Rolf Seljelid

Rainbow trout macrophages in vitro: Morphology and phagocytic activity

The properties of macrophages from the pronephros of Rainbow trout (Salmo gairdneri Richardson) were studied in vitro. We found that phagocytes obtained from the pronephros constitute a non-homogeneous cell population. Three populations with different adherence properties were examined with special emphasis on morphology and phagocytic capacity. The differentiation of the three populations in culture was similar morphologically, and their phagocytic activity showed only small variations. The methods for cell separation and culture reported here are a useful tool for gaining better understanding of how Rainbow trout macrophages function in the immune response.

Macrophages produce blood coagulation factors

2 .l . Preparation of macrophage cultures Macrophages were obtained from hybrid C3DZF1 (C3H/Tifo9 X DBA/2 d) mice by peritoneal washing with phosphate-buffered saline without anticoagulant. Cells were routinely cultured in MEM (minimum essential medium with Earle’s salts, Gibco Bio-cult, Paisley, Scotland) supplemented with 0.05% (w/v) heat inactivated (lOO’C, 10 min) la&albumin hydrolysate (Nutritional Biochemicals Corp., Cleveland, OH) and 100 IU/ml of penicillin and streptomycin (Gibco Biocult). The cells were either seeded in Costar plastic plates (Costar, Broadway, Cambridge, MA) in 16 mm circular wells on glass coverslips (0.7 X lo6 cells seeded/well) or in 100 mm Falcon plastic dishes (20 X lo6 cells seeded/dish). After 2 h in culture non-adherent cells were washed away and new medium containing lactalbumin hydrolysate was added. The incubation took place at 37OC in an atmosphere of 5% CO2 in air. The medium collected after 24 h in culture was centrifuged at 500 X g and the supernatant tested for coagulation factors or subjected to chromatography on dextran sulphateSepharose for partial separation of the factors. In the experiments with [35S]methionine, cells were cultures in medium supplemented with 50 /.&i [35S]methionine/ml. After 24 h incubation the

Echinoid phagocytes in vitro

Abstract A method is described for obtaining pure monolayers of phagocytes from the sea urchin Strongylocentrotus droebachiensis in vitro. The coelomic fluid contains four types of cells. About 67% of the cells are phagocytes, the rest is comprised of the red and white morula cells and the vibratile cells. The different cell types could be separated by centrifugation on a discontinuous gradient of sodium metrizoate. Release of granula from the vibratile cells was found to be responsible for rapid and extensive clotting of the coelomic fluid immediately after its removal from the animal. Clotting was prevented by adding a mixture of 50 mM mercaptoethanol, 3 mM caffeine and 2 mM TAME ( p -tosyl- l -arginine methyl ester) to the coelomic fluid. The phagocytes were isolated from other cell types by their attachment to glass, and were grown at 10 °C in a simple peptone-sea water medium. The phagocytes are very motile cells and spread rapidly on glass, accompanied by a complete change of their morphology to flattened cells with peripheral ruffling. After few hours in vitro the cells fuse to form monolayer-syncytia, and later still cell clusters and free floating balls of cells are formed. During a culture period of 10 days there was no change in the DNA content per culture, while a small increase in protein was found.

Role of mast cells in zymosan-induced peritoneal inflammation in Balb/c and mast cell-deficient WBB6F1 mice

Zymosan‐induced peritonitis was investigated in mast cell‐deficient WBB6F1 mice and in Balb/c mice pretreated with mast cell stabilizer (cromolyn) or antagonists of histamine receptors (mepyramine, triprolidine, cimetidine, or ranitidine). The inherited mast cell deficiency in W/Wv knockouts of WBB6F1 mice impaired significantly the level of histamine and plasma exudation (measured 30 min after stimulation) as well as the influx of exudatory leukocytes, accumulation of plasma and exudate chemoattractants, and the release of proinflammatory cytokines (TNF‐α, IL‐1β, and IL‐6) measured at 6 h of inflammation. All of those factors were fully restored after selective intraperitoneal reconstitution of W/Wv mice with bone marrow‐derived mast cells from their control +/+ counterparts. Cromolyn pretreatment of Balb/c mice reduced exclusively the early plasma exudation and histamine influx. Blocking of histamine receptors inhibited not only the early plasma exudation but also temporarily diminished primary leukocyte influx and levels of MCP‐1 and IL‐1β. In conclusion, mast cells play an important role in the initiation of zymosan‐induced peritonitis and modulate its further course.

