Institute of Medical Microbiology and Immunology, University of Copenhagen, Panum Institute, 3C Blegdamsvej, DK-2200 Copenhagen N, Denmark1
Author for correspondence: Allan Randrup Thomsen.Fax +45 35327874. e- mail a.r.thomsen{at}sb.immi.ku.dk
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Abstract |
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Introduction |
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Induction of iNOS expression has been found not to be restricted solely to the T cell-dependent late phase of infection, but up- regulation of iNOS may also be an important component of the innate host response. Thus, iNOS-knockout (iNOS-/-) mice are highly susceptible to ectromelia virus, yet show no impairment of specific antiviral immune responses (Karupiah et al., 1998 a ). Moreover, in leishmania-infected mice, where iNOS is focally induced by IFN-
/ß within the first 24 h of infection, lack of iNOS leads to a reduction in cytotoxic activity of natural killer (NK) cells, decreased expression of IL-12 mRNA and the complete absence of up-regulation of IFN-
(Diefenbach et al., 1998
).
Regarding neuroimmunological functions, iNOS expression is indicated to be one of the factors contributing to the expansion of the brain damage that occurs following an ischaemic insult (Iadecola et al. , 1995 , 1997
). In contrast, iNOS appears to play a predominantly protective role in the development of experimental allergic encephalitis (Bogdan, 1998
). Local iNOS expression has also been suggested to influence the outcome of lymphocytic choriomeningitis (LCM) by mediating protective functions (Campbell, 1996
). The development of fatal LCM in mice occurs as a response to intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and is characterized at the pathological level by marked recruitment and extravasation of immunoinflammatory cells to the sites of virus replication in the meninges and ependyma (Doherty et al., 1990
). This results eventually in death, 68 days post-infection (p.i.). Experiments have shown that CD8+ MHC class I-restricted cytotoxic T lymphocytes (Tc cells), but not CD4+ T lymphocytes, are pivotal in the development of LCM (Doherty et al., 1990
; Kagi et al., 1994
), and the temporal and spatial expression of iNOS in the infected brain is clearly associated with the development of the virus-induced inflammatory infiltrate (Campbell et al., 1994
b). High levels of IFN-
are present in the cerebrospinal fluid (CSF) (Frei et al., 1988
; Nansen et al., 1998
), but the outcome of this infection in iNOS-/- mice has not been investigated previously. In addition, iNOS-dependent NO production may be one important pathway through which IFN-
mediates its antiviral action (Croen, 1993
; Karupiah et al., 1993
), and previous studies by our group have revealed a pivotal role of IFN-
in controlling both NO production in vitro and virus clearance after systemic infection with viscerotropic strains of LCMV. A consequence of the latter is severely exacerbated Tc-mediated immunopathology in IFN-
-/- mice. In fact, lack of IFN-
results in a fatal outcome of intravenous (i.v.) infection in the majority (~85%) of mice infected with LCMV Traub (unpublished results).
Therefore, in the present study, we have investigated the role of iNOS/NO in LCMV-induced meningitis, as well as in the T cell-mediated immune response to LCMV in general. This also included an analysis of LCMV-induced immune suppression, which is known to reflect an increased propensity of T lymphocytes to undergo apoptosis upon T-cell receptor (TCR) stimulation (activation-induced cell death; AICD) (Razvi & Welsh, 1993 ), a phenomenon that has recently been found to be dependent partly on IFN-
(Lohman & Welsh, 1998
).
By using mice lacking iNOS (Wei et al., 1995 ; MacMicking et al., 1995
; Laubach et al., 1995
), we found a minimal role for iNOS/NO in the host response to LCMV. Thus, besides a reduced local oedema observed in the knockout mice, iNOS seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.
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Methods |
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Virus.
LCMV of the Traub strain, produced and stored as described previously, was used in most experiments (Marker & Volkert, 1973 ). Mice to be infected received a dose of 103 LD 50 in an i.v. injection of 0·3 ml. Infection by this route is followed by transient immunizing infection (Marker & Volkert, 1973
; Thomsen & Marker, 1989
). LCMV of the Armstrong clone 53b strain was kindly provided by M. B. A. Oldstone (Scripps Clinic and Research Foundation, La Jolla, CA, USA) and used for i.c. infection. Mice were infected with 200 p.f.u. in an i.c. injection of 0·03 ml. This inoculation induces a fatal, T cell-mediated meningitis, to which the animals succumb days 68 p.i. (Campbell et al., 1994a
).
