Shawn J. Green

Cytokine-induced synthesis of nitrogen oxides in macrophages: a protective host response to Leishmania and other intracellular pathogens.

Fixed macrophages within tissues and the mononuclear phagocytes cabled to sites of inflammation are premier scavenger cells able to eliminate most infectious threats by a wide array of toxic effector molecules and hydrolytic enzymes. Paradoxically, a variety of protozoa, bacteria, fungi, and viruses preferentially infect and replicate within these same scavenger cells. Sites of replication include not only the phagobysosome, but also the cytoplasm of infected cells. In many instances, these microorganisms can also be killed by the infected macrophage host cell. Complex signals generated through the cytokine network of local and systemic immune reactions induce a state of activation in the infected cell which kills the intracellular parasite (presumably without killing the infected macrophage). Effector molecules that such activated macrophages use to kill and eliminate these pathogens have always been a mystery. In this review, we examine the experimental approaches that identified nitrogen oxides derived from L-arginine as essential components in the microbicidab activity of cytokineactivated macrophages, and discuss the fascinating, but complex interactions of host cells, cytokines, and infectious pathogens that regulate production and action of toxic nitrogen oxides. Details of the biochemical pathways for nitrogen oxidation of L-arginine and its regulation within mammabian cells are now emerging [1-3]. Although not fully characterized, it is known that nitric oxide (NO) is a short-lived intermediate product of this novel pathway

Nitric oxide: Cytokine-regulation of nitric oxide in host resistance to intracellular pathogens

To discover how nitric oxide (NO) synthesis is controlled in different tissues as cells within these tissues combat intracellular pathogens, we examined three distinctively different experimental murine models designed for studying parasite-host interactions: macrophage killing of Leishmania major; nonspecific protection against tularemia (Francisella tularensis) by Mycobacterium bovis (BCG); and specific vaccine-induced protection against hepatic malaria with Plasmodium berghei. Each model parasite and host system provides information on the source and role of NO during infection and the factors that induce or inhibit its production. The in vitro assay for macrophage antimicrobial activity against L. major identified cytokines involved in regulating NO-mediated killing of this intracellular protozoan. L. major induced the production of two competing cytokines in infected macrophages: (1) the parasite activated the gene for tumor necrosis factor (TNF), and production of TNF protein was enhanced by the presence of interferon-gamma (IFN-gamma). TNF then acted as a autocrine signal to amplify IFN-gamma-induced production of NO; and (2) the parasite upregulated production of transforming growth factor-beta (TGF-beta), which blocked IFN-gamma-induced production of NO. Whether parasite-induced TNF (parasite destruction) or TGF-beta (parasite survival) prevailed depended upon the presence and quantity of IFN-gamma at the time of infection. The relationship between NO production in vivo and host resistance to infection was demonstrated with M. bovis (BCG).(ABSTRACT TRUNCATED AT 250 WORDS)

Cellular mechanisms of nonspecific immunity to intracellular infection: Cytokine-induced synthesis of toxic nitrogen oxides from l-arginine by macrophages and hepatocytes

Nitric oxide (NO) produced by cytokine-treated macrophages and hepatocytes plays a vital role in protective host responses to infectious pathogens. NO inhibits iron-sulfur-dependent enzymes involved in cellular respiration, energy production, and reproduction. Synthesis of L-arginine-derived nitrite (NO2-), the oxidative end product of NO, directly correlates with intracellular killing of Leishmania major, an obligate intracellular protozoan parasite of macrophages: the level of NO2- production is a quantitative index for macrophage activation. The competitive inhibitor of NO synthesis, monomethylarginine (NGMMLA), inhibits both parasite killing and NO2- production. For Leishmania, the parasite itself participates in the regulation of this toxic effector mechanism. This participation is mediated by parasite induction of tumor necrosis factor alpha (TNF alpha), an autocrine factor of macrophages: NO synthesis by interferon-gamma (IFN-gamma)-treated cells can be blocked by monoclonal antibodies to TNF alpha. NO production by IFN gamma-treated hepatocytes is of special interest in malaria infections: sporozoite-infected hepatocytes kill the intracellular malaria parasite after treatment with IFN gamma; this killing is inhibited by NGMMLA.

Tumor Necrosis Factor-Alpha: Central Regulatory Cytokine in the Induction of Macrophage Antimicrobial Activities

Expression of activated macrophage resistance to infection requires the cooperative interaction of interferon-gamma (IFN-gamma) and either interleukin-2 (IL-2), interleukin-4 or granulocyte/macrophage-colony-stimulating factor: no single cytokine is effective. For IFN-gamma and IL-2, the effector activity can be suppressed by the presence of anti-tumor necrosis factor-alpha (TNF-alpha) antibodies in the reaction mixture. IFN-gamma and IL-2, only in combination, induce TNF-alpha-specific mRNA and secretion of this cytokine by macrophages. Development of intracellular killing activity by activated macrophages also requires the autocrine effects of TNF-alpha. IFN-gamma provides the first signal for the production of nitric oxide (NO), the effector molecule for intracellular destruction of parasites. When IFN-gamma-treated cells are infected with pathogens, they are stimulated to make TNF-alpha. Expression of intracellular killing, as well as production of NO, is inhibited by anti-TNF-alpha antibody.

Cytokines that regulate macrophage production of nitrogen oxides and expression of antileishmanial activities.

Resident tissue and inflammatory macrophages produce low but detectable levels of nitrogen oxides de novo, and serve successfully as host cells for Leishmania major, a protozoan parasite that replicates in phagolysosomes (Fortier et al., 1983). In contrast, activated macrophages release markedly increased levels of NO 2 and NO~ (Stuehr and Marietta, 1987; Drapier et al., 1988), and the intracellular environment of these cells is extremely inhospitable for L. major survival (Nacy et al., 1981). We recently correlated the intracellular microbicidal capacity of activated macrophages with production of nitrogen oxides (Green et al., 1990a): in all cases, macrophages that kill intracellular amastigotes release high levels of NO 2 into the culture medium. The effector molecule for destruction of the parasite is NO, the highly reactive and unstable intermediate of the nitrogen oxidation of L-arginine (Hibbs eta!., !987; Green et al., 1990a; Liew et al., 1990a). A competitive inhibitor of L-arginine, N~-monomethyl-L arginine (NGMMLA), completely blocks synthesis of NO and inhibits killing of L. major (Green et al., 1990a).

Interleukin-2 and the Regulation of Activated Macrophage Cytotoxic Activities

In 1976, Morgan and colleagues reported that conditioned media from mitogen-stimulated mononuclear cells contained a factor which maintained the exponential proliferative growth of human leukemic blood or bone marrow cells (1); the proliferative cells were identified as normal T lymphocytes (2). Isolation, characterization, and subsequent purification of this factor in the conditioned medium lead to the identification of a T cell growth factor (TCGF) now known as IL-2 (3), a glycoprotein of 15.5 kD with a slightly basic isoelectric point (4). Shortly thereafter, Taniguchi et al. (5) isolated a cDNA clone for IL-2. Although IL-2 was initially described as the ultimate mitogenic signal for both antigenically and polyclonally activated T cells, aiding in their cell cycle transition from G1 to S phase (6), subsequent studies showed that IL-2 stimulates NK and LAK cell activity (7, 8), induces B cell differentiation and proliferation (9), and activates macrophage cytotoxicity (10). IL-2 also participates in induction of T cell synthesis of cytokines, such as IFN-γ (11) and B cell growth factor-1, or IL-4 (12).