiments, the secondary immune response against antigens is studied after priming with liposome associated antigens and booster injections with the antigen alone, in order to study the effect of liposomes on the generation of immunological memory against the associated antigens. Liposomes show adjuvant activity with respect to both the primary and secondary immune response against associated human serum albumin (HSA). When the injected dose of liposome associated HSA was too low to elicit a primary immune response, generation of immunological memory against the antigen could not be detected. Horse radish peroxidase (HRP) associated with liposomes did not elicit a primary immune response, but immunological memory against the antigen was established.

Localization of PNA-binding and nonbinding thymus cells in peripheral lymphoid organs

Abstract Thymus cells were labeled in vitro with FITC and injected into syngeneic recipients. In cell suspensions of lymphoid organs green cells were inspected for PNA receptors with double immunofluorescence. A striking preference of PNA-negative cells to localize in lymph nodes and the lymphoid compartment of the spleen was demonstrated. Incubation with anti-Ly sera revealed that Ly 1 + PNA-negative cells homed in popliteal lymph nodes and Peyers patch but not in mesenteric lymph nodes.

SOME ASPECTS OF IMMUNOHISTOPEROXIDASE REACTIONS IN THE AGAROSE BEAD MODEL SYSTEM

Agarose beads that serve as matrices for antigens can be useful tools, not only for studying the quantitative aspects of immunohistoenzyme reactions but also for detecting antibodies. The present study focuses on localization of the ultimate reaction product within the beads; this factor is important when quantitative aspects of immunohistoenzyme reactions are under investigation. Localization varies with respect to the concentration of CNBr used to activate the beads. In addition, a method is described that facilitates the use of indirect immunohistochemical bead procedures and reduces the amount of antigen (-coupled beads) needed. These advantages may be particularly helpful when these methods are used for serologic detection of specific antibodies on a large scale.

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells

SummaryIntracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP antibody producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin (μ or γ chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin antibody-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.