Virus titration.
Virus titrations were carried out by i.c. inoculation of 10-fold dilutions of a 10% organ suspension into young adult Swiss mice. Titration end-points were calculated by the Kärber method and expressed as LD50.
Severity of LCMV-induced meningitis.
Mortality and CSF cell numbers were used to evaluate the clinical severity of acute LCMV-induced meningitis (Doherty et al., 1990 ; Marker et al., 1995
). Mice were checked twice daily for a period of up to 14 days after i.c. inoculation.
Clinical severity of systemic LCMV infection.
Mice were monitored daily for a period of 4 weeks. Weight loss was measured as the percentage difference between initial weight and weight on the indicated day.
Assay of LCMV-specific delayed-type hypersensitivity (DTH).
Mice were primed by i.v. injection of LCMV Armstrong (200 p.f.u.) and, in addition, were inoculated in their right-hind footpad with 30 µl virus (200 p.f.u.). Footpad thickness was measured with a dial calliper (Mitutoyo 7309) daily from day 6 p.i. until termination of the experiment and virus-specific swelling was determined as the difference in thickness of the infected right and the uninfected left feet (Thomsen & Marker, 1989 ). LCMV- specific DTH was also evaluated by footpad challenge 8 days after an i.v. infection with an immunodominant class I-restricted viral peptide (LCMV GP 3341) (Nansen et al., 1998
). Mice were injected with 30 µl of a solution containing 50 µg/ml peptide.
Cell preparations.
Spleen single-cell suspensions were obtained by pressing the organ through a fine steel mesh and erythrocytes were lysed by treatment with 0·83% NH4Cl (Gey's solution). CSF cells were obtained from the fourth ventricle of mice that had been ether- anaesthetized and exsanguinated; background levels in uninfected mice are <100 cells/µl (Marker et al., 1995 ).
Cytotoxicity assays.
Virus-specific Tc cell activity was assayed in a standard 51 Cr-release assay by using EL-4 cells incubated for 1 h at 37 °C with either of two different immunodominant class I- restricted viral peptides (LCMV GP 3341 and NP 396404); EL-4 cells incubated without peptide served as control targets. NK cell activity was assayed in a standard 51Cr-release assay by using NK-sensitive YAC-1 cells; spleen cells from uninfected mice were used as controls (Thomsen & Marker, 1989 ). The time of assay was 4 and 5 h for NK cell activity and Tc cell activity, respectively, and percentage specific release was calculated as described previously (Marker & Volkert, 1973
).
T cell proliferation.
Splenocytes from individual mice were plated at 2x105 , 1x105 and 0·5x105 cells per well in 96-well flat-bottomed microtitre plates. IL-2 responsiveness was evaluated as proliferation after 48 h incubation in the presence of 1000 IU/ml murine recombinant IL-2 (R&D Systems) (Andersson et al., 1995 ), anti- CD3 responsiveness as proliferation after 48 h incubation in the presence of 1 µg/ml anti-CD3 (PharMingen) and ConA responsiveness as proliferation after 72 h incubation in the presence of 2·5 µg/ml ConA (Pharmacia). Cultures were labelled by adding 1 µCi [3H]TdR per well (specific activity 2 Ci/mmol) during the last 6 h of incubation.
MAb for flow cytometry.
The following MAbs were purchased from PharMingen as rat anti-mouse antibodies: FITC-conjugated anti-CD49d (common -chain of LPAM-1 and VLA-4), PE-conjugated anti-CD8a, biotin-conjugated anti-CD62L [L- selectin (L-sel), MEL-14], PE-conjugated anti-IL-5 and PE-conjugated anti-IFN-
.
Flow cytometric analysis.
