Augmentation of naive,Th1 and Th2 effector CD4 Responses by IL-6, IL-1 and TNF

The role of antigen-presenting cell (APC)-derived cytokines in T cell activation is still controversial. Highly purified CD4 T cell populations of the naive and short-term Th1 and Th2 effector subsets were examined. Stimulation from anti-CD3 in the absence of APC was used to analyze directly T occurring cell-mediated effects, and the requirement for co-signaling was addressed using anti-CD28. Exogenous IL-6, IL-1 and TNF each enhanced proliferation and IL-2 secretion from naive cells, although IL-6 was most active in this regard. Peak responses, however, were obtained with IL-1 or TNF in combination with IL-6 resulting in up to 11-fold increases in IL-2 secretion. Enhanced naive T cell responses were only observed with anti-CD3 and anti-CD28, suggesting that co-signaling through surface-bound receptors was required to initiate IL-2 production. Although the cytokines enhanced naive activation, little effect was seen on differentiation into effector populations. IL-6 alone, or in combination, partially suppressed effectors secreting IFN-γ, but did not promote generation of effectors secreting IL-4. In contrast to reports on cloned cell lines, IL-6, TNF and IL-1 had enhancing activities on all cytokines elicited from already generated Th1 and Th2 effector populations. Again combinations of IL-6, TNF and IL-1 were most effective and generally required CD28 signaling. Induced responses with preexisting effector cells were far less than with naive cells and predominantly directed at augmenting IFN-γ and IL-5 secretion rather than IL-2 and IL-4. These studies show that APC-derived cytokines can promote T cell responses directly but largely after co-stimulation from accessory molecule co-receptors, that the effect is not specific for one T cell subset or cytokine, and that the naive T cell is the main target of action.     

CD28 activation promotes Th2 subset differentiation by human CD4+ cells

Ligation of CD28 provides a costimulatory signal to T cells necessary for their activation resulting in increased interleukin (IL)-2 production in vitro, but its role in IL-4 and other cytokine production and functional differentiation of T helper (Th) cells remains uncertain. We studied the pattern of cytokine production by highly purified human adult and neonatal CD4⁺ T cells activated with anti-CD3, phorbol 12-myristate 13-acetate (PMA) and ionomycin, or phytohemagglutinin (PHA) in the presence or absence of anti-CD28 in repetitive stimulation-rest cycles. Initial stimulation of CD4⁺ cells with anti-CD3 (or the mitogens PHA or PMA+ionomycin) and anti-CD28 monoclonal antibodies induced IL-4, IL-5 and interferon-γ (IFN-γ) production and augmented IL-2 production (6- to 11-fold) compared to cells stimulated with anti-CD3 or mitogen alone. The anti-CD28-induced cytokine production corresponded with augmented IL-4 and IL-5 mRNA levels suggesting increased gene expression and/or mRNA stabilization. Most striking, however, was the progressively enhanced IL-4 and IL-5 production and diminished IL-2 and IFN-γ production with repetitive consecutive cycles of CD28 stimulation. The enhanced Th2-like response correlated with an increased frequency of IL-4-secreting cells; up to 70% of the cells produced IL-4 on the third round of stimulation compared to only 5% after the first stimulation as determined by ELISPOT. CD28 activation also promoted a Th2 response in naive neonatal CD4⁺ cells, indicating that Th cells are induced to express a Th2 response rather than preferential expansion of already established Th2-type cells. This CD28-mediated response was IL-4 independent, since enhanced IL-5 production with repetitive stimulation cycles was not affected in the presence of neutralizing anti-IL-4 antibodies. These results indicate that CD28 activation may play an important role in the differentiation of the Th2 subset in humans.     

