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. 2002 Dec 10;99(25):16186-91.
doi: 10.1073/pnas.252464599. Epub 2002 Nov 25.

Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor beta 2 expression via cGMP

Wanda Niedbala  1 , Xiao-Qing Wei, Carol Campbell, Duncan Thomson, Mousa Komai-Koma, Foo Y Liew
Affiliations

Nitric oxide preferentially induces type 1 T cell differentiation by selectively up-regulating IL-12 receptor beta 2 expression via cGMP

Wanda Niedbala et al. Proc Natl Acad Sci U S A. .

Abstract

Nitric oxide plays an important role in immune regulation. We have shown that although high concentrations of NO generally were immune-suppressive, low concentrations of NO selectively enhanced the differentiation of T helper (Th)1 cells but not Th2 cells. This finding provided an explanation for the crucial role of NO in defense against intracellular pathogens. However, the mechanism for the selective induction of Th1 cells was unknown. We report here that at low concentrations, NO activates soluble guanylyl cyclase, leading to the up-regulation of cGMP, which selectively induces the expression of IL-12 receptor beta2 but has no effect on IL-4 receptor. Because IL-12 and IL-4 are the key cytokines for induction of Th1 and Th2 cells, respectively, these results, therefore, provide the mechanism for the selective action of NO on T cell subset differentiation. Furthermore, this selectivity also applies to CD8+ cytotoxic and human T cells and, thus, demonstrates the general implication of this observation in immune regulation. Our results also provide an example of the regulation of cytokine receptor expression by NO. The selectivity of such action via cGMP suggests that it is amenable to therapeutic intervention.

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Figures

Fig 1.
Fig 1.
Low concentrations of NO enhance differentiation of Tc1 cells and human Th1 cells. (a and b) CD8+ T cells were purified from pooled BALB/c spleen and lymph node cells and cultured with immobilized anti-CD3 antibodies and IL-12 + anti-IL-4 (Tc1), or IL-4 + anti-IL-12/anti-IFN-γ (Tc2), in the presence of graded concentrations of the NO donor (NOC-18) for 4 days. T cell proliferation was determined by [3H]thymidine incorporation and cytokine production by ELISA. (c) CD4+ T cells were purified from human cord blood and cultured with phytohemagglutinin and recombinant human IL-12 and anti-human IL-4 antibodies. IFN-γ concentrations in the culture supernatant were determined on day 4. Results, representative of three to four experiments, are mean ± SD, n = 4–5. *, P < 0.05 compared with culture without NO.
Fig 2.
Fig 2.
The enhancing effect of low concentrations of NO on type 1 cell differentiation results not from affecting apoptosis but via cGMP. (a) Tc1 cells were purified from BALB/c mice and cultured in the presence of graded concentrations of NO as described in the legend for Fig. 1. Apoptosis (annexin V+ and positive propidium iodide) were determined on day 4. Results are representative of three experiments. Similar results also were obtained with Th1 cells (not shown). (b) Negatively purified CD4+ T cells from BALB/c mice were polyclonally activated for 30 min with anti-CD3 under Th1 conditions in the presence of graded concentrations of NOC-18 and 3-isobutyl-1-methylxantine. Intracellular cGMP was extracted and assayed. (c) CD4+ T cells were negatively purified from pooled spleen and lymph nodes of OVA-TCRαβ transgenic mice (D0.11) and cultured with APC and OVA peptide under Th1 or Th2 conditions as described in Methods. NO had no effect on Th2 cells whose activation also was not affected by the presence of ODQ (data not shown). (d) CD4+ T cells from BALB/c mice were polyclonally activated with plate-bound anti-CD3 antibodies under Th1 conditions. NOC-18 and ODQ were added at the beginning of culture, and cellular proliferation and cytokine production were determined on day 4 of culture. For clarity, in c and d, only results for 20 μM NOC-18 and 10 μM ODQ were shown. Data, representative of four experiments, are mean ± SD, n = 4–5, *, P < 0.05, compared with culture without NO.
Fig 3.
Fig 3.
8-Br-cGMP selectively enhanced type 1 cell differentiation. CD4+ T cells from D0.11 mice were negatively purified and cultured under Th1 or Th2 conditions as in Fig. 2 b and c. Graded concentrations of 8-Br-cGMP were added at the beginning of cultures, which were harvested on day 4. Results, representative of four similar experiments, are mean ± SD, n = 4, *, P < 0.05, compared with culture without 8-Br-cGMP. Similar results were obtained with polyclonally activated (anti-CD3 antibody-treated BALB/c CD4+ T cells) Th1 and Th2 cells and Tc1 and Tc2 cells (data not shown).
Fig 4.
Fig 4.
Low concentrations of NO selectively enhanced IL-12Rβ2 induction. Purified CD4+ and CD8+ T cells from BALB/c mice were cultured under type 1 or type 2 conditions with immobilized anti-CD3 antibody for 16 h. Cells were harvested, mRNA was extracted, and the expression of IL-12Rβ2, IL-18Rα, and IL-4R was determined by quantitative PCR as described in Methods. Results, representative of three experiments, are mean ± SD, n = 3, *, P < 0.05, compared with culture without NO.
Fig 5.
Fig 5.
cGMP enhanced and ODQ inhibited IL-12Rβ2 expression during type 1 cell differentiation. Purified CD4+ T cells from BALB/c mice were cultured under Th1 driving conditions in the presence of 8-Br-cGMP (120 μM), NOC-18 (10 μM), or NOC-18 (10 μM) + ODQ (10 μM). Cells were harvested at 16 h, and mRNA was extracted for quantitative PCR analysis for IL-12Rβ2 expression. Results, representative of two experiments, are mean ± SD, n = 5, *, P < 0.05.
Fig 6.
Fig 6.
Schematic representation of the mechanism by which low concentrations of NO selectively enhance type 1 T cell differentiation. Type 1 cells differentiate from Tp cells through TCR activation by antigen (peptide) presented by the MHC of the APC (e.g., dendritic cells and macrophages) in the presence of IL-12. NO produced by the APC in response to infection or present exogenously in the microenvironment can up-regulate the levels of cGMP in the Tp cells by activating sGC. cGMP, in turn, selectively enhances the expression of IL-12Rβ2. This then increases the response of Tp cells to IL-12 and, hence, preferentially promotes the differentiation of type 1 T cells.
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