Efficient induction of an antigen-specific, T helper type 1 immune response by interleukin-12-secreting fibroblasts

To determine whether the paracrine secretion of interleukin (IL)-12 can efficiently convert immune responses characterized by high levels of synthesis of IL-4 and immunoglobulin E (IgE) into T helper 1 (Th1)-dominated responses, 3T3 fibroblasts were stably transfected to secrete IL-12 (480 units/10(6) cells/48 hr). Their effects on the T helper cell-mediated immune response were investigated in ovalbumin (OVA)-primed mice. Free mouse recombinant IL-12 was included as a control group. IL-12-secreting fibroblasts (3T3/IL-12) were more effective than free recombinant IL-12 at increasing OVA-specific interferon-gamma (IFN-gamma) production and decreasing OVA-specific IL-4 production in CD4+ T cells. In addition, injection with 3T3/IL-12 cells significantly increased anti-OVA immunoglobulin G2a (IgG2a) levels and decreased anti-OVA IgE levels in OVA-primed mice. This work suggests that IL-12-secreting fibroblasts can efficiently induce an antigen-specific Th1 response and may be beneficial in the treatment of diseases caused by undesirable T helper 2 (Th2)-dominated responses, including allergic diseases.     

MAGE-1与IL-18共表达DNA疫苗在小鼠体内诱导特异性免疫应答能力的研究

目的：观察已构建的peIL-18-MAGE共表达质粒疫苗接种小鼠所引起的免疫应答情况。方法：质粒大量制备肌注小鼠，每隔10天加强免疫1次，共免疫3次。末次免疫后7天，收集血清及脾细胞，流式细胞术检测小鼠T细胞亚群情况及血清中抗MAGE-1多抗的产生，MTT法检测小鼠CTL杀伤活性。结果：peIL-18、pcMAGE-1、peIL-18＋pcMAGE-1及peIL-18-MAGE免疫组CD3^＋、CD4^＋、CD8^＋、CD4^＋／CD8^＋及MAGE-1抗体的产生均高于peDNA3免疫组（P〈0．05），且peIL-18-MAGE共表达免疫组高于peIL-18＋peMAGE-1共同注射组（P〈0．05）。pdL-18-MAGE免疫组对SMMC-7721的杀伤活性最高。结论：pelL-18-MAGE共表达基因疫苗接种小鼠与其它实验小鼠相比，可以有效产生更好的特异性免疫应答。     

Role of beta1 and beta2 subunits of the interleukin-12 receptor in determining T helper 1/T helper 2 responses in vivo in the rat

Interleukin-12 (IL-12) responsiveness, and hence capacity to mount a T helper type 1(Th1) immune response, may be regulated via differential expression of the IL-12 receptor beta2 subunit at least in vitro in human and murine cells. To test whether a similar phenomenon operates in vivo in the rat we cloned and sequenced partial cDNAs for rat IL-12Rbeta1 and IL-12Rbeta2 subunits and analysed expression of these genes in vivo in two rat strains with different Th1/Th2 bias. After treatment with mercuric chloride (HgCl2), Brown-Norway rats develop Th2-biased autoimmunity whereas Lewis rats do not develop autoimmunity, instead becoming resistant to Th1-biased diseases to which they are normally susceptible. We report close sequence homology between the segments of the rat IL-12R genes sequenced and corresponding mouse genes (95.6% and 92% for IL-12Rbeta1 and IL-12Rbeta2, respectively). Both Brown-Norway and Lewis rats express both beta1 and beta2 subunits of IL-12 receptor in vivo in spleen; Brown-Norway rats express the beta2 subunit at a lower level than Lewis rats. After HgCl2 treatment, IL-12Rbeta1 expression was not altered but there was down-regulation of IL-12Rbeta2 expression in both strains. We conclude that relative under-expression of IL-12Rbeta2 by Brown-Norway rats contributes to their Th2 bias, and that down-regulation of IL-12Rbeta2 after HgCl2 administration in Lewis rats underlies subsequent resistance to induction of Th1-biased diseases.     