Ebastine inhibits T cell migration, production of Th2-type cytokines and proinflammatory cytokines

BACKGROUND: Cytokine imbalance and cellular migration to inflammatory sites are critical components of allergic diseases. Redirecting cytokine imbalance and inhibiting cell migration therefore represent important therapeutic strategies for the treatment of these disorders. OBJECTIVES: To study the in vitro effect of ebastine, a novel non-sedating H1 receptor antagonist, on cytokine secretion and migration of activated T cells, as well as production of pro-inflammatory cytokines by macrophages. METHODS: Peripheral T cells obtained from healthy volunteers were cultured in wells coated with the combination of anti-CD3 monoclonal antibody (mAb) and anti-CD26 mAb, anti-CD3 mAb and anti-CD28 mAb, or anti-CD3 mAb with PMA, in the presence or absence of ebastine. T cell proliferation and the production of cytokines were measured by [3H]thymidine incorporation assay and ELISA, respectively. In addition, transendothelial migration of T cells and production of pro-inflammatory cytokines by macrophages were examined. RESULTS: Ebastine inhibited T cell proliferation and the production of IL-4, IL-5, IL-6, and TNF-alpha by T cells under each co-stimulatory condition tested, whereas it exhibited no effect on the production of IL-2 or IFN-gamma. In addition, T cell migration and the production of such pro-inflammatory cytokines as TNF-alpha and IL-6 by macrophages were inhibited by ebastine. CONCLUSIONS: These results indicate that ebastine has a specific inhibitory effect on Th2-type cytokine production. Moreover, ebastine inhibited T cell migration and pro-inflammatory cytokine production by T cells and macrophages, suggesting that ebastine might be useful for the treatment of T cell-mediated allergic inflammatory disorders, including asthma, atopic dermatitis, and Th2-type autoimmune diseases.     

Glucocorticoids drive human CD8(+) T cell differentiation towards a phenotype with high IL-10 and reduced IL-4, IL-5 and IL-13 production

Glucocorticoids are highly effective in the treatment of allergy and asthma and inhibit the synthesis of IL-4, IL-5 and IL-13 by disease-promoting CD4(+) Th2 cells. CD8(+) T cells also synthesize these cytokines, and the aim of this study was to investigate how glucocorticoids effect cytokine production by these cells. When CD8(+) T cells are stimulated with anti-CD3 and IL-2 plus IL-4 or dexamethasone, production of the anti-inflammatory cytokine IL-10 is low in both primary and secondary cultures restimulated with anti-CD3 and IL-2 alone. However, when both are present, a synergistic effect on IL-10 synthesis is observed. The additional presence of antigen-presenting cells (APC) in the priming culture maintains IL-10 levels, but inhibits IL-4 and IL-5 production. CD4(+) T cells develop a similar glucocorticoid-induced phenotype. These cells demonstrate regulatory activity and inhibit CD4(+) T cell activation in an IL-10-dependent manner. Earlier reports show glucocorticoids promote a Th2 phenotype by effects on purified naive T cells or pretreatment of APC. This study demonstrates, more critically, that when APC are present, glucocorticoids induce CD4 and CD8 T cell populations synthesizing high levels of IL-10, but greatly reduced amounts of disease-promoting IL-4 and IL-5.     

Lipid A directly inhibits IL-4 production by murine Th2 cells but does not inhibit IFN-gamma production by Th1 cells

Lipopolysaccharide (LPS) is known to be an immunopotentiator but its effect on cytokine production by Th1 and Th2 cells is unknown. We found that high amounts of LPS, its lipid A moiety, and a lipid A analog all induced a decrease in IL-4 production and an increase in IFN-gamma production when given to keyhole limpet hemocyanin (KLH)-restimulated lymph node cells prepared from KLH-primed mice. Lipid A was similarly found to inhibit IL-4 production by purified CD4+ T cells and Th2 clones activated with immobilized anti-CD3epsilon and anti-CD28 antibodies, suggesting that the inhibition is not indirectly mediated through effects on antigen-presenting cells. No inhibitory effect of lipid A was observed on IFN-gamma production by a Th1 clone. Production of both IL-4 by the Th2 clones and IFN-gamma by the Th1 clone were inhibited by the immunosuppressive agent cyclosporin A. These findings indicate that lipid A can directly inhibit IL-4 production by CD4+ T cells without inhibiting the production of IFN-gamma. Lipid A may therefore become a useful tool to study the intracellular events that differentiate Th1 and Th2 cells.     

