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Reactive Nitrogen Intermediates in Immunology
METHODS: A Companion to Methods in Enzymology 10, 1–7 (1996) Article No. 0071
EDITORIAL
Reactive Nitrogen Intermediates in Immunology Nitric oxide synthase (NOS) is an ubiquitous enzyme capable of affecting a multitude of mechanisms in the immune response. The contributions in this issue of Methods reflect that diversity and include descriptions of the intracellular signaling function of NO, factors governing NO synthesis and its effects in central nervous system pathology, xenobiotic-induced lung injury, arthritic joint pathology, diabetes induced by b-cell destruction, tissue destruction during graft-versus-host disease, the hepatic response to infectious agents, and host defense against parasites. The modulatory influence of NO must be examined in light of the fact that either small amounts (constitutive enzyme) or large amounts (inducible enzyme) can be produced and that biphasic effects of NO are apparent in many systems (1). As will be described, many observations of the effects of NO studied under apparently similar conditions reveal vastly different results. Some of the variation could be due to the amount of NO produced by the different isoforms of NOS. The endothelial cell (ec) and neuronal cell (nc) constitutive NOS enzymes are responsible for production of small amounts of NO that function as signal transduction molecules. In contrast, the high amount of NO produced by inducible nitric oxide synthase (iNOS) can result in inhibition of cellular function by virtue of NO-mediated inactivation of proteins with iron sulfur centers or heme moieties. (See current reviews on control (2), function (3), biochemistry (4), and targets (5) of the NO synthases.) A paradoxical effect of NO that is pertinent to the role of NO in the immune response is that production via iNOS can result in beneficial or detrimental outcome for the organism. For example, NO produced acutely, directed against a pathogen, is beneficial, while NO produced over a long time period, as might be the case with chronic conditions such as arthritis, is detrimental. Another remarkable feature governing induction of iNOS is that inflammatory mediators, such as TNFa, LPS, IL-1b, and IFNg, can stimulate NO synthesis, while other mediators, such as TGFb, IL-4, and IL-10, can downregulate NO synthesis. This separation of cytokines into promoters and inhibitors of NO synthesis resembles closely the dichotomy observed in cytokines produced by T-helper 1 lympho-
cytes (IFNg, IL-2, TNFa), in contrast to T-helper 2 lymphocytes (IL-4, IL-5, IL-10). Thus, NO appears to be a player in the inflammatory milieu that accompanies delayed type hypersensitivity (DTH) reactions and not the milieu that promotes humoral immunity. The following is a brief accounting of how NO has been demonstrated to influence major pathways in the immune response. Development of T- and B-lymphocyte repertoires. Tcell precursors migrate from the bone marrow to the thymus and progress through a series of maturational stages, whereby expression of CD4 or CD8 as well as the T-cell receptor is acquired. Interaction of the developing T cell with thymic stroma results in either positive selection, in which T cells that can respond to foreign antigen in the context of self MHC are selected, or negative selection, in which those cells that are specific for self peptides bound to self MHC (autoreactive cells) are eliminated. The thymic stroma is composed of cell types known to be capable of NO synthesis such as epithelial cells, dendritic cells, and macrophages. Sato et al. (6) have demonstrated by immunohistochemistry that cells in the medulla but not the cortex of unstimulated rat thymus stained positively for iNOS. Fehsel et al. (7, 8) have demonstrated that after LPS administration both increased iNOS expression and DNA fragmentation were seen in thymus tissue sections and that NO could be both a protective and an inducing agent for thymocyte apoptosis in vitro. B-cell maturation from progenitors in the bone marrow is influenced by cytokines as well as contact with bone marrow stromal cells such as endothelial cells, macrophages, fibroblasts, and fat cells. Deletion and functional anergy occurs during the B-cell maturational process. Genaro et al. (9) have described that NO prevented B-cell apoptosis, indicating a possible role for NO in B-cell maturation. NO has also been shown in vitro to induce differentiation of a monocytic cell line, accompanied by altered gene expression (10), indicating a possible role of NO in hematopoietic differentiation. However, contrasting results by Maciejewski et al. (11) described NO-induced inhibition of growth and induction of apoptosis in human bone marrow cells. Since iNOS knockout mice, described by MacMicking et al. (12) and Wei et al. (13), do not demonstrate any 1
1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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obvious abnormality in the lymphocyte populations of thymus, spleen, or lymph node, it is unlikely that NO plays a role in the development of lymphocyte repertoires. These animals are housed under specific pathogen-free conditions and the possibility exists that defects in lymphocyte positive and negative selection could become apparent if the iNOS knockout mice were exposed to inflammatory stimuli. T-Cell-mediated immunity. The activation of a naive T cell requires interaction with a professional antigen presenting cell (APC). A professional APC possesses processed antigen in the cleft of either MHC Class I or Class II molecules as well as costimulatory molecules, such as B7. Professional APCs include dendritic cells, B cells, and macrophages. Dendritic cells are the most potent APC because of their constitutive expression of Class II as well as costimulatory molecules (14). Class I molecules are present on all nucleated cells and Class II antigen is constitutively expressed on dendritic cells and B cells. However, Class II antigen and costimulatory molecule expression must be induced on macrophages and costimulatory molecule expression must be induced on B cells for effective antigen presentation to occur (see Table 1). Naive CD4 T cells interact with Class II-bearing APCs, while CD8 T cells interact with Class I-bearing APCs. NO synthesis by macrophages is well documented, NO synthesis by dendritic cells has recently been discerned (Angus W. Thomson, personal communication), and B cells have not been reported to produce NO, except for transformed B cells (15). The obvious concern is whether induction of iNOS will adversely affect expression of Class II and costimulatory molecules. Some reports documented no effect on Class II expression and some reports documented an inhibition by NO (16– 19). These differences could be due to different stimuli
TABLE 1 Comparison of the Ability of Various Antigen-Presenting Cells to Synthesize NO Class II antigen
Cell type Professional antigen presenting cells Dendritic cells B lymphocytes Macrophage Nonprofessional antigen presenting cells Vascular endothelial cells Epithelial cells
Costimulatory NO molecules synthesis
Constitutive Constitutive Constitutive Inducible Inducible Inducible
Yesa Nob Yes
Inducible Inducible
Yes Yes
Constitutive ?