Cells (1x106) were stained with directly labelled MAb in staining buffer (1% rat serum, 1% BSA, 0·1% NaN3 in PBS) for 20 min in the dark at 4 °C and then washed. Biotin-conjugated antibodies were additionally incubated with streptavidinTri-color (Caltag Laboratories), washed and fixed with 1% paraformaldehyde (Christensen et al., 1996 ). To detect intracellular IFN-
, splenocytes were cultured at 37 °C in 96-well round-bottomed plates at a concentration of 1x106 cells per well in 0·2 ml complete RPMI medium supplemented with 10 U per well murine recombinant IL- 2 (R&D Systems) and 3 µM monensin (Sigma), either with or without 0·1 µg/ml LCMV GP 3341 peptide. After 5 h incubation, cells were washed once in staining buffer (1% BSA, 0·1% NaN3 and 3 µM monensin) and subsequently incubated in the dark for 30 min at 4 °C with relevant antibodies to cell-surface antigens. Cells were washed twice in staining buffer and resuspended in the dark for 20 min at 4 °C in fixation buffer (1% paraformaldehyde in PBS). Cells were then washed in staining buffer, resuspended in permeabilization buffer (1% rat serum, 1% BSA, 0·1% NaN3 and 0·5% saponin in PBS) and incubated for 30 min at 4 °C in the dark with the relevant cytokine-specific antibodies (Christensen et al., 1996
). Finally, cells were washed in permeabilization buffer and resuspended in staining buffer.
Samples were analysed with a Becton Dickinson FACSCalibur, and 12x104 viable mononuclear cells were gated by using a combination of forward angle and side scatter to exclude dead cells and debris. Data analysis was carried out using the PC-LYSIS program and results are presented as dot plots.
Detection of cytokine and chemokine gene expression by RNase protection assays.
Mice were exsanguinated under ether anaesthesia and brains were removed and immediately frozen in liquid nitrogen. Total RNA was isolated and gene expression was evaluated by RNase protection assays (Campbell et al., 1994a ). Total RNA was extracted from homogenized organs by use of the RNeasy Midi kit (Qiagen). Detection of cytokine and chemokine mRNA was done by using the following kits obtained from PharMingen: RiboQuant in vitro Transcription kit, mouse Multi-Probe Template sets mCK-3 and mCK-5 and the RiboQuant ribonuclease protection assay kit. All analyses were carried out according to manufacturer's instructions.
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Results |
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Discussion |
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We analysed the role of NO during LCMV infection by using mice deficient in iNOS (Laubach et al., 1995 ), and compared the experimental results with those of wild-type mice. We found that LCMV-induced NK cell activation was comparable in iNOS -/- and wild-type mice, and FACS analysis revealed equal numbers of NK cells 4 days after i.v. infection with LCMV. Thus, in contrast to the situation in leishmania-infected mice (Diefenbach et al., 1998
), iNOS-derived NO appears not to be involved in the up-regulation of NK cell activity following virus infection (similar results were obtained in mice infected with vesicular stomatitis virus; unpublished observations). Regarding the generation of effector T cells, our results clearly demonstrate that the generation of LCMV-specific Tc1 cells is unaffected by the absence of iNOS. No difference was observed in either clonal expansion or effector cell differentiation, as evaluated both functionally and by flow cytometry, and similar results were obtained by flow-cytometric evaluation of Tc1 cells induced by i.v. infection with vesicular stomatitis virus (data not shown). Thus, NO does not seem to exert the same marked feedback inhibition on Tc1 cells as has recently found for antiviral Th1 cells in influenza virus-and HSV-1-infected mice (Karupiah et al., 1998b
; MacLean et al., 1998
).
We found no involvement of iNOS in the immunodeficiency induced by systemic LCMV infection, despite the recent finding that IFN- plays an important role (Lohman & Welsh, 1998
). Thus, our findings confirm and extend previous results obtain by treatment with N-monomethyl l-arginine (Butz et al., 1994
).
Analysis in vivo revealed identical kinetics of virus clearance (which is an indirect measure of Tc1 effector capacity; Kagi et al., 1994 ), and no clinical signs of illness, measured in terms of weight loss and survival rate, were observed in either strain. Since impaired virus clearance and severe wasting is observed in similarly infected IFN-
-/- mice, it may be concluded that the protective effect of IFN-
is not mediated through the up-regulation of iNOS.
Quantitative and qualitative analyses of CSF exudate cells indicated that cell migration into the inflammatory site can proceed to the same extent in the absence of NO; this is in keeping with analogous findings in IFN--/- mice, in which up-regulation of iNOS cannot be detected (Nansen et al., 1998
). Likewise, the up-regulation of proinflammatory cytokine and chemokine genes in the brain after i.c. infection with LCMV seems to be regulated independently of NO. This result may seem to conflict with data from mice treated with aminoguanidine (Campbell, 1996
), a NOS inhibitor with relative selectivity for iNOS, which indicated a role for NO in the up-regulation of TNF-
, IL-1
and IL-1ß in the brain 3 days after i.c. LCMV infection. Furthermore, time to death was slightly shortened in mice treated in this way (Campbell, 1996
). In the same study, however, ruffled fur and diarrhoea were noted in drug-treated control animals, suggesting that toxic levels of aminoguanidine had been administered. Therefore, our results with iNOS-/- mice probably provide a more valid indication of the role of iNOS in LCMV-induced meningitis.