Differences in regulatory pathways identify subgroups of T cell-derived Th2 cytokines

We analysed regulatory mechanisms involved in the production of Th2 cytokines by freshly isolated human T cells. We used an in vitro culture system in which the primary signal was provided by a cross-linking anti-CD3 MoAb presented on the Fc receptors of P815 cells. Both CD80 and CD86, expressed on transfected P815 cells, were able to provide efficient costimulation for the production of IL-4, IL-5 and IL-13. IL-2 was also highly important for induction of all three Th2 cytokines. However, differences between IL-4 on the one hand and IL-5 and IL-13 on the other hand were observed when sensitivity to cyclosporin A (CsA) was studied. CsA (an inhibitor of calcineurin phosphatase activity) strongly inhibited IL-4 production, but it did either not affect or even increased IL-5 and IL-13 production. In accordance with this, CD80 and phorbol myristate acetate (PMA) (without anti-CD3 or calcium ionophore) were sufficient to induce production of IL-5 and IL-13, but not of IL-4. The subgrouping of Th2 cytokines was further confirmed at another level on the basis of differences in cell sources: IL-4 was predominantly produced by CD4+ T cells, while IL-5 and IL-13 were produced by both CD4+ and CD8+ T cells. Thus, differences in cell sources and in the requirement of the calcium/calcineurin-signalling pathway allowed us to identify two subgroups (IL-4 and IL-5/IL-13) among human Th2-type T cell cytokines.     

Prostaglandin E2 primes naive T cells for the production of anti-inflammatory cytokines

In addition to their capacity to induce pain, vasodilatation and fever, prostaglandins E (PGE) exert anti-inflammatory activities by inhibiting the release of pro-inflammatory cytokines by macrophages and T cells, and by increasing interleukin (IL)-10 production by macrophages. We here report that PGE₂, the major arachidonic acid metabolite released by antigen-presenting cells (APC), primes naive human T cells for enhanced production of anti-inflammatory cytokines and inhibition of pro-inflammatory cytokines. Unfractionated as well as CD45RO^?CD31⁺ sort-purified neonatal CD4 T cells acquire the capacity to produce a large spectrum of cytokines after priming with anti-CD3 and anti-CD28 monoclonal antibodies (mAb), in the absence of both APC and exogenous cytokines. PGE₂ primes naive T cells in a dose-dependent fashion for production of high levels of IL-4, IL-10 and IL-13, and very low levels of IL-2, interferon (IFN)-γ, tumor necrosis factor (TNF)-α, and TNF-β. PGE₂ does not significantly increase IL-4 production in priming cultures, whereas it suppresses IL-2 and IFN-γ. Addition of a neutralizing mAb to IL-4 receptor in primary cultures, supplemented or not with PGE₂, prevents the development of IL-4-producing cells but does not abolish the effects of PGE₂ on IL-10 and IL-13 as well as T helper (Th)1-associated cytokines. Addition of exogenous IL-2 in primary cultures does not alter the effects of PGE₂ on naive T cell maturation. Thus PGE₂ does not act by increasing IL-4 production in priming cultures, and its effects are partly IL-4 independent and largely IL-2 independent. Together with the recent demonstration that PGE₂ suppresses IL-12 production, our results strongly suggest that this endogenously produced molecule may play a significant role in Th subset development and that its stable analogs may be considered for the treatment of Th1-mediated inflammatory diseases.     

