Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.

Previous studies with inhibitors of inducible nitric oxide synthase (iNOS) suggested that high-output production of nitric oxide (NO) is an important antimicrobial effector pathway in vitro and in vivo. Here, we investigated the tissue expression of iNOS in mice after infection with Leishmania major. Immunohistochemical staining with an iNOS-specific antiserum revealed that in the cutaneous lesion and draining lymph nodes (LN) of clinically resistant mice (C57BL/6), iNOS protein is found earlier during infection and in significantly higher amounts than in the nonhealing BALB/c strain. Similar differences were seen on the mRNA level as quantitated by competitive polymerase chain reaction. Anti-CD4 treatment of BALB/c mice not only induced resistance to disease, but also restored the expression of iNOS in the tissue. In situ, few or no parasites were found in those regions of the skin lesion and the draining LN which were highly positive for iNOS. By double labeling experiments, macrophages were identified as iNOS expressing cells in vivo. In the lesions of BALB/c mice, cells staining positively for transforming growth factor beta (TGF-beta), a potent inhibitor of iNOS in vitro, were strikingly more prominent than in C57BL/6, whereas no such difference was found for interleukin 4 or interferon gamma (IFN-gamma). In vitro, production of NO was approximately threefold higher in C57BL/6 than in BALB/c macrophages after stimulation with IFN-gamma. We conclude that the pronounced expression of iNOS in resistant mice is an important mechanism for the elimination of Leishmania in vivo. The relative lack of iNOS in susceptible mice might be a consequence of macrophage deactivation by TGF-beta and reduced responsiveness to IFN-gamma.

Reactivation of latent leishmaniasis by inhibition of inducible nitric oxide synthase.

Nitric oxide (NO) synthase (iNOS) is required for the resolution of acute cutaneous leishmaniasis in resistant C57BL/6 mice. As is the case in several other infections, the clinically cured host organism still harbors small amounts of live Leishmania major parasites. Here, we demonstrate lifelong expression of iNOS at the site of the original skin lesion and in the draining lymph node of long-term-infected C57BL/6 mice. iNOS activity in the lymph node was dependent on CD4+, but not on the CD8+ T cells. By double labeling techniques, iNOS and L. major were each found in macrophages (F4/80+, BM-8+, and/or MOMA-2+) and dendritic cells (NLDC-145+), but not in granulocytes or endothelial cells. In situ triple labeling of lymph node sections revealed that approximately 30-40% of the L. major foci were associated with iNOS-positive macrophages or dendritic cells. The majority of the L. major foci (60-70%), however, was located in areas that were negative for both iNOS and the macrophage and dendritic cell markers. In L. major-infected C57BL/6 mice, which had cured their cutaneous lesions, administration of L-N6-iminoethyl-lysine (L-NIL), a potent inhibitor of iNOS, led to a 10(4)-10(5)-fold increase of the parasite burden in the cutaneous and lymphoid tissue and caused clinical recrudescence of the disease. Persistent expression of iNOS and resumption of parasite replication after application of L-NIL was also observed in resistant C3H/HeN and CBA/J mice. We conclude that iNOS activity is crucial for the control of Leishmania persisting in immunocompetent hosts after resolution of the primary infection. Failure to maintain iNOS activity might be the mechanism underlying endogenous reactivation of latent infections with NO-sensitive microbes during phases of immunosuppression.

L-N6-(1-iminoethyl)-lysine potently inhibits inducible nitric oxide synthase and is superior to NG-monomethyl-arginine in vitro and in vivo.

L-N6-(1-iminoethyl)-lysine is a novel inhibitor of nitric oxide (NO) synthase, which similar to aminoguanidine but unlike NG-monomethyl-L-arginine is 30-fold more selective for the inducible than for the constitutive isoform of the enzyme. Here, we characterized this inhibitor for the first time in intact cells and during infection of mice with a NO-sensitive parasite (Leishmania major). L-N6-(1-iminoethyl)-lysine potently inhibited the activity of inducible NO-synthase in primary macrophages. After stimulation by interferon-gamma the IC50 of L-N6-(1-iminoethyl)-lysine was 0.4 +/- 0.1 mu M and 10- or 30-fold lower than that of NG-monomethyl-L-arginine or aminoguanidine, respectively. In vivo, L-N6-(1-iminoethyl)-lysine (0.4-9 mM in the drinking water) suppressed inducible NO-synthase activity and caused a dramatic exacerbation of leishmaniasis, despite a counterregulatory increase of inducible NO-synthase protein in the tissue. In contrast, considerably higher concentrations of NG-monomethyl-L-arginine (20-50 mM) were required in order to achieve comparable effects. NG-monomethyl-L-arginine, but not L-N6-(1-imino-ethyl)-lysine led to weight loss, reduced water and food consumption. We conclude that L-N6-(1-iminoethyl)-lysine should be used instead of NG-monomethyl-L-arginine for potent suppression of inducible NO-synthase in vitro and in vivo.

Backbone dynamics of ribonuclease T1 and its complex with 2'GMP studied by two-dimensional heteronuclear NMR spectroscopy.

The backbone dynamics of free ribonuclease T1 and its complex with the competitive inhibitor 2'GMP have been studied by (15)N longitudinal and transverse relaxation experiments, combined with {(1)H, (15)H} NOE measurements. The intensity decay of individual amide cross peaks in a series of ((1)H, (15)N)-HSQC spectra with appropriate relaxation periods (Kay, L.E. et al. (1989) Biochemistry, 28, 8972-8979; Kay, L.E. et al. (1992) J. Magn. Reson., 97, 359-375) was fitted to a single exponential by using a simplex algorithm in order to obtain (15)N T(1) and T(2) relaxation times. These experimentally obtained values were analysed in terms of the 'model-free' approach introduced by Lipari and Szabo (Lipari, G. and Szabo, A. (1982) J. Am. Chem. Soc., 104, 4546-4559; 4559-4570). The microdyramical parameters accessible by this approach clearly indicate a correlation between the structural flexibility and the tertiary structure of ribonuclease T1, as well as restricted mobility of certain regions of the protein backbone upon binding of the inhibitor. The results obtained by NMR are compared to X-ray crystallographic data and to observations made in molecular dynamics simulations.