Overexpression of interleukin-12 and T helper 1 predominance in lupus nephritis

Imbalance of cytokine homeostasis is a prominent feature of both experimental and human systemic lupus erythematosus (SLE). Because interleukin (IL)-12 promotes interferon (IFN)-gamma production leading to polarization of peripheral cells toward a T helper (Th) 1 phenotype, we investigated its role in lupus nephritis (LN). Soluble Th1 and Th2 cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in sera and urines of SLE patients and controls. Th1/Th2 peripheral lymphocyte polarization was determined by flow cytometry. Glomerular accumulation of IL-12 was evaluated by immunohistochemistry, whereas urinary IL-12 was evaluated by ELISA. Higher serum IL-12 levels in SLE were associated with LN, whereas IL-4 was unrelated to the renal damage. Peripheral cells from LN patients showed a Th1 phenotype with a high IFN-gamma expression that paralleled the severity of renal damage. IL-12 was present within glomerular mononuclear cells in classes IV and V LN, and its accumulation was correlated strongly with urinary levels. IL-12 overexpression in SLE may contribute to the development of LN. Both serum and urinary IL-12 elevation reflect its glomerular production and parallel Th1 polarization of peripheral T cells and high IFN-gamma production. In SLE patients, IL-12 measurement may thus be predictive of the development of LN.     

Interleukin-12 is produced by dendritic cells and mediates T helper 1 development as well as interferon-γ production by T helper 1 cells

Interleukin-12 (IL-12), a 70-kDa heterodimeric cytokine composed of covalently linked p35 and p40 chains, is to date the most critical factor for skewing the immune response towards a T helper 1 (Th1) of cytokine profile [high interferon-γ (IFN-γ), low IL-4]. Established sources of IL-12 are stimulated macrophages, neutrophils and B cells. As dendritic cells (DC) process antigen in the periphery and then migrate to lymphoid organs to sensitize T cells and induce cell-mediated immunity, we reasoned that DC should constitute a critical source of IL-12. The criteria used to detect IL-12 in DC were the demonstration of p40 and p35 mRNA (semiquantitative polymerase chain reaction, Northern blotting, and in situ hybridization) as well as IL-12 protein (p70 enzyme-linked immunosorbent assay, p70 antigen capture followed by IFN-γ bioassay, free p40 chain radioimmunoassay or immunoprecipitation). We found that conventional stimuli such as Staphylococcus aureus induced production of IL-12 bymurine as well as human DC in amounts comparable to spleen cells, peritoneal macrophages or peripheral blood mononuclear cells. DC exhibited, however, features that had not been seen with other antigen-presenting cells: they produced bioactive IL-12 upon antigen-specific interaction with T cells without any other stimuli; in an allogeneic mixed leukocyte reaction model, neutralizing anti-IL-12 antibodies showed that DC-derived IL-12 was critical for optimal proliferation and IFN-γ production by activated Th1 blasts; and finally, the priming of resting, naive allogeneic T cells by DC, followed by restimulation of primed T blasts by DC, skewed the response to Th1 without the need for any exogenous cytokines or stimuli such as microorganisms. This skewing to Th1 cytokine production, which depended on DC-derived IL-12, but did not require anti-IL-4, exogenous IL-12, or microbes, might be a major function of DC.     

Regulation of the immune response by nitric oxide differentially produced by T helper type 1 and T helper type 2 cells

The balance between T helper type 1 (Th1) and T helper type 2 (Th2) cells determines the outcome of many important diseases. Using cloned murine T cell lines, evidence is provided that Th 1, but not Th 2, cells can be activated by specific antigens or a T cell mitogen, concanavalin A, to produce large amounts of nitric oxide (NO). Furthermore, NO can inhibit the secretion of interleukin (IL)-2 and interferon-γ by Th 1 cells but has no effect on IL-4 production by Th 2 cells. Th 1 and Th 2 cells can, thus, be distinguished by their differential production of and susceptibility to NO. NO exerts a self-regulatory effect on Th 1 cells which are implicated in immunopathology.     

Administration of plasmids expressing interleukin-4 and interleukin-10 causes BALB/c mice to induce a T helper 2-type response despite the expected T helper 1-type response with a low-dose infection of Leishmania major

BALB/c mice are susceptible to developing an infection with Leishmania major as a result of a fatal T helper 2 (Th2)-type response. However, mice infected with a low dose of parasites are reported to be able to overcome the lesions associated with a T helper 1 (Th1)-type response. To clarify why a difference in the dose of parasites induces a difference in the polarization of the Th phenotype, we first attempted to measure cytokine production. Soon after infection, the mice given high doses of parasites produced elevated levels of both Th1 [interferon-gamma (IFN-gamma)] and Th2 [interleukin (IL)-4 and IL-10] cytokines. However, when assessed at 1 and 2 weeks after infection, no significant difference in the balance of Th1 and Th2 cytokines could be detected between mice infected with low or high doses of L. major. These results support the notion that the Th2 cytokine levels at an early phase of infection could be a key factor for the induction of a Th2 response. In order to assess the efficacy of Th2 cytokines, the mice infected with low doses of L. major were co-administered IL-4 plasmid and IL-10 plasmid. Consequently, the mice (which originally exhibited a Th1 response) showed progressive disease and developed a Th2 response. However, administration of these plasmids at 7 days postinfection could not alter the Th polarization. Furthermore, production of IL-12 from the spleen cells stimulated by L. major was suppressed in the presence of IL-4 and IL-10. These results strongly suggest that the susceptibility to L. major in BALB/c mice depends on the persistence of Th2 cytokine levels at an early phase of infection.     

