T cell expansion is regulated by activated Gr-1+ splenocytes.

CD4+ T cell proliferation depends on the balance between NO and extra-cellular superoxide (O2-). By reducing NO bio-availability, O2- promotes splenic T cell proliferation and immune response intensity. Here, we show that spleen cells from naïve mice produced neither NO nor O2- during T cell activation, but Gr-1+ splenocytes from primed mice regulated Ag-specific T cell expansion via production of both molecules. Purified splenic Gr-1+ cells included mostly granulocytes at various stages of maturation, as well as monocytes. Activation or recruitment of regulatory Gr-1+ cells was dependent on immunization with CFA. Importantly, these regulatory cells were not detected in draining lymph nodes. These data suggest that innate Gr-1+ splenic cells regulate adaptive immunity.

Superoxide prevents nitric oxide-mediated suppression of helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide adenine dinucleotide phosphate oxidase knockout mice.

NO, which suppresses T cell proliferation, may be inactivated by superoxide (O2-) due to their strong mutual affinity. To examine this possibility, preactivated Th clones were cocultured with stimulated macrophages. PMA neutralized the inhibitory activity of NO, which was dependent on extracellular O2- production. In contrast, macrophages from p47phox -/- (pKO) mice, which lack functional NADPH oxidase, retained their NO-dependent inhibition of T cell proliferation upon stimulation with PMA, indicating that NADPH oxidase is the major source of NO-inactivating O2- in this system. To examine the NO-O2- interaction in vivo, the role of NADPH oxidase in experimental autoimmune encephalomyelitis was studied in pKO mice. No clinical or histological signs were observed in the pKO mice. Neither a bias in Th subsets nor a reduced intensity of T cell responses could account for the disease resistance. Although spleen cells from pKO mice proliferated poorly in response to the immunogen, inhibition of NO synthase uncovered a normal proliferative response. These results indicate that NO activity may play a critical role in T cell responses in pKO mice and that in normal spleens inhibition of T cell proliferation by NO may be prevented by simultaneous NADPH oxidase activity.

Macrophage-derived nitric oxide inhibits the proliferation of activated T helper cells and is induced during antigenic stimulation of resting T cells.

To examine how macrophage-derived nitric oxide (NO) affects T helper (Th) cell activity, T cell clones representing Th1 and Th2 subsets were activated before exposure to stimulated peritoneal macrophages or microglia. Both Th subsets were similarly sensitive to inhibition by NO, indicating that macrophage-derived NO regulates the proliferation of activated Th1 and Th2 cells equally well. Since IFN-gamma production remained intact in NO-treated Th1 cells, we studied whether NO was produced during antigen-specific activation of Th1 cells by unstimulated macrophages. Indeed, T cell proliferation only occurred when a NO synthase inhibitor was included, while IFN-gamma was essential for the induction of NO. These studies demonstrate that macrophages produce NO following antigen presentation to Th1 cells and that macrophage-derived NO inhibits Th1 and Th2 cell proliferation without inhibiting cytokine production.

Antigen presentation to Th1 but not Th2 cells by macrophages results in nitric oxide production and inhibition of T cell proliferation: interferon-gamma is essential but insufficient.

The induction and role of nitric oxide (NO) during antigen presentation by macrophages to T helper (Th) cell subsets was examined. When cultured with Th1 clones, macrophage APC produced NO only in the presence of cognate Ag, which in turn suppressed T cell proliferation. IFN-gamma production by the activated Th1 cells was essential for the induction of NO. Th2 cells presented with the same cognate Ag did not induce NO production and proliferated uninhibited. Coactivation of Th1 and Th2 cells specific for the same Ag indicated that Th2 cells did not inhibit NO production, but were sensitive to NO induced by stimulated Th1 cells. Antigenic activation of Th2 cells in the presence of rIFN-gamma resulted in NO-mediated inhibition of proliferation. Th2 cells provided only a cell-associated cofactor, whereas Th1 cells secreted a soluble cofactor for IFN-gamma as well, i.e., TNF-alpha. Finally, a role for IFN-gamma and NO during immune responses was studied in spleen cells obtained from immunized IFN-gamma(-/-) mice. NO production and subsequent inhibition of Ag-specific proliferation ex vivo was observed only after the addition of rIFN-gamma. These studies suggest an IFN-gamma-dependent regulatory role for NO during Ag-specific Th cell activation involving macrophages, with obvious implications for Th subset-dependent immune responses in general.

Nitric oxide inhibits the proliferation of T-helper 1 and 2 lymphocytes without reduction in cytokine secretion.