a
Personal communication, Angus W. Thomson. EBV-transformed lymphocytes and Burkitt’s lymphoma lines produce low levels of NO. b
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and use of primary macrophages versus macrophage tumor cell lines. Effective inhibition/downregulation of a primary immune response would be achieved by NOmediated downregulation of Class II expression. An example of this modulatory influence described by Holt et al. was that alveolar macrophage NO synthesis functions to downregulate pulmonary dendritic cell APC function in vitro coincident with inhibition of Class II expression (20). Differential regulation of costimulatory molecule expression, B7.1 versus B7.2, was reported by treatment of macrophages with LPS, which caused upregulation of both B7.1 and 2, while IFNg increased B7.2 and downregulated B7.1 expression (21). Whether these differential effects were due to NO has not been described. Additionally, many investigators using varied systems where suppression of lymphocyte proliferation was seen have shown that an NO synthesis inhibitor permitted enhanced lymphocyte proliferation (22–28). Thus, NO may have a dual role in inhibiting lymphocyte activation: (1) prevention of upregulation of Class II antigen as well as costimulatory molecule expression on the APC and (2) inhibition of proliferation by the direct action of NO on the lymphocyte (Table 2). Once activated, T cells do not require costimulatory signals to interact with an APC, but do require T-cell receptor interaction with the peptide:MHC complex. Thus, since activated CD8 T cells can interact with any cell expressing Class I molecules, this cell population would include many NO-producing cells, such as fibroblasts, hepatocytes, tumor cells, etc. (see list in (29)). Activated CD4 T cells interact with APC possessing Class II even without costimulatory molecule expression. Modulation of activated CD4 or CD8 T-cell expansion or effector function could occur via target cell production of NO. An example of a paracrine regulatory effect is that described by Stefani et al. (30), in which Leishmania major-specific CD8/ cells, stimulated with L. major-infected macrophages, produced IFNg, which in turn caused NO synthesis in the macrophages. This NO synthesis caused downregulation of CD8 T-cell proliferation and IFNg synthesis. The successful interaction of either a naive or a previously activated T cell with APC results in cytokine synthesis by the T cell, some of which can influence NO synthesis positively (IFNg, IL-2, TNFa) or negatively (IL-4, IL-10, TGFb). The cytokines that elicit NO synthesis are products of Th1 cells and facilitate the inflammatory, DTH type of reaction, while cytokines that downmodulate NO synthesis facilitate humoral immunity. As suggested by the work of Taub and Cox (31) interaction of Th1 or Th2 cells with macrophages may represent a potent paracrine regulatory network controlling the activation of macrophages. Taylor-Robinson et al. (32) have demonstrated that cloned Th1 but not Th2 lymphocytes produced NO and that Th1
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cloned lymphocyte synthesis of IL-2 and IFN-g was inhibited by NO, while Th2 cloned lymphocyte synthesis of IL-4 was not inhibited. It is evident that NO can modulate T-cell-mediated immunity at several levels encompassing initiation as well as the propagation of the immune response.
SIGNAL TRANSDUCTION Initial studies on how NO affects cells focused on how NO inhibits various pathways in the cell, such as mitochondrial respiration and proliferation. Subsequently, the intracellular signaling pathways triggered by NO were discovered and are now being investigated in many different cells. For example, NO activates iron response element binding (33) and modulates AP-1 transcriptional factors (34). Lander et al. (35, 36) have found that NO enhanced the rate of glucose transport and induced NF-kB binding activity and secretion of TNFa by human peripheral blood mononuclear cells. Recent work (37) has shown that NO can activate T lymphocyte p21ras, a member of the Ras superfamily, which are proteins that relay signals from receptor tyrosine kinases to the nucleus. Further elucidation of the effects of NO on T-lymphocyte intracellular signaling will provide a clearer picture of how NO might affect an immune response.
AUTOIMMUNITY Autoimmune responses are thought to be the result of T-lymphocyte responses to self antigen because that antigen is recognized as foreign, not as self. This can be the result of newly exposed antigen, defective negative selection in the thymus, or exposure to self antigens under inflammatory conditions where costimulatory molecule induction can result in lymphocyte activation. The autoimmune response results in cytokine synthesis by activated T cells, which can result in activation of B cells to synthesize antibodies and macrophages to secrete inflammatory molecules. Since the response is directed to self, there is an ample supply of antigen to produce a chronic inflammatory response. The role of NO in arthritis, a chronic inflammation of the joints, has been well studied. NO is produced by chondrocytes (38) and synovial fibroblasts (39). Inhibition of joint swelling by administration of NO synthesis inhibitors has been reported in animal models of arthritis (40–43) as well as the autoimmune disease seen in MRL-lpr/lpr mice (44). Since inhibition of NO is accompanied by decreased joint inflammation, it would appear that NO does not function to downregulate lym-
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phocyte activation during the arthritic process, since inhibition of NO might then potentiate lymphocyte responses, worsening the disease. Rather, NO appears to be functioning in promotion of the inflammatory response. In fact, Ialenti et al. (40) have demonstrated enhanced lymphoproliferative responses in L-argininefed arthritic rats and decreased responses in animals fed an NO synthesis inhibitor. The autoimmune disease insulin-dependent diabetes mellitus is characterized by destruction of the pancreatic b cell, due to islet infiltration by inflammatory macrophages and T cells. Ample evidence documents the role of NO in inhibition of b-cell function (45–47) due to IL-1-induced NO synthesis by b cells (48) or by NO synthesized by infiltrating macrophages (49). NO has been shown to inhibit mitochondrial oxidation in purified b cells (50) and to induce DNA strand breaks in islet cells (51). Administration of the NO synthesis inhibitor aminoguanidine delayed the development of diabetes in NOD mice (52). Since NO has also been shown to stimulate the activity of both cCOX and iCOX (53), NO apparently functions as an inflammatory molecule during induction of b-cell dysfunction, actually promoting production of another mediator, PGE2 . The role of NO in central nervous system pathology has also been investigated utilizing a model of autoimmune encephalomyelitis (54, 55). NO was localized to the spinal cord of infected mice and administration of an NO synthesis inhibitor ameliorated the disease process. Rat oligodendrocyte cell death has been documented in vitro by microglia activated to produce NO (56). iNOS activity has been demonstrated in human fetal astrocytes but not in microglia (57), in contrast to what has been observed in the rodent, where both cell types can produce NO. Frozen sections of human multi-
TABLE 2 Effect of NO on Lymphocyte Functions Function
Effect
Signal transduction NF-kB p21ras Apoptosis Proliferation Cytolytic function Acquisition of Direct effect on Cytokine synthesis IFNg IL-2
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Activates Activates Promotes Inhibits Inhibits
(35) (37) (7) (7, 9, 15) (22–24, 98)
Inhibits No effect
(98–100) Unpublished observationa
Inhibits No effect Inhibits No effect
(30, 32) (98, 100, 101) (32) (32)
Hoffman and Simmons.
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ple sclerosis lesions, examined for evidence of NO activity by NADPH diaphorase staining ((34), Lee and Brosnan, this issue), showed intense staining in acute and chronic-active lesions. It is, as yet, unclear whether NO exerts immunomodulatory effects on inflammatory cells or effects tissue damage in central nervous system pathology.
TOLERANCE Tolerance can be induced by interaction of a T cell with a nonprofessional APC (lacks costimulatory molecule expression) which can result in anergy or apoptosis of the T cell. Recent work by Fehsel et al. (7) and our unpublished observations demonstrate that NO can induce T-cell apoptosis. NO can also induce apoptosis in macrophages (58, 59). These data indicate that NO could play a role in tolerance induction since deleterious effects on both the APC and the T cell can be seen. A paucity of information is available on the role of NO in tolerance induction. NO may be a factor contributing to the immunologically privileged site of the eye, since retinal pigment epithelial cells secreted NO in response to cytokines (60). Introduction of antigen into the portal venous circulation can induce a state of antigen-specific tolerance. This process can be reversed by administration of gadolinium chloride, an agent which enhances the ability of the liver Kupffer cells to produce NO. This enhanced NO synthesis may function to prevent the initial expansion of lymphocytes thought to be necessary for tolerance induction (61, 62). These results suggest that NO impacts negatively on the induction of tolerance.
