Biological activity of a lipopolysaccharide extracted from Treponema hyodysenteriae.

A lipopolysaccharide (LPS) was obtained from pathogenic Treponema hyodysenteriae by hot phenol-water extraction. Various effects of the LPS on host cells were examined in vitro. Toxicity for mouse peritoneal macrophages was observed after 10 h of incubation at concentrations as low as 15 micrograms of the LPS per ml. Marked enhancement of both complement (C3) and immunoglobulin G-Fc receptor-mediated internalization was noted in macrophages obtained from mice injected 6 days previously with 75 micrograms of the material. Incorporation of [3H]thymidine into murine splenocytes was elevated approximately fourfold when splenocytes were treated with 5 to 10 micrograms of LPS per ml. Incubation of the LPS with normal porcine serum resulted in the generation of a factor(s) that stimulated the migration of porcine leukocytes. Generation of the chemotactic activity was inhibited by heating the serum at 56 degrees C for 30 min before treatment with LPS. The results suggest that T. hyodysenteriae contains an LPS that is biologically active.

Generation of chemotactic factor for granulocytes and monocytes from serum by fractions of Brucella abortus.

Several fractions isolated from Brucella abortus were examined for their ability to generate chemotactic factor from normal serum. Phenol phase lipopolysaccharides exhibited activity equivalent to that obtained with E. Coli lipopolysaccharide. A carbohydrate-rich aqueous methanol fraction was inhibitory at high concentrations, but a non-dialysable component of this fraction contained a potent stimulator of chemotactic activity. Protein-rich fractions from both strain 19 and strain 2308 were inactive. Preheating the serum at 56 degrees for 30 min prevented generation of chemotactic activity by the various fractions, suggesting a role for serum complement. No chemotactic activity was produced by Brucella fractions in C5-deficient DBA/2J mouse serum.

Antigen presentation to T cell hybridomas by a macrophage cell line: an inducible function.

We have investigated the ability of an Ia-, nonantigen-presenting macrophage tumor cell line, P388D, (H-2d), to present antigen to T cell hybridomas after incubation in a lymphokine-containing preparation. P388D, cells were incubated in microtiter wells with various concentrations of Con A-stimulated spleen cell supernatants. Antigen-specific stimulation of H-2d-restricted, KLH-specific T cell hybridomas was observed by P388D1 incubated with SUP.P388D1 cells incubated for 3 days in medium or control SUP did not present antigen. In addition, no stimulation of T hybridomas was seen by P388D1 in the inhibited by the appropriate monoclonal anti-Ia reagents. These results demonstrate that a macrophage tumor cell line can be induced to present antigen and provides for large numbers of readily available, homogeneous macrophages for studying the cellular biochemical requirements for antigen processing and presentation.

Characterization of macrophage functions in mice infected with Brucella abortus.

Macrophage spreading, surface receptor density/avidity, phagocytosis, random migration, chemotactic responsiveness, and serum lysozyme were examined during the course of infection (up to 60 days) of mice with Brucella abortus strain 19. Markedly enhanced in vitro spreading activity was observed throughout the period of study. The density/avidity of cell surface immunoglobulin G Fc receptors was increased for up to 60 days postinfection. Internalization of sheep erythrocytes via C3 receptors was significantly enhanced. Random locomotion and chemotactic responsiveness to lymphocyte-derived chemotactic factor and N-formyl-L-methionyl-L-leucyl-L-phenylalanine were markedly stimulated. Serum lysozyme was also elevated in infected animals. These changes indicated significant and prolonged enhancement of macrophage activity during Brucella infection. These findings are discussed in relation to previous reports describing macrophage activation by Brucella.

Detection of cell surface immunoglobulin on leucocytes by agglutination.

The detection of immunoglobulin (Ig) on murine peritoneal cell (PC) surfaces by an agglutination test is described. Addition of anti-mouse IgG (amIgG) to a suspension of PC results in agglutination of the cells. Trypsin or pronase treatment of the cells abrogated agglutination upon addition of amIgG. Incubation of washed murine PC with porcine IgG (pIgG) for 30 minutes resulted in agglutination of the cells upon addition of anti-porcine IgG (apIgG) to the cell suspension. This suggests that the heterologous pIgG is also able to bind to the murine cell surface. The implications of these findings are discussed and compared with respect to the agglutination test described herein and other techniques for detection of cytophilic antibody.

The isolation, long-term cultivation and characterization of bovine peripheral blood monocytes.

Bovine peripheral blood monocytes were isolated from buffy coats of ACD-anticoagulated whole blood. Cells cultivated on plastic surfaces for 20 h were judged to be 95% monocytes based on nonspecific esterase-1 staining, uptake of latex particles and surface receptor characteristics. Bovine monocytes were maintained up to 80 days in vitro in Dulbecco's modified Eagle medium with 15% horse serum and 15% foetal bovine serum. Several morphological and physiological features of bovine monocytes were examined during the course of culture. Cell size and cytoplasmic spreading, granulation and vacuolization increased progressively. Multinucleated giant cells predominated during the latter stages of in vitro culture. A high percentage of bovine monocytes possessed C3 and IgG Fc receptors, whereas IgM Fc and sheep erythrocyte receptors were not detected. Phagocytosis was mediated by the IgG receptor, but not by the C3 receptor. Peroxidase activity declined in a linear fashion, with cells essentially negative after 8 days of culture. Total cell protein and acid phosphatase increased during cultivation. Lysozyme activity was undetectable in both lysates and supernatants of bovine monocyte. These findings are consistent with the concept of maturation of mononuclear phagocytes. The procedures for isolation and cultivation described in this paper will provide methodology for detailed study of bovine mononuclear phagocytes.

A method for obtaining swine alveolar macrophages by segmental pulmonary lavage.

A technique for obtaining alveolar macrophages (AM) from anesthetized swine is described. Animals were intubated and segmental pulmonary lavage was performed utilizing a double lumen catheter (DLC). An average of 98% of the initial 100 ml lavage fluid was recovered with a typical yield of 1 x 10(8) free alveolar cells (FAC). AM were then separated from other FAC by their adherence to plastic. The final adherent cell population consisted of greater than 95% macrophage as determined by morphology and non-specific esterase activity. The technique described had no adverse effects on the animals even when repeated on the same animal several days later.