Mechanism of mercury-induced autoimmunity: both T helper 1- and T helper 2-type responses are involved

Mercury can induce a systemic autoimmune disease in susceptible mouse strains. H-2s mice are particularly susceptible to mercury-induced autoimmunity and other mouse strains are more or less resistant. T helper 1/T helper 2 (Th1/Th2) dichotomy has been proposed for resistance or susceptibility, respectively. In the current study we show that mercury treatment induced a full autoimmune response in both C57BL/6 (H-2b) wild-type and interleukin-4 (IL-4)-deficient mice. Antibody production of all isotypes were induced, except that in IL-4-deficient mice there was no immunoglobulin E (IgE) and very low levels of immunoglobulin G1 (IgG1) antibody synthesis. Autoantibodies of different specificities were produced. The granular pattern of all IgG subclasses deposits were detected in the kidneys. In contrast to mercury-treated H-2s seconds mice, we did not detect any anti-nucleolar autoantibodies in the sera of mercury-treated wild-type or IL-4-deficient mice. To further explore the role of Th1/Th2 cytokines in the mercury model, we performed anti-interferon-gamma antibody treatment in IL-4-deficient mice together with mercury treatment and found that the production of IgG2a and IgG3, but not IgG2b, antibodies was downregulated. This indicated that besides Th2-type cytokines, Th1-type and other cytokines were involved as well in mercury-induced autoimmune response. Thus, C57BL/6 mice with H-2b genotype are highly susceptible to mercury-induced autoimmunity, and the genetic susceptibility to mercury involves more than a predisposition of a Th1-or Th2-type response.     

CD8+ immunoregulatory cells in the graft-versus-host reaction: CD8 T cells activate dendritic cells to secrete interleukin-12/interleukin-18 and induce T helper 1 autoantibody

Initiation of cell-mediated immunity or autoimmunity requires secretion of interleukin (IL)-12 from dendritic cells (DC), which drives the generation of T helper 1 (Th1) effector cells in synergy with IL-18. Induction of IL-12 can be triggered by microbial stimuli but also requires signals from activated T cells. We investigated interactions between alloreactive CD4 and CD8 T cells in mixed lymphocyte reactions (MLR) in vitro and in the graft-versus-host reaction (GVHR) in vivo. In a parent-into-F1 model of GVHR, donor CD8 cells were found to suppress the hyper-immunoglobulin E (IgE) syndrome, anti-DNA immunoglobulin G1 (IgG1) autoantibodies and donor CD4-cell expansion, but were essential for Th1-dependent immunoglobulin G2a (IgG2a) autoantibody production and release of serum IL-12 p40. In vitro, addition of alloreactive CD8 cells to CD4 cells and mature DC enhanced Th1 development. CD4 and CD8 T cells induced IL-18 from DC and primed for IL-12 p70 secretion via interferon-gamma (IFN-gamma) or tumour necrosis factor-alpha (TNF-alpha). However CD8 T cells, but not CD4 cells, released IFN-gamma/TNF-alpha after primary stimulation. The data suggest that rapid release of inflammatory cytokines from central memory-type CD8 cells early in immunity is critical for induction of Th1 cells via DC activation and IL-12 production. This pathway could provide a means for amplification of cell-mediated autoimmunity in the absence of microbial stimuli.     

Interleukin-33 alleviates psoriatic inflammation by suppressing the T helper type 17 immune response