HOST DEFENSE The most well-studied role of NO is that of a cytotoxic/cytostatic force against microbial invaders. NO has been shown to facilitate destruction/elimination of both intra- and extracellular bacteria, viruses, and parasites. Several investigators have shown that NO played a major effector role in resistance of murine hosts to Leishmania (63) and Schistosoma mansoni (64) and that iNOS expression is increased in mouse strains resistant to Leishmania (65) (see review (66) and Oswald and James, this issue). An anti-viral effect of NO has also been demonstrated in vitro and in vivo (67, 68). As expected, iNOS knockout mice proved to be highly susceptible to Leishmania infection (13). NO has also been shown to be induced in the rat lung after exposure to a xenobiotic agent such as ozone. Laskin et al. (69) have demonstrated iNOS mRNA and protein
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expression in alveolar and intestinal macrophages as well as type II epithelial cells. Thus, NO plays a role in host defense against not only microbial invaders, but also various noxious agents (reviewed by Laskin and Laskin, this issue). However, the determination of whether NO plays a role in acquired as well as innate immunity cannot always be made. In the innate arm of host defense, NO may serve as a cytostatic/cytotoxic agent as well as an inflammatory mediator produced by macrophages and endothelial cells upon exposure to a microbial byproduct such as LPS and inflammatory cytokines such as TNFa and IL-1b. Additionally, another effector cell of innate immunity, NK cells, can produce IFNg —a potent stimulator/costimulator of NO synthesis by many cell types. Some investigators have made a distinction between the role of NO in innate versus acquired immunity. Beckerman et al. (70) demonstrated that SCID mice, which possess NK cells but lack T and B cells, were compromised in their ability to deal with a Listeria monocytogenes challenge when an NO synthesis inhibitor was also administered. These data provided evidence in vivo that, in the absence of acquired immunity mediated by T cells, NO played a role in host defense. NO may also play a role in the T-cell-dependent, acquired immunity to microbial challenge in that cytokines produced by activated T cells might also be expected to promote NO synthesis. Whether NO synthesis functions to promote eradication of the organism only or whether it may also function to suppress the primary immune response, as described by Gregory et al. (71), has not been fully elucidated. The role of NO in host defense in human cells has not been elucidated, partly because NO synthesis by human monocytes is not consistently observed. However, Nussler et al. (72) demonstrated that human hepatocytes produce NO and subsequently described the antiplasmodial activity of human hepatocyte-derived NO (73).
TUMOR IMMUNITY The cellular effectors of host defense against tumor cells are T cells, NK cells, and macrophages. Tumor cells can evade T-cell-mediated immunity if Class I and Class II antigen expression and costimulatory molecule expression are downregulated. The initial description by Hibbs of the cytotoxic molecule produced by macrophages utilizing the amino acid L-arginine demonstrated inhibition of proliferation and enzyme activity in a tumor target cell (74). Of course, the cytostatic/ cytotoxic effects of NO are exerted upon normal cells as well as transformed cells, but the potential of NO as an anti-tumor tool has been further studied.
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The role of NO in host defense against tumor has been reported to depend on the pathway that macrophages use to metabolize L-arginine (75) and on whether infiltrating macrophage NO production or Tlymphocyte activation is downregulated by factors produced by the tumor such as IL-10, TGFb, and PGE2 (76, 77). Inhibition of tumor growth in vivo has been achieved by pretreatment of mice with BCG (78) as a mechanism of inducing NO synthesis. In contrast, Yim et al. have demonstrated enhanced tumor growth by administration of an NO synthesis inhibitor to tumorbearing mice (79). An inverse correlation between expression of iNOS and metastatic potential (80, 81) has been described. Paradoxically, Jenkins et al. (82) transfected the DLD-1 human colon adenocarcinoma line with iNOS and noted that while tumor growth in vitro was slowed, tumor growth in nude mice was faster, accompanied by an increase in vascularization. Similarly, Ghigo et al. (83) demonstrated iNOS production by an endothelioma cell line transformed by polyoma virus middle T antigen and that in vivo growth of this tumor was inhibited by administration of NO synthesis inhibitors, suggesting that NO is necessary to tumor development. The role of NO in host defense against tumor as well as in tumorigenesis is, at this time, incompletely defined.
TRANSPLANT IMMUNITY Effector mechanisms in allograft rejection include lysis of graft tissue by cytolytic T lymphocytes (CTL), Tcell recruitment of macrophages initiating graft injury via a DTH reaction, and antibody-mediated graft destruction. NO is produced during rejection of a variety of organ grafts as well as graft- versus-host disease in rat and mouse species (84–87). Nitrosyl complexes, detected by electron paramagnetic resonance imaging, have been detected in allografted tissue but not in syngeneic grafts (88, 89). Additionally, evidence of iNOS induction in human liver allografts has been described (90, 91). In rejecting rat heart allografts, Yang et al. (92) have demonstrated that iNOS is present in the infiltrating macrophages, microvascular endothelial cells, cardiac muscle fibers, as well as the cardiac myocytes. Pinsky et al. (93) have shown that induction of NO in myocytes results in myocyte death, providing evidence for a role for NO in direct toxicity to grafts. Maciejewski et al. (11) have demonstrated NO induction followed by growth inhibition and apoptosis in human bone marrow cells treated with IFNg and TNFa, suggesting that NO can promote hematopoietic suppression and thus might exacerbate GvHD. We have shown that some of the immunosuppressive characteristics of GvHD were due to the production of NO (85).
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As in other experimental models, seemingly opposite effects have been seen when the effect of administration of NO synthesis inhibitors was evaluated. Variations in the strength of the allograft response (genetic disparity) and differences in the inhibitor and animal species studied could account for these variations. Bastian et al. (94) saw very little difference in survival of heart allografts in mice and, similarly, Winlaw et al. (95) observed slight prolongation of heart allografts in rats. In contrast, Worrall et al. (96) have demonstrated prolonged survival of rat heart allografts by administration of aminoguanidine, coincident with a decrease in the inflammatory infiltrate. We have shown a decreased destruction of host tissue during GvHD by administration of an NO synthesis inhibitor (86). Conversely, Drobyski et al. (97) have shown enhanced lethality by administration of an NO synthesis inhibitor during GvHD, indicating that NO may play a role in hematopoietic engraftment. NO could be affecting at least three different aspects of graft rejection: NO could be downregulating T-lymphocyte effector function and thus NO synthesis inhibition might facilitate graft rejection; NO could be acting directly on the graft, contributing to graft dysfunction and thus administration of NO synthesis inhibitors might promote graft survival; and finally, NO could be contributing to the inflammatory cascade during graft rejection and thus NO synthesis inhibitors would also prolong graft survival. Studies examining the contribution of NO to graft rejection will hopefully be able to delineate the contribution of inflammation vs cell-mediated immunity in this process.