To study the effect of nitric oxide (NO) on the activity of Th subsets, cloned Th1 and Th2 lymphocytes were stimulated in the presence of an NO donor. NO, when present from the start of incubation, inhibited the proliferation of both Th subsets dose-dependently, achieving complete inhibition at a relatively low level. The addition of NO 24 h after the onset of T cell stimulation also resulted in reduced proliferation of both Th subsets, suggesting that NO affects a late process during T cell activation. Stimulation of T cells in the presence of NO did not induce apoptosis at the concentrations that completely inhibited proliferation, although apoptosis became evident at higher NO concentrations. The secretion of several cytokines (i.e., IFN-gamma, IL-4, and IL-5) was slightly upregulated, while IL-2 production was modestly inhibited in the presence of NO. However, exogenous IL-2 did not reverse the NO-induced inhibition of T cell proliferation, nor did additional stimulation with phorbol esters. Finally, expression of IL-2R was modestly decreased in the presence of NO, although TCR expression was not affected. These studies demonstrate that relatively low concentrations of NO induce a strong and specific inhibition of T cell proliferation in both Th subsets, suggesting that local NO production may regulate Th-mediated tissue inflammation.

Mouse hepatitis virus nucleocapsid protein as a translational effector of viral mRNAs.

The mouse hepatitis virus (MHV) nucleocapsid protein stimulated translation of a chimeric reporter mRNA containing an intact MHV 5'-untranslated region and the chloramphenicol acetyltransferase (CAT) coding region. The nucleocapsid protein binds specifically the tandemly repeated-UCYAA- of the MHV leader. This RNA sequence is the same as the intergenic motif found in the genome RNA. Preferential translation of viral mRNA in MHV infected cells is stimulated in part by this interaction and represents a specific, positive translational control mechanism employed by coronaviruses.

Coronavirus translational regulation: leader affects mRNA efficiency.

Cells infected with the murine coronavirus, mouse hepatitis virus (MHV), show decreased host protein synthesis concomitant with an increase in viral protein synthesis. We examined the in vitro translation property of the conserved MHV 5'-leader RNA sequence by constructing chimeric mRNAs in which the 72-nt 5'-leader of M protein mRNA (A59 strain) was positioned upstream of the human alpha-globin coding region in a T7 expression vector. Synthetic 5'-capped transcripts of these mRNA constructs were translated in cell-free extracts prepared from uninfected and MHV-infected murine DBT cells. Nonviral mRNAs translated readily in both uninfected and infected cell-free extracts. By contrast, replacement of the human alpha-globin 5'-untranslated region (UR) with the MHV 5'-leader increased translation ca. three- to fourfold in cell-free extracts from MHV-infected cells versus translation in extracts from uninfected cells. Chimeric globin mRNA containing the reverse complementary sequence of the viral leader RNA in the 5'-UR showed no such increase in translation, indicating sequence specificity for the effect. A 13-nt region (-UCUAAUCCAAACA-) immediately proximal to the start codon was found to be important for the increased translation of the MHV leader-containing mRNAs. These data indicate that the apparent down-regulation of host translation is not primarily due to an inhibition of host translation but also involves a significant stimulation of viral translation in cis by a structural feature of the MHV 5'-leader RNA sequence in conjunction with a virus-specified or virus-induced factor.

Effect of eukaryotic initiation factor 4F on AUG selection in a bicistronic mRNA.

Artificial bicistronic mRNAs based on rabbit beta-globin and bacterial chloramphenicol acetyltransferase protein-coding sequences were tested for translation activity in a mouse astrocytoma cell-free extract. This cell extract exhibited an apparent preference for 5'-distal or internal initiation over 5'-proximal ("first AUG") initiation. 5'-Distal initiation appeared to be 5'-cap independent, suggesting that nonstandard initiation was responsible. This conclusion was based on a lack of inhibition of internal initiation by added cap analog and insensitivity of internal initiation to the presence or absence of a 5'-cap structure. Exogenous reticulocyte initiation factors were tested for effect on 5'-proximal initiation. The only factor with a significant effect was found to be eukaryotic initiation factor 4F, or the cap-binding protein. Addition of this factor promoted 5'-end initiation as evident by a general increase in 5'-proximal open reading frame (ORF) product relative to 5'-distal ORF product. The relative expression of 5'-proximal to 5'-distal ORFs in bicistronic or multicistronic mRNAs may very well be dependent on activity levels of eukaryotic initiation factor 4F and possibly other mRNA-dependent initiation factors.