Inge E. A. Flesch

MIP-1alpha, MIP-1beta, RANTES, and ATAC/lymphotactin function together with IFN-gamma as type 1 cytokines.

We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstrate in vitro and in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1α, MIP-1β, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)/lymphotactin are cosecreted to a high degree with IFN-γ by activated individual natural killer (NK), CD8+ T, and CD4+ T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-γ in the up-regulation of CD40, IL-12, and tumor necrosis factor-α, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1α, MIP-1β, RANTES, and ATAC are not only chemoattractants but also coactivators of macrophages, (ii) MIP-1α, MIP-1β, RANTES, and ATAC constitute together with IFN-γ a group of “type 1 cytokines,” and (iii) these cytokines act together as a functional unit that is used by NK cells in the innate phase and then “handed over” to CD8+ T cells in the antigen-specific phase of the immune defense, thus bridging the two components of a Th1 immune reaction.

Role of cytokines in tuberculosis.

Mycobacterium tuberculosis and Mycobacterium bovis are facultative intracellular pathogens which preferentially utilize the macrophage as their host cell. Acquired resistance against mycobacteria depends on T cells which activate antimicrobial macrophage functions via the release of cytokines. The data summarized below suggest an important role for interferon-gamma (IFN-gamma) as well as the B cell-stimulatory factors interleukin-4 (IL-4) and IL-6 in the induction of tuberculostatic macrophage functions. Growth inhibition of mycobacteria by cytokine-stimulated macrophages is mediated by reactive nitrogen intermediates (RNI) derived from L-arginine. Tumor necrosis factor-alpha (TNF-alpha) and IL-10 act as autocrine regulators in the induction of the enzyme NO-synthase. Both cytokines are produced by macrophages stimulated with IFN-gamma and infected with M. bovis. While TNF-alpha mediates activation of the NO-synthase and production of RNI, IL-10 suppresses this enzyme activity. The outcome of mycobacterial infection is probably regulated by a complex network between stimulatory and inhibitory cytokines.

The role of T cell — Macrophage interactions in tuberculosis

Acquired resistance against tuberculosis paradigmatically depends on specific T lymphocytes and mononuclear phagocytes. The etiological agent,Mycobacterium tuberculosis is capable of replicating in mononuclear phagocytes which act both as habitat and as effectors of protection. Upon interaction with antigen-specific T lymphocytes infected mononuclear phagocytes acquire tuberculosis activities. Here, data from experimental tuberculosis studies in mice are summarized which show that: interleukins produced by cloned T cells and recombinant interferon-γ are capable of activating tuberculostatic capacities in macrophages; both CD4 and CD8 T cells, after adequate stimulation, produce interferon-γ; CD8 T cells lyse macrophages in an antigen-specific way; not only CD8 but also CD4 T cells possess an antigen-specific cytolytic potential; lysis of infected macrophages results in mycobacterial growth inhibition. Evidence is also presented that tuberculostatic activities of activated macrophages depend on phagosome-lysosome fusion and are independent of reactive oxygen metabolites and that some strains ofM. tuberculosis are resistant against interferon-γ activated macrophages. These findings suggest that both helper and cytolytic T cells participate in the immune response to tuberculosis and that similar T cell mechanisms contribute to resistance as well as pathogenesis. Protection against tuberculosis, therefore, depends on subtle coordination of the immune response.

Interleukin‐4 and listeriosis

Summary: Experimental infection of mice with Listeria monocytogenes (L. monocytogenes) has served as an appropriate model for analyzing Thl nil driven immune responses. Generally, Th2 responses are absent and IL‐4 is not detectable. Here, we describe experimental settings under which IL‐4 is detectable in listeriosis. Our data suggest that IL‐4 is rapidly produced after infection. This prompt IL‐4 burst seems to stimulate chemokine responses and, therefore, may participate in the regulation of the early antilisterial host response. Soon thereafter, lL‐4 production wanes. At least partially this seems to be caused by downregulation of IL‐4–producing CD4+ NK1+ TCRαβint lymphocytes by IL‐12. In the absence of IFN‐γ responsiveness, IL‐4 production is demonstrable during acquired immunity against L. monocytogenes, and this elevated IL‐4 production apparently contributes to disease exacerbation. In conclusion, the data are consistent with a detrimental role of IL‐4 in listeriosis and active control of IL‐4 synthesis by the antilisterial immune response. The rapid, but transient, IL‐4 burst in listeriosis probably contributes to host defense without impairing development of the acquired T‐cell response because of its shortness.

Effect of cellular fatty acid composition on the phospholipase A2 activity of bone marrow-derived macrophages, and their ability to induce lucigenin-dependent chemiluminescence

Mouse bone marrow macrophages were obtained by cultivation in serum-free medium. Addition of specific fatty acids to the medium leads to macrophage populations which differ in their fatty acid composition. The fatty acid composition of the cellular membranes directly modulates functional abilities of the macrophages such as the generation of superoxide anion and phospholipase A2 activity in response to phorbol ester and zymosan. Both capacities were lowest in macrophages cultured serum-free without lipids. Incorporation of unsaturated fatty acids into macrophage phospholipids leads to an increase of O2- production as measured by lucigenin-dependent chemiluminescence and to an increased phospholipase A2 activity after challenge with phorbol ester or zymosan.

Macrophages and hepatocytic cells as chemokine producers in murine listeriosis.