CONCLUSION Many more pieces of the puzzle need to be fitted before the role of NO in the immune response can be fully appreciated. More critical examination of how NO might affect the major players in the immune response, such as APC and T cells, is needed. This information may reveal under what circumstances NO functions to benefit the host by destroying an invader or when NO functions to destroy host tissue via inflammatory responses.
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33. Pantopoulos, K., and Hentze, M. W. (1995) Proc. Natl. Acad. Sci. USA 92, 1267–1271. 34. Bo, L., Dawson, T. M., Wesselingh, S., Mork, S., Choi, S., Kong, P. A., Hanley, D., and Trapp, B. D. (1994) Ann. Neurol. 36, 778–786. 35. Lander, H. M., Sehajpal, P., Levine, D. M., and Novogrodsky, A. (1993) J. Immunol. 150, 1509–1516. 36. Lander, H. M., Sehajpal, P. K., and Novogrodsky, A. (1993) J. Immunol. 151, 7182–7187. 37. Lander, H. M., Ogiste, J. S., Pearce, S. F. A., Levi, R., and Novogrodsky, A. (1995) J. Biol. Chem. 270, 7017–7020. 38. Stadler, J., Stefanovic-Racic, M., Billiar, T. R., Curran, R. D., McIntyre, L. A., Georgescu, H. I., Simmons, R. L., and Evans, C. H. (1991) J. Immunol. 147, 3915–3920. 39. Stefanovic-Racic, M., Stadler, J., Georgescu, H. I., and Evans, C. H. (1994) J. Rheumatol. 21, 1892–1898. 40. Ialenti, A., Moncada, S., and Di Rosa, M. (1993) Br. J. Pharmacol. 110, 701–706. 41. McCartney-Francis, N., Allen, J. B., Mizel, D. E., Albina, J. E., Xie, Q., Nathan, C. F., and Wahl, S. M. (1993) J. Exp. Med. 178, 749–754. 42. Stefanovic-Racic, M., Meyers, K., Meschter, C., Coffey, J. W., Hoffman, R. A., and Evans, C. H. (1994) Arthritis Rheum. 37, 1062–1069. 43. Connor, J. R., Manning, P. T., Settle, S. L., Moore, W. M., Jerome, G. M., Webber, R. K., Tjoeng, F. S., and Currie, M. G. (1995) Eur. J. Pharmacol. 273, 15–24. 44. Weinberg, J. B., Granger, D. L., Pisetsky, D. S., Seldin, M. F., Misukonis, M. A., Mason, S. N., Pippen, A. M., Ruiz, P., Wood, E. R., and Gilkeson, G. S. (1994) J. Exp. Med. 179, 651–660. 45. Southern, C., Schulster, D., and Green, I. C. (1990) FEBS Lett. 276, 42–44. 46. Corbett, J. A., Lancaster, J. R., Jr., Sweetland, M. A., and McDaniel, M. L. (1991) J. Biol. Chem. 266, 21351–21354. 47. Bergmann, L., Kroncke, K.-D., Suschek, C., Kolb, H., and KolbBachofen, V. (1992) FEBS Lett. 299, 103–106. 48. Corbett, J. A., and McDaniel, M. L. (1995) J. Exp. Med. 181, 559–568. 49. Kolb, H., Burkart, V., Appels, B., Hanenberg, H., KantwerkFunke, G., Kiesel, U., Funda, J., Schraermeyer, U., and KolbBachofen, V. (1990) J. Autoimmun. 3(Suppl), 117–120. 50. Corbett, J. A., Wang, J. L., Sweetland, M. A., Lancaster, Jr., J. R., and McDaniel, M. L. (1992) J. Clin. Invest. 90, 2384– 2391. 51. Fehsel, K., Jalowy, A., Qi, S., Burkart, V., Hartmann, B., and Kolb, H. (1993) Diabetes 42, 496–500. 52. Corbett, J. A., Mikhael, A., Shimizu, J., Frederick, K., Misko, T. P., McDaniel, M. L., Kanagawa, O., and Unanue, E. R. (1993) Proc. Natl. Acad. Sci. USA 90, 8992–8995. 53. Corbett, J. A., Kwon, G., Turk, J., and McDaniel, M. L. (1993) Biochemistry 32, 13767–13770. 54. Lin, R. F., Lin, T.-S., Tilton, R. G., and Cross, A. H. (1993) J. Exp. Med. 178, 643–648. 55. Cross, A. H., Misko, T. P., Lin, R. F., Hickey, W. F., Trotter, J. L., and Tilton, R. G. (1994) J. Clin. Invest. 93, 2684–2690. 56. Merrill, J. E., Ignarro, L. J., Sherman, M. P., Melinek, J., and Lane, T. E. (1993) J. Immunol. 151, 2132–2141. 57. Lee, S. C., Dickson, D. W., Liu, W., and Brosnan, C. F. (1993) J. Neuroimmunol. 46, 19–24. 58. Sarih, M., Souvannavong, V., and Adam, A. (1993) Biochem. Biophys. Res. Comm. 191, 503–508.