By using another model system of LCMV-induced, T cell-mediated inflammation, virus-induced footpad swelling, we found a significant role for NO. Thus, footpad swelling was significantly reduced in iNOS -/- mice compared with both IFN- -/- and wild-type mice. This agrees well with the results of a study on the role of NO in contact hypersensitivity reactions, which suggested that epidermal cell-derived NO contributed to the ear-swelling reaction (Ross et al., 1998
). As the swelling reaction is more a measure of the induced oedema than of cellular infiltration per se, and assuming that our findings on cell infiltration and expression of cytokine/chemokine genes can be extrapolated to apply also to the footpad, the reduced reaction in iNOS -/- mice would suggest that NO may be involved in regulating secondary effects of the inflammatory response; vascular reactivity is a strong possibility (Wei et al., 1995
; Peng et al., 1998
). The apparent redundancy of IFN-
in the swelling reaction (Nansen et al. , 1998
) is puzzling if NO indeed plays a role. However, this could be the result of a compensatory phenomenon. Thus, we find a higher antigenic load and more extensive virus dissemination in IFN-
-/- mice (Nansen et al., 1998
), which may accelerate and augment the specific T cell response in these mice compared with matched wild-type mice. Indeed, we have previously found some indications that this might be the case (Nansen et al., 1998
), and the observed redundancy of IFN-
in the swelling reaction should therefore be interpreted with some care.
In summary, this study discloses a minimal role for iNOS/NO in LCMV- induced, T cell-mediated protective immunity and immunopathology. Our results indicate that NO plays a role in regulating the magnitude of the inflammatory reaction but is otherwise redundant in LCMV infection.
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Acknowledgments |
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References |
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Akaike, T. , Noguchi, Y. , Ijiri, S. , Setoguchi, K. , Suga, M. , Zheng, Y. M. , Dietzschold, B. & Maeda, H. (1996). Pathogenesis of influenza virus- induced pneumonia: involvement of both nitric oxide and oxygen radicals. Proceedings of the National Academy of Sciences, USA 93, 2448-2453 .
Albina, J. E. , Abate, J. A. & Henry, W. L.Jr (1991). Nitric oxide production is required for murine resident peritoneal macrophages to suppress mitogen-stimulated T cell proliferation. Role of IFN-gamma in the induction of the nitric oxide-synthesizing pathway. Journal of Immunology 147, 144-148.
Andersson, E. C. , Christensen, J. P. , Scheynius, A. , Marker, O. & Thomsen, A. R. (1995). Lymphocytic choriomeningitis virus infection is associated with long-standing perturbation of LFA-1 expression on CD8+ T cells. Scandinavian Journal of Immunology 42, 110-118.[Medline]
Bogdan, C. (1997). Of microbes, macrophages and nitric oxide. Behring Institute Mitteilungen, 5872.
Bogdan, C. (1998). The multiplex function of nitric oxide in (auto)immunity. Journal of Experimental Medicine 187, 1361-1365 .
Butz, E. A. , Hostager, B. S. & Southern, P. J. (1994). Macrophages in mice acutely infected with lymphocytic choriomeningitis virus are primed for nitric oxide synthesis. Microbial Pathogenesis 16, 283-295.[Medline]
Campbell, I. L. (1996). Exacerbation of lymphocytic choriomeningitis in mice treated with the inducible nitric oxide synthase inhibitor aminoguanidine. Journal of Neuroimmunology 71, 31-36.[Medline]
Campbell, I. L. , Hobbs, M. V. , Kemper, P. & Oldstone, M. B. (1994a). Cerebral expression of multiple cytokine genes in mice with lymphocytic choriomeningitis. Journal of Immunology 152, 716 -723.
Campbell, I. L. , Samimi, A. & Chiang, C. S. (1994b). Expression of the inducible nitric oxide synthase. Correlation with neuropathology and clinical features in mice with lymphocytic choriomeningitis. Journal of Immunology 153, 3622 -3629.