Nickel-induced IL-10 down-regulates Th1- but not Th2-type cytokine responses to the contact allergen nickel

Whereas the involvement of Th1- and Th2-type cytokines in contact allergy to nickel (Ni) is well documented, the role of the regulatory cytokine IL-10 is less clear. We therefore investigated the impact of IL-10 on Ni-induced Th1- (IFN-gamma) and Th2-type (IL-4 and IL-13) cytokine responses in human peripheral blood mononuclear cells (PBMC). PBMC from 15 blood donors with reactivity to Ni (Ni-PBMC) and 8 control donors devoid of reactivity (control PBMC) were stimulated with Ni and the frequency of cytokine-producing cells and the levels of secreted cytokines were analysed by ELISpot (IL-4, IL-13 and IFN-gamma) and ELISA (IL-10, IL-13 and IFN-gamma), respectively. The Ni-induced response was further assessed in the presence of recombinant IL-10 (rIL-10) or neutralizing antibody to IL-10 and the phenotype of the Ni-specific cytokine-producing cells regulated by IL-10 was determined by cell depletion experiments. Ni induced IL-10 production in Ni-PBMC (mean, (range); 33.1 pg/ml (0-93.4 pg/ml)) but not control PBMC (2.2 pg/ml (0-14.9 pg/ml)) (P = 0.002). Ni also induced significant production of IL-4, IL-13 and IFN-gamma that correlated with the IL-10 response. Addition of rIL-10 down-regulated the Ni-induced production of all cytokines but with a more pronounced effect on IFN-gamma. However, neutralization of Ni-induced IL-10 enhanced the levels of IFN-gamma induced by Ni (P = 0.004) but did not affect the number of IFN-gamma-producing cells or the production of other cytokines. Cell depletion experiments suggested that the Ni-specific IFN-gamma (and Th2-type cytokine) producing cells were CD4(+) T cells. The impact of IL-10 on Ni-induced IFN-gamma responses by CD4(+) T cells suggests that an important role of IL-10 in vivo is to counteract the allergic reactions mediated by Th1-type cytokines.     

In vivo administration of IL-18 can induce IgE production through Th2 cytokine induction and up-regulation of CD40 ligand (CD154) expression on CD4+ T cells

IL-18 is considered to be a strong cofactor for CD4+ T helper 1 (Th1) cell induction. We have recently reported that IL-18 can induce IL-13 production in both NK cells and T cells in synergy with IL-2 but not IL-12, suggesting IL-18 can induce Th1 and Th2 cytokines when accompanied by the appropriate first signals for T cells. We have now found that IL-18 can act as a cofactor to induce IL-4, IL-10 and IL-13 as well as IFN-gamma production in T cells in the presence of anti-CD3 monoclonal antibodies (mAb). IL-18 can rapidly induce CD40 ligand (CD154) mRNA and surface expression on CD4+ but not CD8+ T cells. The administration of IL-18 alone in vivo significantly increased serum IgE levels in C57BL/6 (B6) and B6 IL-4 knockout mice. Furthermore, the administration of IL-18 plus IL-2 induced approximately 70-fold and 10-fold higher serum levels of IgE and IgG1 than seen in control B6 mice, respectively. IgE and IgG1 induction in B6 mice by administration of IL-18 plus IL-2 was eliminated by the pretreatment of mice with anti-CD4 or anti-CD154, but not anti-CD8 or anti-NK1.1 mAb. These results suggest that IL-18 can induce Th2 cytokines and CD154 expression, and can contribute to CD4+ T cell-dependent, IL-4-independent IgE production.     

Immunomodulatory effects of peptide T on Th 1/Th 2 cytokines.

Peptide T is an octapeptide from the V2 region of HIV-1 gp120. It has been shown to resolve psoriatic lesions--an inflammatory skin disease. The mechanisms of anti-inflammatory actions of peptide T are not well understood. Th1 cytokines such as IL-2, and IFN-gamma are upregulated in psoriasis. These cytokines play a key role in the inflammatory and proliferative processes of psoriasis. The effects of peptide T on Th1 and Th2 cytokines were studied in order to elucidate the mechanisms of antiinflammatory actions of peptide T. It was observed that peptide T at 10(-8) M induces IL-10 production by the human Th2 cell line and PBMC (P < 0.05, ANOVA). Also peptide T at 10(-9) M concentration significantly inhibited IFN-gamma production by PBMC (P < 0.001, ANOVA). Anti IL-10 antibody inhibited the anti-IFN-gamma effect of peptide T (P < 0.05, t-test). Our study shows that peptide T induces IL-10 production and inhibits IFN-gamma production. IL-10 is a potent anti-inflammatory cytokine. It inhibits IL-2 and IFN-gamma production from the T cells and downregulates the expression of TNF-alpha in the antigen presenting cells. Recently, IL-10 has been shown to resolve psoriatic lesions. The effects of peptide T on IL-10 and IFN-gamma production provides a plausible explanation for its clinical efficacy in psoriasis.     