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EDITORIAL 59. Albina, J. E., Cui, S., Mateo, R. B., and Reichner, J. S. (1993) J. Immunol. 150, 5080–5085. 60. Liversidge, J., Grabowski, P., Ralston, S., Benjamin, N., and Forrester, J. V. (1994) Immunol. 83, 404–409. 61. Roland, C. R., Mangino, M. J., and Flye, M. W. (1993) J. Surg. Res. 54, 401–410. 62. Roland, C. R., Walp, L., Stack, R. M., and Flye, M. W. (1994) J. Immunol. 153, 5453–5464. 63. Evans, T. G., Thai, L., Granger, D. L., and Hibbs, J. B., Jr. (1993) J. Immunol. 151, 907–915. 64. Wynn, T. A., Oswald, I. P., Eltoum, I. A., Caspar, P., Lowenstein, C. J., Lewis, F. A., James, S. L., and Sher, A. (1994) J. Immunol. 153, 5200–5209. 65. Stenger, S., Thuring, H., Rollinghoff, M., and Bogdan, C. (1994) J. Exp. Med. 180, 783–793. 66. Oswald, I. P., Wynn, T. A., Sher, A., and James, S. L. (1994) Comp. Biochem. Physiol. 108C, 11–18. 67. Croen, K. D. (1993) J. Clin. Invest. 91, 2446–2452. 68. Karupiah, G., Xie, Q.-W., Buller, R. M. L., Nathan, C., Duarte, C., and MacMicking, J. D. (1993) Science 261, 1445–1448. 69. Laskin, D. L., Pendino, K. J., Punjabi, C. J., Rodriguez del Valle, M., and Laskin, J. D. (1994) Env. Hlth. Persp. 102(Suppl. 10), 61–64. 70. Beckerman, K. P., Rogers, H. W., Corbett, J. A., Schreiber, R. D., McDaniel, M. L., and Unanue, E. R. (1993) J. Immunol. 150, 888–895. 71. Gregory, S. H., Wing, E. J., Hoffman, R. A., and Simmons, R. L. (1993) J. Immunol. 150, 2901–2909. 72. Nussler, A., Di Silvio, M., Billiar, T., Hoffman, R., Geller, D., Selby, R., Madariaga, J., and Simmons, R. L. (1992) J. Exp. Med. 176, 261–264. 73. Mellouk, S., Hoffman, S. L., Liu, Z.-Z., de la Vega, P., Billiar, T. R., and Nussler, A. K. (1994) Infect. Immun. 62, 4043–4046. 74. Hibbs, J. B., Jr., Vavrin, Z., and Taintor, R. R. (1987) J. Immunol. 138, 550–565. 75. Mills, C. D., Shearer, J., Evans, R., and Caldwell, M. D. (1992) J. Immunol. 149, 2709–2714. 76. Lejeune, P., Lagadec, P., Onier, N., Pinard, D., Ohshima, H., and Jeannin, J.-F. (1994) J. Immunol. 152, 5077–5083. 77. Alleva, D. G., Burger, C. J., and Elgert, K. D. (1994) J. Immunol. 153, 1674–1686. 78. Farias-Eisner, R., Sherman, M. P., Aeberhard, E., and Chaudhuri, G. (1994) Proc. Natl. Acad. Sci. USA 91, 9407–9411. 79. Yim, C.-Y., Bastian, N. R., Smith, J. C., Hibbs, J. B., Jr., and Samlowski, W. E. (1993) Cancer Res. 53, 5507–5511. 80. Dong, Z., Staroselsky, A. H., Qi, X., Xie, K., and Fidler, I. J. (1994) Cancer Res. 54, 789–793. 81. Xie, K., Huang, S., Dong, Z., Juang, S.-H., Gutman, M., Xie, Q.-W., Nathan, C., and Fidler, I. J. (1995) J. Exp. Med. 181, 1333–1343. 82. Jenkins, D. C., Charles, I. G., Thomsen, L. L., Moss, D. W., Holmes, L. S., Baylis, S. A., Rhodes, P., Westmore, K., Emson, P. C., and Moncada, S. (1995) Proc. Natl. Acad. Sci. USA 92, 4392–4396.
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83. Ghigo, D., Arese, M., Todde, R., Vecchi, A., Silvagno, F., Costamagna, C., Dong, Q. G., Alessio, M., Heller, R., Soldi, R., Trucco, F., Garbarino, G., Pescarmona, G., Mantovani, A., Bussolino, F., and Bosia, A. (1995) J. Exp. Med. 181, 9–19. 84. Langrehr, J. M., Murase, N., Markus, P. M., Cai, X., Neuhaus, P., Schraut, W., Simmons, R. L., and Hoffman, R. A. (1992) J. Clin. Invest. 90, 679–683. 85. Hoffman, R. A., Langrehr, J. M., Wren, S. M., Dull, K. E., Ildstad, S. T., McCarthy, S. A., and Simmons, R. L. (1993) J. Immunol. 151, 1508–1518. 86. Hoffman, R. A., Langrehr, J. M., Berry, L. M., White, D. A., Schattenfroh, N. C., McCarthy, S. A., and Simmons, R. L. (1996) Transplantation 61, 610–618. 87. Winlaw, D. S., Schyvens, C. G., Smythe, G. A., Rainer, S. P., Keogh, A. M., Mundy, J. A., Lord, R. S. A., Spratt, P. M., and MacDonald, P. S. (1994) Transplantation 58, 1031–1036. 88. Lancaster, J. R., Jr., Langrehr, J. M., Bergonia, H. A., Murase, N., Simmons, R. L., and Hoffman, R. A. (1992) J. Biol. Chem. 267, 10994–10998. 89. Langrehr, J. M., Mu¨ller, A. R., Bergonia, H. A., Jacobs, T. D., Lee, T. K., Schraut, W. H., Lancaster, J. R., Jr., Hoffman, R. A., and Simmons, R. L. (1992) Surgery 112, 395–402. 90. Ohdan, H., Suzuki, S., Kanashiro, M., Amemiya, H., Fukuda, Y., and Dohi, K. (1994) Transplantation 57, 1674–1677. 91. Devlin, J., Palmer, R. M. J., Gonde, C. E., O’Grady, J., Heaton, N., Tan, K. C., Martin, J. F., Moncada, S., and Williams, R. (1994) Transplantation 58, 592–595. 92. Yang, X., Chowdhury, N., Cai, B., Brett, J., Marboe, C., Sciacca, R. R., Michler, R. E., and Cannon, P. J. (1994) J. Clin. Invest. 94, 714–721. 93. Pinsky, D. J., Cai, B., Yang, X., Rodriguez, C., Sciacca, R. R., and Cannon, P. J. (1995) J. Clin. Invest. 95, 677–685. 94. Bastian, N. R., Xu, S., Shao, X. L., Shelby, J., Granger, D. L., and Hibbs, J. B., Jr. (1994) Biochim. Biophys. Acta 1226, 225– 231. 95. Winlaw, D. S., Schyvens, C. G., Smythe, G. A., Du, Z. Y., Rainer, S. P., Lord, R. S. A., Spratt, P. M., and MacDonald, P. S. (1995) Transplantation 60, 77–82. 96. Worrall, N. K., Lazenby, W. D., Misko, T. P., Lin, T.-S., Rodi, C. P., Manning, P. T., Tilton, R. G., Williamson, J. R., and Ferguson, T. B., Jr. (1995) J. Exp. Med. 181, 63–70. 97. Drobyski, W. R., Keever, C. A., Hanson, G. A., McAuliffe, T., and Griffith, O. W. (1994) Blood 84, 2363–2373. 98. Hoffman, R. A., Langrehr, J. M., Billiar, T. R., Curran, R. D., and Simmons, R. L. (1990) J. Immunol. 145, 2220–2226. 99. Langrehr, J. M., Hoffman, R. A., Billiar, T. R., Lee, K. K. W., Schraut, W. H., and Simmons, R. L. (1991) Surgery 110, 335– 342. 100. Langrehr, J. M., Dull, K. E., Ochoa, J. B., Billiar, T. R., Ildstad, S. T., Schraut, W. H., Simmons, R. L., and Hoffman, R. A. (1992) Transplantation 53, 632–640. 101. Huot, A. E., Moore, A. L., Roberts, J. D., and Hacker, M. P. (1993) Immunol. Invest. 22, 319–327.