Early Vascular Permeability in Murine Experimental Peritonitis Is Comediated by Resident Peritoneal Macrophages and Mast Cells: Crucial Involvement of Macrophage-Derived Cysteinyl-Leukotrienes

The initial phase of zymosan-induced peritonitis involves an increase of vascular permeability (peak at 30 min) that is correlated with high levels of vasoactive eicosanoids, namely, prostaglandins (PGI2 and PGE2) of cyclooxygenase-1 origin (as estimated by RT-PCR) and cysteinyl-leukotrienes. Previously, we showed that the increase of vascular permeability can be attributed only partially to mast cells and their histamine, as seen in mast cell–deficient WBB6F1-W/Wv mice. Thus we aimed to identify the major cellular source(s) that mediate vasopermeability, as well as particular vasoactive mediators operating in this model. For this purpose, some mice were selectively depleted of either peritoneal macrophages or mast cells, and/or they were treated with several pharmacologic inhibitors of cyclooxygenase- and lipoxygenase-metabolic pathways. Moreover, macrophage-depleted mast cell–deficient WBB6F1-W/Wv mice and their controls (+/+) were used. The macrophage depletion always caused a profound decrease of both vascular permeability and lipid-mediator levels, which was particularly pronounced for leukotrienes, whereas the effects of mast-cell depletion were less severe. The macrophage/mast-cell comediation of vasopermeability was also revealed in thioglycolate-induced peritonitis, as well as the macrophage origin of cysteinyl-leukotrienes. Taken together, these findings demonstrate that the resident peritoneal macrophages are in fact the main contributors to the vasopermeability at the early stages of zymosan-induced peritonitis.

In vitro biosynthesis of cold insoluble globulin (fibronectin) by mouse peritoneal macrophages.

Previous studies [ I,2 J have implicated that cold insoluble globulin (GIG) [3,4] plays an important role in the phagocytic function of the reticuloendothelial system (RES). It has been reported that the phagocytic dysfunction of the RES often seen in patients after trauma, surgery or in sepsis, is concomitant with a decreased plasma concentration of CIG [5,6] and can be reversed by the intravenous infusion of plasma cryoprecipitate 17,s J which contains GIG as a major component. GIG is the plasma form of Bbronectin, a glycoprotein found in connective tissue and reported to be produced by fibroblasts, endothelial and glial cells grown in vitro (reviewed [9] ). Most of the fibronectin produced by fibroblasts in vitro is deposited as a matrix adherent to the tissue culture dish [lo]. Previous studies [l 11 have demonstrated that peritoneal macrophages do not synthesize a fibronect~~onta~ing adhesive matrix. However, here we present evidence that CIG is produced by macrophages, and is secreted to the culture medium.

On the mechanism of internalization of opsonized particles by rat Kupffer cells in vitro

Abstract The attachment and internalization of opsonized sheep red blood cells by cultured rat Kupffer cells were studied with phase-contrast and scanning electron microscopy (SEM) as well as timelapse microcinematography. We observed that sheep red cells coated with IgG attached over the entire Kupffer cell surface at random, whereas those coated with IgM and complement attached all over the cell with the exception of the extreme periphery. When the Fc and C3 receptors were given appropriate stimuli to internalize the attached red cells, they functioned very differently. In Fc internalization, the Kupffer cell membrane rose above the main cell body and wrapped tightly around the attached red cell, eventually surrounding it entirely. In the C3 internalization, triggered by new-born calf serum, the membrane activity was less spectacular; the folds that did sometimes rise up were coarser and did not fit tightly around the red cell, which was eventually interiorized by a sinking, deep into the cytoplasm of the Kupffer cell. These two mechanisms of internalization also showed different sensitivities to cytochalasin B (CB); the Fc internalization being far more vulnerable to this inhibitor of microfilament activities. Studies with colchicine, however, did not show any clear-cut difference in sensitivity between the two cases.