The major target organ of systemic infection with the intracellular bacterium Listeria monocytogenes is the liver, to where inflammatory leukocytes are rapidly recruited. We determined by reverse transcriptase polymerase chain reaction the early chemokine response in the liver after systemic infection of mice with listeriae, and in parallel compared chemokine release from macrophages and hepatocytic cells in vitro. Murine bone marrow-derived macrophages (BMM) grown in fetal calf serum-supplemented medium were used as macrophages and the TIB75 cell line as hepatocytic cells. Within 1-3 hours, gene expression of monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1 alpha, MIP-2, KC, and interferon-gamma inducible protein-10 (IP-10) was upregulated in liver tissue of infected mice. BMM infected in vitro with L. monocytogenes showed a generalized chemokine response, and readily released MCP-1, MIP-1 alpha, MIP-2, and KC, as measured by enzyme-linked immunosorbent assay. In contrast, the chemokine response of hepatocytic cells was more restricted, and infection induced MCP-1 and KC, but not MIP-2 and MIP-1 alpha. Interferon gamma enhanced chemokine release from hepatocytic cells, but unexpectedly had either no or a negative effect on chemokine secretion by BMM cultured in serum-supplemented medium. Listeriolysin (Hly)-negative avirulent listeriae as well as listeriae killed by heat or gentamycin initiated a similar chemokine response in BMM and hepatocytic cells as did wild-type L. monocytogenes. Stimulation of hepatocytic cells with the monokines, tumor necrosis factor alpha and interleukin (IL-)1 alpha, but not IL-6, augmented liberation of chemokines. Together, our data demonstrate an early hepatic chemokine response to L. monocytogenes in murine listeriosis. Probably, not only macrophages but also parenchymal cells participate in chemokine production.

Growth requirements of murine bone marrow macrophages in serum-free cell culture.

A serum-free medium for mass production of pure murine bone marrow-derived macrophages in liquid culture was developed. Iscoves modified Dulbeccos medium (IMDM) was used as a basal nutrient medium supplemented with 2-mercaptoethanol, transferrin and a source of colony-stimulating factor (CSF). Addition of exogenous lipids or bovine serum albumin was not required. The macrophages can be kept viable for at least eight days in the serum-free culture medium, and they are able to incorporate tritiated thymidine. The tritiated thymidine incorporation can be enhanced by retinoic acid, phorbol myristate acetate (PMA), colony-stimulating factor (CSF) and transferrin. Prostaglandin E1, prostaglandin E2 and dexamethasone inhibited tritiated thymidine incorporation. The serum-free culture of bone marrow-derived macrophages has some important implications. First, signal molecules can be defined which enhance or inhibit the proliferative capacity of bone marrow-derived macrophages. Second, the fatty acid composition of membrane phospholipids can be altered to study the relationship between macrophage function and membrane lipid composition.

Functional comparison of bone marrow-derived macrophages obtained by cultivation in serum-free or serum-supplemented medium

Bone marrow-derived macrophages obtained by cultivation in a serum-free or in a serum-supplemented medium were compared in terms of the activation of the respiratory burst and the activation of tumor cytotoxicity. Serum-free-cultured macrophages responded to interferon-gamma (IFN-gamma) and to lipopolysaccharide (LPS) by an enhancement of the respiratory burst. Macrophages obtained in a serum-supplemented medium are characterized by a diminished capacity to release O2-. These cells did not respond to IFN-gamma unless the stimulation was performed in a serum-containing medium. In terms of activation of tumor cell cytotoxicity, serum-supplemented macrophage cultures seem to be primed by unknown serum constituents because they only need one signal (IFN-gamma or LPS) to become fully cytotoxic. Serum-free cultivated macrophages can be rendered cytotoxic only after exposure to combinations of IFN-gamma and LPS.

Tumoricidal Effector Molecules of Murine Macrophages

Activated macrophages secrete a variety of factors affecting proliferation or viability of neoplastic cells. Factors described previously are discussed for their relevance as effector molecules for the selective destruction of tumor cells by activated macrophages and compared to a recently characterized high-molecular-weight tumoricidal activity secreted by activated murine bone-marrow-derived macrophages.

Acylchain specificity and kinetic properties of phospholipase A1 and A2 of bone marrow-derived macrophages

Abstract The fatty acyl specificity of phospholipase A1 and A2 in homogenates of mouse bone marrow-derived macrophages was determined using phosphatidylcholine and phosphatidylethanolamine of different acylchain composition. Phosphatidylcholine with arachidonoyl at position 2 was cleaved preferentially by an alkaline phospholipase A2 (pH-optimum 9.0) leading to selective liberation of arachidonic acid. In contrast, phosphatidylcholines with oleoyl or linoleoyl at position 2 were degraded mainly by an acid phospholipase A1 (pH-optimum 4 -5) resulting in a conservation of these fatty acids esterified in lysophosphatides. Substrate kinetics of the alkaline phospholipase A2 revealed a 30 fold higher affinity (Km = 3.8 x 10-7 ᴍ) for 1-acyl-2-arachidonoyl-glycerophosphocholine compared to 1-acyl-2-oleoyl-glycerophosphocholine. The kinetic data were not influenced by endogenous lipids indicating that exogenous substrates do not equilibrate with cellular lipids. These results are suitable to explain a selective liberation of arachidonic acid from a mixture of phospholipids.