Christensen, J. P. , Stenvang, J. P. , Marker, O. & Thomsen, A. R. (1996). Characterization of virus-primed CD8+ T cells with a type 1 cytokine profile. International Immunology 8, 1453-1461.[Abstract]
Corbett, J. A. , Mikhael, A. , Shimizu, J. , Frederick, K. , Misko, T. P. , McDaniel, M. L. , Kanagawa, O. & Unanue, E. R. (1993). Nitric oxide production in islets from nonobese diabetic mice: aminoguanidine- sensitive and -resistant stages in the immunological diabetic process. Proceedings of the National Academy of Sciences, USA 90, 8992-8995 .[Abstract]
Croen, K. D. (1993). Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. Journal of Clinical Investigation 91, 2446-2452 .[Medline]
Diefenbach, A. , Schindler, H. , Donhauser, N. , Lorenz, E. , Laskay, T. M. , MacMicking, J. , Rollinghoff, M. , Gresser, I. & Bogdan, C. (1998). Type 1 interferon (IFN/ß) and type 2 nitric oxide synthase regulate the innate immune response to a protozoan parasite. Immunity 8, 77-87.[Medline]
Doherty, P. C. , Allan, J. E. , Lynch, F. & Ceredig, R. (1990). Dissection of an inflammatory process induced by CD8+ T cells. Immunology Today 11, 55-59.[Medline]
Fang, F. C. (1997). Perspectives series: host/pathogen interactions. Mechanisms of nitric oxide-related antimicrobial activity. Journal of Clinical Investigation 99, 2818-2825 .
Fehsel, K. , Kroncke, K. D. , Meyer, K. L. , Huber, H. , Wahn, V. & Kolb-Bachofen, V. (1995). Nitric oxide induces apoptosis in mouse thymocytes. Journal of Immunology 155, 2858-2865 .[Abstract]
Frei, K. , Leist, T. P. , Meager, A. , Gallo, P. , Leppert, D. , Zinkernagel, R. M. & Fontana, A. (1988). Production of B cell stimulatory factor-2 and interferon gamma in the central nervous system during viral meningitis and encephalitis. Evaluation in a murine model infection and in patients. Journal of Experimental Medicine 168, 449-453.[Abstract]
Green, I. C. , Cunningham, J. M. , Delaney, C. A. , Elphick, M. R. , Mabley, J. G. & Green, M. H. (1994). Effects of cytokines and nitric oxide donors on insulin secretion, cyclic GMP and DNA damage: relation to nitric oxide production. Biochemical Society Transactions 22, 30-37.[Medline]
Hoffman, R. A. , Langrehr, J. M. , Billiar, T. R. , Curran, R. D. & Simmons, R. L. (1990). Alloantigen-induced activation of rat splenocytes is regulated by the oxidative metabolism of l-arginine. Journal of Immunology 145, 2220-2226 .
Iadecola, C. , Zhang, F. & Xu, X. (1995). Inhibition of inducible nitric oxide synthase ameliorates cerebral ischemic damage. American Journal of Physiology 268, R286-R292 .
Iadecola, C. , Zhang, F. , Casey, R. , Nagayama, M. & Ross, M. E. (1997). Delayed reduction of ischemic brain injury and neurological deficits in mice lacking the inducible nitric oxide synthase gene. Journal of Neuroscience 17, 9157-9164 .
Kagi, D. , Ledermann, B. , Burki, K. , Seiler, P. , Odermatt, B. , Olsen, K. J. , Podack, E. R. , Zinkernagel, R. M. & Hengartner, H. (1994). Cytotoxicity mediated by T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 369, 31-37.[Medline]
Karupiah, G. , Xie, Q. W. , Buller, R. M. , Nathan, C. , Duarte, C. & MacMicking, J. D. (1993). Inhibition of viral replication by interferon-gamma-induced nitric oxide synthase. Science 261, 1445-1448 .[Medline]
Karupiah, G. , Chen, J. H. , Nathan, C. F. , Mahalingam, S. & MacMicking, J. D. (1998a). Identification of nitric oxide synthase 2 as an innate resistance locus against ectromelia virus infection. Journal of Virology 72, 7703 -7706.
Karupiah, G. , Chen, J. H. , Mahalingam, S. , Nathan, C. F. & MacMicking, J. D. (1998b). Rapid interferon gamma-dependent clearance of influenza A virus and protection from consolidating pneumonitis in nitric oxide synthase 2-deficient mice. Journal of Experimental Medicine 188, 1541 -1546.