Three-dimensional solution structure of human angiogenin determined by 1H,15N-NMR spectroscopy--characterization of histidine protonation states and pKa values.

Human angiogenin is a member of the pancreatic ribonuclease superfamily that induces blood vessel formation. Its three-dimensional solution structure has been determined to high resolution by heteronuclear NMR spectroscopy. 30 structures were calculated, based on a total of 1441 assigned NOE correlations, 64 coupling constants and 50 hydrogen bonds. The backbone atomic rms difference from the mean coordinates is 0.067 +/- 0.012 nm and 0.13 nm from the previously determined crystal structure. The side-chain of Gln117 was found to obstruct the active site as observed in the crystal state. There was no evidence of an alternative open form of angiogenin, although two sets of chemical shifts were observed for some residues, mainly around the active site and in the C-terminal segment. The topology of the ribonucleolytic active site is described with a particular emphasis on the conformation and protonation of active-site His residues. The side-chain of His114 adopts two main conformations in solution. In contrast to pancreatic ribonuclease A, His13 was shown to be more basic than His114, with pKa values of 6.65 and 6.05 respectively. The His47 residue is located in an environment very resistant to protonation with a pKa lower than 4.

Two-dimensional 1H, 15N-NMR investigation of uniformly 15N-labeled ribonuclease T1. Complete assignment of 15N resonances.

Uniformly 15N-enriched ribonuclease T1 (RNase T1) was obtained from Escherichia coli by recombinant techniques. Heteronuclear 1H, 15N-shift correlation spectra were recorded utilizing proton detection. Direct 1H, 15N connectivities were established applying the heteronuclear multiple-quantum coherence technique. Additional 1H, 1H-TOCSY or 1H, 1H-NOESY transfer steps allowed for sequential assignments. Nitrogen atoms without directly bonded protons were detected by means of the heteronuclear multiple-bond correlation experiment. Signals emerging from 15NH and 15NH2 groups were distinguished by heteronuclear triple-quantum filtering methods. 119 nitrogen resonances out of the expected 127 were assigned unambiguously; in addition, previously obtained proton assignments were extended. Preliminary 1H, 15N NMR investigation were performed on the RNase-T1-3'GMP inhibitor complex. Results were interpreted with respect to nucleotide binding.

Lack of inducible nitric oxide synthase activity in T cell clones and T lymphocytes from naive and Leishmania major-infected mice.

Nitric oxide (NO) generated by the inducible isoform of nitric oxide synthase (iNOS) is implicated in a number of immunological processes including killing of intracellular parasites, suppression of T cell proliferation, production of cytokines and destruction of tissue in autoimmune diseases. Considering that cytokine-activated mouse macrophages, fibroblasts and endothelial cells are potent producers of NO, we investigated whether T cells, as central participants in immune responses, can also be activated for the release of NO. Neither thymocytes nor type 1 or type 2 T helper cell clones generated significant amounts of nitrite (the stable end product of NO in culture supernatants) when stimulated by T cell mitogens, cytokines or antigen in the presence of irradiated antigen-presenting cells. Similarly, T cells freshly isolated from mice acutely infected with the intracellular pathogen Leishmania major did not produce NO upon restimulation in vitro. The lack of NO production was not due to the expression of enzymatically inactive iNOS, as we were unable to detect any iNOS protein in activated T helper clones or in freshly isolated T cells from infected mice by Western (protein) blot analysis. Finally, we tested whether iNOS expression in T cells might be restricted to a minor subpopulation and therefore only detectable on a single cell level. After immunofluorescence staining of lymph node or spleen cells from infected mice with antibodies against iNOS, F4/80- or Thy-1-antigen, macrophages, but no T cells, were found to express iNOS. Thus, we have no evidence that activated T helper cell clones or T cells from L. major-infected mice are high producers of NO.

Mechanism of suppression of macrophage nitric oxide release by IL-13: influence of the macrophage population.

IL-13 is a cytokine produced by T lymphocytes, mast cells, basophils, and certain B cell lines that up-regulates or inhibits various macrophage functions. In the present study we analyzed the mechanisms of suppression of nitric oxide (NO) release by IL-13 in the macrophage cell line J774A.1 and in thioglycolate-elicited mouse peritoneal macrophages. In both cell types efficient reduction (>80%) of NO production required treatment of the macrophages with IL-13 for at least 7 h before stimulation with IFN-gamma and LPS. In J774A.1 cells, increasing concentrations of IFN-gamma partially antagonized the suppression mediated by IL-13, whereas in peritoneal macrophages, the inhibitory effect of IL-13 was largely independent of the concentrations of IFN-gamma and LPS. In J774A.1 cells, IL-13 strongly reduced both the mRNA and protein levels of inducible nitric oxide synthase (iNOS, NOS-2), as determined by Northern blot analysis and immunoprecipitation. In peritoneal macrophages, in contrast, IL-13 decreased iNOS protein and enzyme activities after 8 to 48 h of stimulation, without altering the expression of iNOS mRNA. Pulse labeling with [35S]methionine revealed that IL-13 caused a 4.7-fold reduction of the de novo synthesis of iNOS protein in these cells. These data demonstrate for the first time that IL-13 is capable of regulating iNOS at both the mRNA and translational levels and underline the important influence of the macrophage population when studying mechanisms of cytokine functions.