Effect of Soluble Aminated β‐1,3‐D‐Polyglucose on Human Monocytes: Stimulation of Cytokine and Prostaglandin E2 Production but Not Antigen‐Presenting Function

Glucans are insoluble polymers of β‐1,3‐linked glucose derived from yeast cell walls that effectively activate macrophages. Recently, aminated derivatives of β‐1,3‐D‐polyglucose have been developed that are soluble but also activate murine macrophages. The current studies were undertaken to determine whether soluble aminated β‐1,3‐D‐polyglucose (AG) would also stimulate human monocytes. The AG employed contained <2 ng endotoxin/mg. AG induced the production of intracellular, membrane‐associated, and secreted forms of interleukin 1 (IL1) in a dose‐dependent manner, with 50 µg/ml yielding maximal responses. AG also induced tumor necrosis factor‐α (TNFα) secretion by human monocytes. Prostaglandin E2 (PGE2) production was also stimulated in a concentration‐dependent manner. Quantitatively, optimal stimulatory concentrations of AG were comparable to endotoxin in the capacity to induce production of these various mediators. In contrast to its capacity to induce production of IL1, TNFα, and PGE2, AG did not stimulate monocytes to become more effective antigen presenting cells. These results indicate that AG is a potent inducer of proinflammatory mediators from human monocytes but does not enhance their capacity to initiate immune responses.

A water-soluble aminated beta 1-3D-glucan derivative causes regression of solid tumors in mice.

Meth A sarcoma, when inoculated in the skin, grew progressively in hybrid CB6 Fl(Balb/c×C57B1/6) mice. When water-soluble aminated β1-3D-glucan (AG) was injected intravenously or intraperitoneally on day 7 of tumor growth, the tumors underwent complete regression. When the injection was performed on day 3 there was regression of tumors in only about half of the cases. When the injection was performed on day 14 there was no apparent effect on tumor growth. Tumors in thymectonized animals did not appear to respond to treatment with AG on day 7. The relatively simple chemistry and low toxicity of AG, together with its solubility in biological fluids, makes it a promising tool in experimental—and possibly clinical—tumor therapy.

Receptors for Complement on Echinoid Phagocytes. I. The Opsonic Effect of Vertebrate Sera on Echinoid Phagocytosis

The ingestion of sheep erythrocytes (SRBC) by echinoid phagocytes was greatly increased after treatment of SRBC with sera from mouse, human and fish. The opsonic principle in the sera was heat labile (56 degrees C 1/2 hr). Opsonization with mammalian sera was studied more extensively. It was dependent on pre-sensitization of SRBC with specific antibody (IgM), required Ca2+, and was inhibited by low temperature (4 degrees C). The finding that serum depleted of C 3 only opsonized very weakly strongly indicates that the opsonic principles is the complement cascade C 1-4-2-3 activated via the classical pathway, and that the opsonic effect largely coincides with the coating of SRBC with C 3b. A role of C 5 or later components was excluded, since the mouse AKR serum was C 5 deficient, and the human serum was normally treated with zymosan. When variations known to impair the opsonic function of C 3 was introduced in the opsonization procedure, a parallel inhibition was recorded on attachment of SRBC to mouse peritoneal macrophages and on ingestion of SRBC by echinoid cells. Thus C 3 receptors are probably present on echinoid phagocytes.