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Rosemary Hoffman Guest Editor
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Reactive Nitrogen Intermediates in Immunology Nitric oxide synthase (NOS) is an ubiquitous enzyme capable of affecting a multitude of mechanisms in the immune response. The contributions in this issue of Methods reflect that diversity and include descriptions of the intracellular signaling function of NO, factors governing NO synthesis and its effects in central nervous system pathology, xenobiotic-induced lung injury, arthritic joint pathology, diabetes induced by b-cell destruction, tissue destruction during graft-versus-host disease, the hepatic response to infectious agents, and host defense against parasites. The modulatory influence of NO must be examined in light of the fact that either small amounts (constitutive enzyme) or large amounts (inducible enzyme) can be produced and that biphasic effects of NO are apparent in many systems (1). As will be described, many observations of the effects of NO studied under apparently similar conditions reveal vastly different results. Some of the variation could be due to the amount of NO produced by the different isoforms of NOS. The endothelial cell (ec) and neuronal cell (nc) constitutive NOS enzymes are responsible for production of small amounts of NO that function as signal transduction molecules. In contrast, the high amount of NO produced by inducible nitric oxide synthase (iNOS) can result in inhibition of cellular function by virtue of NO-mediated inactivation of proteins with iron sulfur centers or heme moieties. (See current reviews on control (2), function (3), biochemistry (4), and targets (5) of the NO synthases.) A paradoxical effect of NO that is pertinent to the role of NO in the immune response is that production via iNOS can result in beneficial or detrimental outcome for the organism. For example, NO produced acutely, directed against a pathogen, is beneficial, while NO produced over a long time period, as might be the case with chronic conditions such as arthritis, is detrimental. Another remarkable feature governing induction of iNOS is that inflammatory mediators, such as TNFa, LPS, IL-1b, and IFNg, can stimulate NO synthesis, while other mediators, such as TGFb, IL-4, and IL-10, can downregulate NO synthesis. This separation of cytokines into promoters and inhibitors of NO synthesis resembles closely the dichotomy observed in cytokines produced by T-helper 1 lympho-
cytes (IFNg, IL-2, TNFa), in contrast to T-helper 2 lymphocytes (IL-4, IL-5, IL-10). Thus, NO appears to be a player in the inflammatory milieu that accompanies delayed type hypersensitivity (DTH) reactions and not the milieu that promotes humoral immunity. The following is a brief accounting of how NO has been demonstrated to influence major pathways in the immune response. Development of T- and B-lymphocyte repertoires. Tcell precursors migrate from the bone marrow to the thymus and progress through a series of maturational stages, whereby expression of CD4 or CD8 as well as the T-cell receptor is acquired. Interaction of the developing T cell with thymic stroma results in either positive selection, in which T cells that can respond to foreign antigen in the context of self MHC are selected, or negative selection, in which those cells that are specific for self peptides bound to self MHC (autoreactive cells) are eliminated. The thymic stroma is composed of cell types known to be capable of NO synthesis such as epithelial cells, dendritic cells, and macrophages. Sato et al. (6) have demonstrated by immunohistochemistry that cells in the medulla but not the cortex of unstimulated rat thymus stained positively for iNOS. Fehsel et al. (7, 8) have demonstrated that after LPS administration both increased iNOS expression and DNA fragmentation were seen in thymus tissue sections and that NO could be both a protective and an inducing agent for thymocyte apoptosis in vitro. B-cell maturation from progenitors in the bone marrow is influenced by cytokines as well as contact with bone marrow stromal cells such as endothelial cells, macrophages, fibroblasts, and fat cells. Deletion and functional anergy occurs during the B-cell maturational process. Genaro et al. (9) have described that NO prevented B-cell apoptosis, indicating a possible role for NO in B-cell maturation. NO has also been shown in vitro to induce differentiation of a monocytic cell line, accompanied by altered gene expression (10), indicating a possible role of NO in hematopoietic differentiation. However, contrasting results by Maciejewski et al. (11) described NO-induced inhibition of growth and induction of apoptosis in human bone marrow cells. Since iNOS knockout mice, described by MacMicking et al. (12) and Wei et al. (13), do not demonstrate any 1
1046-2023/96 $18.00 Copyright q 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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obvious abnormality in the lymphocyte populations of thymus, spleen, or lymph node, it is unlikely that NO plays a role in the development of lymphocyte repertoires. These animals are housed under specific pathogen-free conditions and the possibility exists that defects in lymphocyte positive and negative selection could become apparent if the iNOS knockout mice were exposed to inflammatory stimuli. T-Cell-mediated immunity. The activation of a naive T cell requires interaction with a professional antigen presenting cell (APC). A professional APC possesses processed antigen in the cleft of either MHC Class I or Class II molecules as well as costimulatory molecules, such as B7. Professional APCs include dendritic cells, B cells, and macrophages. Dendritic cells are the most potent APC because of their constitutive expression of Class II as well as costimulatory molecules (14). Class I molecules are present on all nucleated cells and Class II antigen is constitutively expressed on dendritic cells and B cells. However, Class II antigen and costimulatory molecule expression must be induced on macrophages and costimulatory molecule expression must be induced on B cells for effective antigen presentation to occur (see Table 1). Naive CD4 T cells interact with Class II-bearing APCs, while CD8 T cells interact with Class I-bearing APCs. NO synthesis by macrophages is well documented, NO synthesis by dendritic cells has recently been discerned (Angus W. Thomson, personal communication), and B cells have not been reported to produce NO, except for transformed B cells (15). The obvious concern is whether induction of iNOS will adversely affect expression of Class II and costimulatory molecules. Some reports documented no effect on Class II expression and some reports documented an inhibition by NO (16– 19). These differences could be due to different stimuli
TABLE 1 Comparison of the Ability of Various Antigen-Presenting Cells to Synthesize NO Class II antigen
Cell type Professional antigen presenting cells Dendritic cells B lymphocytes Macrophage Nonprofessional antigen presenting cells Vascular endothelial cells Epithelial cells
Costimulatory NO molecules synthesis
Constitutive Constitutive Constitutive Inducible Inducible Inducible
Yesa Nob Yes
Inducible Inducible
Yes Yes
Constitutive ?