Kolb, H. & Kolb-Bachofen, V. (1998). Nitric oxide in autoimmune disease: cytotoxic or regulatory mediator? Immunology Today 19, 556-561.[Medline]
Laubach, V. E. , Shesely, E. G. , Smithies, O. & Sherman, P. A. (1995). Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide- induced death. Proceedings of the National Academy of Sciences, USA 92, 10688-10692 .[Abstract]
Lohman, B. L. & Welsh, R. M. (1998). Apoptotic regulation of T cells and absence of immune deficiency in virus-infected gamma interferon receptor knockout mice. Journal of Virology 72, 7815-7821 .
MacLean, A. , Wei, X.-Q. , Huang, F.-P. , Al-Alem, U. A. H. , Chan, W. L. & Liew, F. Y. (1998). Mice lacking inducible nitric-oxide synthase are more susceptible to herpes simplex virus infection despite enhanced Th1 cell responses. Journal of General Virology 79, 825-830.[Abstract]
MacMicking, J. D., Nathan, C., Hom, G., Chartrain, N., Fletcher, D. S., Trumbauer, M., Stevens, K., Xie, Q. W., Sokol, K., Hutchinson, N. and others (1995). Altered responses to bacterial infection and endotoxic shock in mice lacking inducible nitric oxide synthase. Cell 81, 641650.[Medline]
MacMicking, J. , Xie, Q. W. & Nathan, C. (1997). Nitric oxide and macrophage function. Annual Review of Immunology 15, 323-350.[Medline]
Marker, O. & Volkert, M. (1973). Studies on cell-mediated immunity to lymphocytic choriomeningitis virus in mice. Journal of Experimental Medicine 137, 1511-1525 .[Medline]
Marker, O. , Scheynius, A. , Christensen, J. P. & Thomsen, A. R. (1995). Virus-activated T cells regulate expression of adhesion molecules on endothelial cells in sites of infection. Journal of Neuroimmunology 62, 35-42.[Medline]
Nansen, A. , Christensen, J. P. , Ropke, C. , Marker, O. , Scheynius, A. & Thomsen, A. R. (1998). Role of interferon- gamma in the pathogenesis of LCMV-induced meningitis: unimpaired leucocyte recruitment, but deficient macrophage activation in interferon-gamma knock-out mice. Journal of Neuroimmunology 86, 202-212.[Medline]
Okuda, Y. , Sakoda, S. , Shimaoka, M. & Yanagihara, T. (1996). Nitric oxide induces apoptosis in mouse splenic T lymphocytes. Immunology Letters 52, 135-138.[Medline]
Peng, H. B. , Spiecker, M. & Liao, J. K. (1998). Inducible nitric oxide: an autoregulatory feedback inhibitor of vascular inflammation. Journal of Immunology 161, 1970-1976 .
Razvi, E. S. & Welsh, R. M. (1993). Programmed cell death of T lymphocytes during acute viral infection: a mechanism for virus- induced immune deficiency. Journal of Virology 67, 5754-5765 .[Abstract]
Ross, R. , Gillitzer, C. , Kleinz, R. , Schwing, J. , Kleinert, H. , Forstermann, U. & Reske-Kunz, A. B. (1998). Involvement of NO in contact hypersensitivity. International Immunology 10, 61-69.[Abstract]
Taylor-Robinson, A. W. , Liew, F. Y. , Severn, A. , Xu, D. , McSorley, S. J. , Garside, P. , Padron, J. & Phillips, R. S. (1994). Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells. European Journal of Immunology 24, 980-984.[Medline]
Thomsen, A. R. & Marker, O. (1989). MHC and non-MHC genes regulate elimination of lymphocytic choriomeningitis virus and antiviral cytotoxic T lymphocyte and delayed-type hypersensitivity mediating T lymphocyte activity in parallel. Journal of Immunology 142, 1333-1341 .
Wei, X. Q. , Charles, I. G. , Smith, A. , Ure, J. , Feng, G. J. , Huang, F. P. , Xu, D. , Muller, W. , Moncada, S. & Liew, F. Y. (1995). Altered immune responses in mice lacking inducible nitric oxide synthase. Nature 375, 408-411.[Medline]
Xie, K. , Dong, Z. & Fidler, I. J. (1996). Activation of nitric oxide synthase gene for inhibition of cancer metastasis. Journal of Leukocyte Biology 59, 797-803.[Abstract]
Received 6 May 1999;
accepted 30 June 1999.