Change in the Th1/Th2 Phenotype of Memory T-Cell Clones from Rheumatoid Arthritis Synovium

The stability of established memory T helper (Th)1/Th2 cells in chronic inflammatory diseases is not clear, and a shift of the cytokine balance could control chronic inflammation. In order to study the regulation of the Th phenotype of memory T cells, polyclonal T-cell lines and clones with a Th1, Th0 or Th2 phenotype were developed from rheumatoid synovial tissue. Th1 [interleukin (IL)-12 + anti-IL-4] and Th2 (IL-4 + anti-IL-12) promoting environments and IL-2 were used to manipulate the cytokine profile. Polyclonal T-cell lines of predominantly Th1 type could be shifted to produce Th2 cytokines, and polyclonal Th2/Th0 lines could be shifted to produce Th1 cytokines. However, this shift was due to an amplification of CD8+ T cells with a memory phenotype and a loss of the CD4+ T cells, giving Tc2 or Tc1 profiles, respectively. Th2 clones cultured repeatedly with IL-2 switched to either a Th0 or a Th1 phenotype, while both Th1 and Th0 memory clones kept a stable phenotype. Addition of Th2-promoting conditions strongly reduced the production of both interferon-gamma and IL-17, while Th1-promoting conditions increased the production of these cytokines. These results demonstrate that RA Th2 clones readily switch, while Th1 and Th0 clones are stable. However, induction of Th2 cytokines can be obtained in polyclonal polarized memory T cells due to amplification of Tc2 cells.     

Massive production of Th2 cytokines by human CD4+ effector T cells transiently expressing the natural killer cell marker CD57/HNK1.

We have reported previously that uncommitted human CD4+ CD45RO- T cells default to the T-helper type 1 (Th1) pathway, if they are costimulated by anti-CD3 plus anti-CD28 monoclonal antibodies (mAb). In contrast, 5% of the uncommitted T cells differentiate into Th2 cells, if they are stimulated by anti-CD28 plus interleukin-2 (IL-2) in the absence of T-cell receptor (TCR) signals. The anti-CD28/IL-2-induced proliferation (and the resulting Th2 commitment) was not affected by neutralizing anti-IL-4 mAb, suggesting a non-conventional IL-4-independent Th2 differentiation pathway. Here we report that the respective CD4+ Th2 cells (but not the Th1 cells) coexpressed the natural killer (NK) cell marker HNK1/CD57. Expression of CD57 on Th2 cells required CD28 stimulation, and was suppressed by CD3/TCR signals. However, Th2 effector cells displayed a TCR V beta-chain usage comparable to that of committed Th1 cells (with V beta 8 dominating). Our data suggest that expression of CD57 on human CD4 T cells may be associated with defined stages of Th2 cell activation/differentiation, and may not necessarily characterize a separate T-cell lineage. The induction of cytokine production and B-cell helper function in both Th1 and Th2 populations required CD3/TCR signalling in costimulation with anti-CD28 or IL-2. Importantly, anti-CD28/IL-2-primed Th2 cells readily secreted IL-4 and induced IgE production by surface IgE- B cells in response to the first TCR signal and independent of previous contact with IL-4. Therefore, CD4+ CD57+ T cells responded comparably to murine CD4+ NK1.1+ T cells, which are critical for the development of Th2/IgE immune responses in vivo. The possible role of human CD4+ CD57/HNK1+ Th2-like cells in cancer, infection and allergy is discussed.     