a
Personal communication, Angus W. Thomson. EBV-transformed lymphocytes and Burkitt’s lymphoma lines produce low levels of NO. b
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and use of primary macrophages versus macrophage tumor cell lines. Effective inhibition/downregulation of a primary immune response would be achieved by NOmediated downregulation of Class II expression. An example of this modulatory influence described by Holt et al. was that alveolar macrophage NO synthesis functions to downregulate pulmonary dendritic cell APC function in vitro coincident with inhibition of Class II expression (20). Differential regulation of costimulatory molecule expression, B7.1 versus B7.2, was reported by treatment of macrophages with LPS, which caused upregulation of both B7.1 and 2, while IFNg increased B7.2 and downregulated B7.1 expression (21). Whether these differential effects were due to NO has not been described. Additionally, many investigators using varied systems where suppression of lymphocyte proliferation was seen have shown that an NO synthesis inhibitor permitted enhanced lymphocyte proliferation (22–28). Thus, NO may have a dual role in inhibiting lymphocyte activation: (1) prevention of upregulation of Class II antigen as well as costimulatory molecule expression on the APC and (2) inhibition of proliferation by the direct action of NO on the lymphocyte (Table 2). Once activated, T cells do not require costimulatory signals to interact with an APC, but do require T-cell receptor interaction with the peptide:MHC complex. Thus, since activated CD8 T cells can interact with any cell expressing Class I molecules, this cell population would include many NO-producing cells, such as fibroblasts, hepatocytes, tumor cells, etc. (see list in (29)). Activated CD4 T cells interact with APC possessing Class II even without costimulatory molecule expression. Modulation of activated CD4 or CD8 T-cell expansion or effector function could occur via target cell production of NO. An example of a paracrine regulatory effect is that described by Stefani et al. (30), in which Leishmania major-specific CD8/ cells, stimulated with L. major-infected macrophages, produced IFNg, which in turn caused NO synthesis in the macrophages. This NO synthesis caused downregulation of CD8 T-cell proliferation and IFNg synthesis. The successful interaction of either a naive or a previously activated T cell with APC results in cytokine synthesis by the T cell, some of which can influence NO synthesis positively (IFNg, IL-2, TNFa) or negatively (IL-4, IL-10, TGFb). The cytokines that elicit NO synthesis are products of Th1 cells and facilitate the inflammatory, DTH type of reaction, while cytokines that downmodulate NO synthesis facilitate humoral immunity. As suggested by the work of Taub and Cox (31) interaction of Th1 or Th2 cells with macrophages may represent a potent paracrine regulatory network controlling the activation of macrophages. Taylor-Robinson et al. (32) have demonstrated that cloned Th1 but not Th2 lymphocytes produced NO and that Th1
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cloned lymphocyte synthesis of IL-2 and IFN-g was inhibited by NO, while Th2 cloned lymphocyte synthesis of IL-4 was not inhibited. It is evident that NO can modulate T-cell-mediated immunity at several levels encompassing initiation as well as the propagation of the immune response.
SIGNAL TRANSDUCTION Initial studies on how NO affects cells focused on how NO inhibits various pathways in the cell, such as mitochondrial respiration and proliferation. Subsequently, the intracellular signaling pathways triggered by NO were discovered and are now being investigated in many different cells. For example, NO activates iron response element binding (33) and modulates AP-1 transcriptional factors (34). Lander et al. (35, 36) have found that NO enhanced the rate of glucose transport and induced NF-kB binding activity and secretion of TNFa by human peripheral blood mononuclear cells. Recent work (37) has shown that NO can activate T lymphocyte p21ras, a member of the Ras superfamily, which are proteins that relay signals from receptor tyrosine kinases to the nucleus. Further elucidation of the effects of NO on T-lymphocyte intracellular signaling will provide a clearer picture of how NO might affect an immune response.
AUTOIMMUNITY Autoimmune responses are thought to be the result of T-lymphocyte responses to self antigen because that antigen is recognized as foreign, not as self. This can be the result of newly exposed antigen, defective negative selection in the thymus, or exposure to self antigens under inflammatory conditions where costimulatory molecule induction can result in lymphocyte activation. The autoimmune response results in cytokine synthesis by activated T cells, which can result in activation of B cells to synthesize antibodies and macrophages to secrete inflammatory molecules. Since the response is directed to self, there is an ample supply of antigen to produce a chronic inflammatory response. The role of NO in arthritis, a chronic inflammation of the joints, has been well studied. NO is produced by chondrocytes (38) and synovial fibroblasts (39). Inhibition of joint swelling by administration of NO synthesis inhibitors has been reported in animal models of arthritis (40–43) as well as the autoimmune disease seen in MRL-lpr/lpr mice (44). Since inhibition of NO is accompanied by decreased joint inflammation, it would appear that NO does not function to downregulate lym-
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phocyte activation during the arthritic process, since inhibition of NO might then potentiate lymphocyte responses, worsening the disease. Rather, NO appears to be functioning in promotion of the inflammatory response. In fact, Ialenti et al. (40) have demonstrated enhanced lymphoproliferative responses in L-argininefed arthritic rats and decreased responses in animals fed an NO synthesis inhibitor. The autoimmune disease insulin-dependent diabetes mellitus is characterized by destruction of the pancreatic b cell, due to islet infiltration by inflammatory macrophages and T cells. Ample evidence documents the role of NO in inhibition of b-cell function (45–47) due to IL-1-induced NO synthesis by b cells (48) or by NO synthesized by infiltrating macrophages (49). NO has been shown to inhibit mitochondrial oxidation in purified b cells (50) and to induce DNA strand breaks in islet cells (51). Administration of the NO synthesis inhibitor aminoguanidine delayed the development of diabetes in NOD mice (52). Since NO has also been shown to stimulate the activity of both cCOX and iCOX (53), NO apparently functions as an inflammatory molecule during induction of b-cell dysfunction, actually promoting production of another mediator, PGE2 . The role of NO in central nervous system pathology has also been investigated utilizing a model of autoimmune encephalomyelitis (54, 55). NO was localized to the spinal cord of infected mice and administration of an NO synthesis inhibitor ameliorated the disease process. Rat oligodendrocyte cell death has been documented in vitro by microglia activated to produce NO (56). iNOS activity has been demonstrated in human fetal astrocytes but not in microglia (57), in contrast to what has been observed in the rodent, where both cell types can produce NO. Frozen sections of human multi-
TABLE 2 Effect of NO on Lymphocyte Functions Function
Effect
Signal transduction NF-kB p21ras Apoptosis Proliferation Cytolytic function Acquisition of Direct effect on Cytokine synthesis IFNg IL-2
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Activates Activates Promotes Inhibits Inhibits
(35) (37) (7) (7, 9, 15) (22–24, 98)
Inhibits No effect
(98–100) Unpublished observationa
Inhibits No effect Inhibits No effect
(30, 32) (98, 100, 101) (32) (32)
Hoffman and Simmons.
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ple sclerosis lesions, examined for evidence of NO activity by NADPH diaphorase staining ((34), Lee and Brosnan, this issue), showed intense staining in acute and chronic-active lesions. It is, as yet, unclear whether NO exerts immunomodulatory effects on inflammatory cells or effects tissue damage in central nervous system pathology.
TOLERANCE Tolerance can be induced by interaction of a T cell with a nonprofessional APC (lacks costimulatory molecule expression) which can result in anergy or apoptosis of the T cell. Recent work by Fehsel et al. (7) and our unpublished observations demonstrate that NO can induce T-cell apoptosis. NO can also induce apoptosis in macrophages (58, 59). These data indicate that NO could play a role in tolerance induction since deleterious effects on both the APC and the T cell can be seen. A paucity of information is available on the role of NO in tolerance induction. NO may be a factor contributing to the immunologically privileged site of the eye, since retinal pigment epithelial cells secreted NO in response to cytokines (60). Introduction of antigen into the portal venous circulation can induce a state of antigen-specific tolerance. This process can be reversed by administration of gadolinium chloride, an agent which enhances the ability of the liver Kupffer cells to produce NO. This enhanced NO synthesis may function to prevent the initial expansion of lymphocytes thought to be necessary for tolerance induction (61, 62). These results suggest that NO impacts negatively on the induction of tolerance.