Apoptotic cells selectively suppress the Th1 cytokine interferon gamma in stimulated human peripheral blood mononuclear cells and shift the Th1/Th2 balance towards Th2

Apoptotic cells are readily recognized and engulfed by phagocytes and usually do not induce inflammation or tissue damage. Furthermore, they can actively suppress a pro-inflammatory response in phagocytes: In the presence of apoptotic cells, activated monocytes/macrophages produce more of the anti-inflammatory and immunoregulatory cytokines IL-10 and TGF-beta, but less of the pro-inflammatory cytokines TNFalpha, IL-1beta and IL-12. This immunoregulatory effect is most likely mediated by several receptors on monocytes/macrophages including the thrombospondin receptor (CD36). In addition to the modulation of cytokine secretion, apoptotic cell material inhibited the expression of MHC class II molecules on the surface of monocytes/macrophages. Decreased MHC II expression appeared to be mediated predominantly by increased IL-10 secretion in a para-/autocrine manner. Here, we show that the functional modulation of antigen-presenting monocytes/macrophages by apoptotic cells also influences T cell activation and function. When human peripheral blood mononuclear cells were stimulated with recall antigens in the presence of apoptotic cells, interferon gamma (IFN gamma) secretion was markedly suppressed, whereas secretion of the Th2 cytokine IL-4 was not significantly altered. Hence, apoptotic cells shift the T cell cytokine secretion pattern towards a Th2-like response. This Th2 shift can largely be prevented by neutralizing IL-10, indicating an important role of this cytokine for modulating T cell cytokine secretion patterns.     

Rapid development of Th2 activity during T cell priming

The paradigm of T helper-1 (Th-1) and Th-2 cells developing from non-committed naive precursors is firmly established. Th1 cells are characterized by IFNgamma production and, in mice, the selective switching to IgG2a. Conversely IL-4 production and selective switching to IgG1 and IgE characterize Th2 cells. Analysis of Th2 induction in vitro indicates that this polarization develops gradually in T cells activated by anti-CD3 in the presence of IL-4; conversely anti-CD3 and IFNgamma induce Th1 cells. In this report, we explore evidence that indicates that the T helper cell polarization in vivo cannot solely be explained by the cytokine environment. This is provided by studying the early acquisition of Th1 and Th2 activities during responses to a mixture of Th1 and Th2-inducing antigens. It is shown that these divergent forms of T cell help can rapidly develop in cells within a single lymph node. It is argued that early polarization to show Th-1 or Th-2 behavior can be induced by signals delivered during cognate interaction between virgin T cells and dendritic cells, in the absence of type 1 or type 2 cytokines. This contrasts with the critical role of the cytokines in reinforcing the Th-phenotype and selectively expanding T helper clones.     

IL-35 is a novel cytokine with therapeutic effects against collagen-induced arthritis through the expansion of regulatory T cells and suppression of Th17 cells

Epstein-Barr virus-induced gene 3 (EBI3) and the p35 subunit of IL-12 have been reported to form a heterodimeric hematopoietin in human and mouse. We have constructed a heterodimeric protein covalently linking EBI3 and p35, to form a novel cytokine which we now call IL-35. The Fc fusion protein of IL-35 induced proliferation of murine CD4(+)CD25(+) and CD4(+)CD25(-) T cells when stimulated with immobilized anti-CD3 and anti-CD28 antibodies in vitro. The IL-35-expanded CD4(+)CD25(+) T cell population expressed Foxp3 and produced elevated levels of IL-10, whereas the IL-35-induced CD4(+)CD25(-) T cells produced IFN-gamma but not IL-4. The in vitro expanded CD4(+)CD25(+) T cells retained their suppressive functions against CD4(+)CD25(-) effector cells. Furthermore, when cultured with soluble anti-CD3 antibody and antigen-presenting cells, IL-35 suppressed the proliferation of CD4(+)CD25(-) effector cells. Moreover, IL-35 inhibited the differentiation of Th17 cells in vitro. In vivo, IL-35 effectively attenuated established collagen-induced arthritis in mice, with concomitant suppression of IL-17 production but enhanced IFN-gamma synthesis. Thus, IL-35 is a novel anti-inflammatory cytokine suppressing the immune response through the expansion of regulatory T cells and suppression of Th17 cell development.     