HOST DEFENSE The most well-studied role of NO is that of a cytotoxic/cytostatic force against microbial invaders. NO has been shown to facilitate destruction/elimination of both intra- and extracellular bacteria, viruses, and parasites. Several investigators have shown that NO played a major effector role in resistance of murine hosts to Leishmania (63) and Schistosoma mansoni (64) and that iNOS expression is increased in mouse strains resistant to Leishmania (65) (see review (66) and Oswald and James, this issue). An anti-viral effect of NO has also been demonstrated in vitro and in vivo (67, 68). As expected, iNOS knockout mice proved to be highly susceptible to Leishmania infection (13). NO has also been shown to be induced in the rat lung after exposure to a xenobiotic agent such as ozone. Laskin et al. (69) have demonstrated iNOS mRNA and protein
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expression in alveolar and intestinal macrophages as well as type II epithelial cells. Thus, NO plays a role in host defense against not only microbial invaders, but also various noxious agents (reviewed by Laskin and Laskin, this issue). However, the determination of whether NO plays a role in acquired as well as innate immunity cannot always be made. In the innate arm of host defense, NO may serve as a cytostatic/cytotoxic agent as well as an inflammatory mediator produced by macrophages and endothelial cells upon exposure to a microbial byproduct such as LPS and inflammatory cytokines such as TNFa and IL-1b. Additionally, another effector cell of innate immunity, NK cells, can produce IFNg —a potent stimulator/costimulator of NO synthesis by many cell types. Some investigators have made a distinction between the role of NO in innate versus acquired immunity. Beckerman et al. (70) demonstrated that SCID mice, which possess NK cells but lack T and B cells, were compromised in their ability to deal with a Listeria monocytogenes challenge when an NO synthesis inhibitor was also administered. These data provided evidence in vivo that, in the absence of acquired immunity mediated by T cells, NO played a role in host defense. NO may also play a role in the T-cell-dependent, acquired immunity to microbial challenge in that cytokines produced by activated T cells might also be expected to promote NO synthesis. Whether NO synthesis functions to promote eradication of the organism only or whether it may also function to suppress the primary immune response, as described by Gregory et al. (71), has not been fully elucidated. The role of NO in host defense in human cells has not been elucidated, partly because NO synthesis by human monocytes is not consistently observed. However, Nussler et al. (72) demonstrated that human hepatocytes produce NO and subsequently described the antiplasmodial activity of human hepatocyte-derived NO (73).
TUMOR IMMUNITY The cellular effectors of host defense against tumor cells are T cells, NK cells, and macrophages. Tumor cells can evade T-cell-mediated immunity if Class I and Class II antigen expression and costimulatory molecule expression are downregulated. The initial description by Hibbs of the cytotoxic molecule produced by macrophages utilizing the amino acid L-arginine demonstrated inhibition of proliferation and enzyme activity in a tumor target cell (74). Of course, the cytostatic/ cytotoxic effects of NO are exerted upon normal cells as well as transformed cells, but the potential of NO as an anti-tumor tool has been further studied.
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The role of NO in host defense against tumor has been reported to depend on the pathway that macrophages use to metabolize L-arginine (75) and on whether infiltrating macrophage NO production or Tlymphocyte activation is downregulated by factors produced by the tumor such as IL-10, TGFb, and PGE2 (76, 77). Inhibition of tumor growth in vivo has been achieved by pretreatment of mice with BCG (78) as a mechanism of inducing NO synthesis. In contrast, Yim et al. have demonstrated enhanced tumor growth by administration of an NO synthesis inhibitor to tumorbearing mice (79). An inverse correlation between expression of iNOS and metastatic potential (80, 81) has been described. Paradoxically, Jenkins et al. (82) transfected the DLD-1 human colon adenocarcinoma line with iNOS and noted that while tumor growth in vitro was slowed, tumor growth in nude mice was faster, accompanied by an increase in vascularization. Similarly, Ghigo et al. (83) demonstrated iNOS production by an endothelioma cell line transformed by polyoma virus middle T antigen and that in vivo growth of this tumor was inhibited by administration of NO synthesis inhibitors, suggesting that NO is necessary to tumor development. The role of NO in host defense against tumor as well as in tumorigenesis is, at this time, incompletely defined.
TRANSPLANT IMMUNITY Effector mechanisms in allograft rejection include lysis of graft tissue by cytolytic T lymphocytes (CTL), Tcell recruitment of macrophages initiating graft injury via a DTH reaction, and antibody-mediated graft destruction. NO is produced during rejection of a variety of organ grafts as well as graft- versus-host disease in rat and mouse species (84–87). Nitrosyl complexes, detected by electron paramagnetic resonance imaging, have been detected in allografted tissue but not in syngeneic grafts (88, 89). Additionally, evidence of iNOS induction in human liver allografts has been described (90, 91). In rejecting rat heart allografts, Yang et al. (92) have demonstrated that iNOS is present in the infiltrating macrophages, microvascular endothelial cells, cardiac muscle fibers, as well as the cardiac myocytes. Pinsky et al. (93) have shown that induction of NO in myocytes results in myocyte death, providing evidence for a role for NO in direct toxicity to grafts. Maciejewski et al. (11) have demonstrated NO induction followed by growth inhibition and apoptosis in human bone marrow cells treated with IFNg and TNFa, suggesting that NO can promote hematopoietic suppression and thus might exacerbate GvHD. We have shown that some of the immunosuppressive characteristics of GvHD were due to the production of NO (85).
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As in other experimental models, seemingly opposite effects have been seen when the effect of administration of NO synthesis inhibitors was evaluated. Variations in the strength of the allograft response (genetic disparity) and differences in the inhibitor and animal species studied could account for these variations. Bastian et al. (94) saw very little difference in survival of heart allografts in mice and, similarly, Winlaw et al. (95) observed slight prolongation of heart allografts in rats. In contrast, Worrall et al. (96) have demonstrated prolonged survival of rat heart allografts by administration of aminoguanidine, coincident with a decrease in the inflammatory infiltrate. We have shown a decreased destruction of host tissue during GvHD by administration of an NO synthesis inhibitor (86). Conversely, Drobyski et al. (97) have shown enhanced lethality by administration of an NO synthesis inhibitor during GvHD, indicating that NO may play a role in hematopoietic engraftment. NO could be affecting at least three different aspects of graft rejection: NO could be downregulating T-lymphocyte effector function and thus NO synthesis inhibition might facilitate graft rejection; NO could be acting directly on the graft, contributing to graft dysfunction and thus administration of NO synthesis inhibitors might promote graft survival; and finally, NO could be contributing to the inflammatory cascade during graft rejection and thus NO synthesis inhibitors would also prolong graft survival. Studies examining the contribution of NO to graft rejection will hopefully be able to delineate the contribution of inflammation vs cell-mediated immunity in this process.
CONCLUSION Many more pieces of the puzzle need to be fitted before the role of NO in the immune response can be fully appreciated. More critical examination of how NO might affect the major players in the immune response, such as APC and T cells, is needed. This information may reveal under what circumstances NO functions to benefit the host by destroying an invader or when NO functions to destroy host tissue via inflammatory responses.
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