Reduced Th1, but not Th2, cytokine production by lymphocytes after in vivo exposure of healthy subjects to endotoxin

Endotoxin (lipopolysaccharide [LPS]) tolerance is characterized by a reduced capacity of monocytes to produce proinflammatory cytokines upon restimulation in vitro. To determine whether LPS exposure induces a change in lymphocyte cytokine production and whether this results in a shift in the T-helper 1 (Th1)/Th2 balance, whole blood obtained from seven healthy subjects before and after an intravenous injection of LPS (4 ng/kg) was stimulated in vitro with the T-cell stimulus anti-CD3/CD28 or staphylococcal enterotoxin B. Whole-blood production of the Th1 cytokines gamma interferon (IFN-gamma) and interleukin-2 (IL-2) was markedly reduced at 3 and 6 h, while the production of the Th2 cytokines IL-4 and IL-5 was not influenced or was slightly increased. The IFN-gamma/IL-4 ratio was strongly decreased at 6 h. Serum obtained after LPS exposure could slightly inhibit the release of IFN-gamma but increased IL-4 production during stimulation of blood drawn from subjects not previously exposed to LPS. Normal serum also inhibited IFN-gamma production, albeit to a lesser extent. LPS exposure influences lymphocyte cytokine production, resulting in a shift toward a Th2 cytokine response, an effect that may be mediated in part by soluble factors present in serum after LPS administration in vivo.     

Nitric oxide inhibits the secretion of T-helper 1- and T-helper 2-associated cytokines in activated human T cells.

Mechanisms regulating the balance of T-helper 1 (Th1) and T-helper 2 (Th2) immune responses are of great interest as they may determine the outcome of allergic and infectious diseases. Recently, in mice, nitric oxide (NO), a powerful modulator of inflammation, has been reported to preferentially down-regulate Th1-mediated immune responses. In the present study, we investigated the effect of NO on the production of Th1- and Th2-associated cytokines by activated human T cells and human T-cell clones. Cytokine secretion was measured in the presence of the NO-donating agents 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP). Both NO-donors markedly inhibited the release of interferon-gamma (IFN-gamma), interleukin-2 (IL-2), IL-5, IL-10 and IL-4 by anti-CD3 activated T cells. A preferential inhibition of Th1-associated cytokines was not observed. Neither was nitrite found in the supernatants of activated T cells, nor was specific mRNA for inducible and constitutive NO synthase detectable, indicating that T cells themselves did not contribute to the observed effect of the NO donors. Costimulation with anti-CD28 monoclonal antibodies (mAb) prevented SIN-1/SNAP-mediated down-regulation of cytokine production only in part. In contrast, when T cells were stimulated by phorbol-ester and ionomycin, they were refractory to SIN-1-induced inhibition of cytokine production. When SIN-1 was added after the onset of anti-CD3 stimulation, the inhibitory effect was found to be less pronounced, indicating that SIN-1 may interfere with early signal transduction events. The addition of superoxide dismutase (SOD) and catalase did not restore the effects of SIN-1, demonstrating that the inhibition of cytokines was due to NO and not to oxygen intermediates. Furthermore, 8-Br-cGMP-mediated increase of intracellular cGMP caused the same pattern of cytokine inhibition as observed with SIN-1 and SNAP. Using a single cell assay, these agents were shown to reduce the frequency of IFN-gamma-producing T cells, suggesting that not all T cells are susceptible to SIN-1/SNAP. However, cytokine production by purified T-cell subpopulations (CD4+, CD8+, CD45RA+, and CD45RO+) was equally impaired by NO donors. In conclusion, in contrast to the murine system, our results do not provide evidence that NO preferentially inhibits Th1-cytokine secretion of activated human T cells in vitro.