Psoriasis is a chronic inflammatory skin disease with unclear pathogenesis. Interleukin-33 (IL-33) is highly expressed in patients with psoriasis, but its role in psoriasis is unknown. The aim of this study was to investigate the possible role of IL-33 in the pathogenesis and treatment of psoriasis. IL-33 expression was determined using enzyme-linked immunosorbent assay, real-time fluorescent quantitative polymerase chain reaction and immunohistochemical staining. CD4⁺ T cells were sorted using magnetic beads and treated with or without IL-33. Imiquimod (IMQ) was used to induce psoriatic inflammation in mice. The frequency of immune cells was determined using flow cytometry. The cytokine level in mouse skin was measured using cytometric bead array. Our results showed that IL-33 was highly expressed in the lesional skin and serum of patients with moderate-to-severe plaque psoriasis. IL-33 inhibited the expression of IL-17 in CD4⁺ T cells of psoriasis patients. Subcutaneous injection of IL-33 alleviated the IMQ-induced psoriatic inflammation in mice, reduced tumor necrosis factor-α and IL-23 expression, and decreased the proportion of T helper type 17 (Th17) cells in the skin-draining lymph nodes in the mice. Our results suggest that IL-33 plays a protective role in the pathogenesis of psoriasis by suppressing Th17 cell differentiation and function. The potential therapeutic effect of IL-33 in treating psoriasis warrants further investigation.     

Negative feedback mechanism suppresses interleukin-12 production by antigen-presenting cells interacting with T helper 2 cells

Interleukin-12 (IL-12) has been shown to be produced by monocytes by the ligation of CD40. In the present experiments, IL-12 is shown to be produced by murine spleen antigen-presenting cells (APC) by interaction with T helper 1 (Th1) 1 clones through CD40-CD40 ligand (CD40L) interaction, but not with Th2 clones. The IL-12 production induced by the Th1 clone interaction was inhibited by the addition of exogenous IL-10. Th2 clones were shown to produce a sufficient amount of IL-10 to inhibit the IL-12 production induced by Th1 clones. In the presence of anti-IL-10 monoclonal antibodies splenic APC interacting with Th2 clones produced IL-12. These results indicate that IL-10 produced by Th2 cells stimulated with antigen suppress IL-12 production of APC interacting with Th2 cells. IL-12 is composed of two subunits, p35 and p40. In our experiments, p40 mRNA accumulation was shown to be affected by IL-10 more severely than the accumulation of p35 mRNA, indicating that IL-10 regulates IL-12 production by APC mainly by affecting p40 mRNA accumulation.     

Resistant mice lacking interleukin-12 become susceptible to Trypanosoma cruzi infection but fail to mount a T helper type 2 response

Interleukin-12 (IL-12) is essential to resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma) that activates macrophages to a parasiticidal effect. Investigation of mice deprived of IL-12 genes (IL-12 knockout mice) has confirmed the important role of IL-12 and IFN-gamma in controlling parasitism in T. cruzi infection. However, it has not yet been addressed whether a shift towards a T helper type 2 (Th2) pattern of cytokine response occurred in these mice that might have contributed to the aggravation of the infection caused by IL-12 deprivation. We examined the course of T. cruzi (Y strain) infection and the regulation of cytokine responses and nitric oxide production in C57BL/6 IL-12 p40-knockout mice. The mutant mice were extremely susceptible to the infection as evidenced by increased parasitaemia, tissue parasitism and mortality in comparison with the control C57BL/6 mouse strain (wild-type) that is resistant to T. cruzi. A severe depletion of parasite-antigen-specific IFN-gamma response, without an increase in IL-4 or IL-10 production, accompanied by reduced levels of nitric oxide production was observed in IL-12 knockout mice. We found no evidence of a shift towards a Th2-type cytokine response. In IL-12 knockout mice, the residual IFN-gamma production is down-regulated by IL-10 but not by IL-4 and nitric oxide production is stimulated by tumour necrosis factor-alpha. Parasite-specific immunoglobulin G1 antibody levels were similar in IL-12 knockout and wild-type mice, whereas IL-12 knockout mice had much higher levels of immunoglobulin G2b.     

Interleukin-12 differentially regulates expression of IFN-gamma and interleukin-2 in human T lymphoblasts.

Interleukin-12 (IL-12) is known to upregulate expression of interferon-gamma (IFN-gamma) by activated T cells. However, the effects of IL-12 on production of other Th1-type cytokines are less well defined. In this study, we examined the effects of IL-12 on expression of several cytokines, including IFN-gamma, IL-2, tumor necrosis factor-alpha (TNF-alpha), and IL-10, by primary human CD3(+) T cells. Although purified resting T cells were largely nonresponsive to IL-12 stimulation, anti-CD3-activated T cell blasts were strongly responsive, as demonstrated by the ability of IL-12 to induce Stat4 DNA-binding activity. Restimulation of T lymphoblasts on immobilized anti-CD3 monoclonal antibodies (mAb) induced rapid expression of TNF-alpha mRNA and more gradual increases in mRNA levels for IL-2, IFN-gamma, and IL-10. IL-12 markedly upregulated expression of IFN-gamma and IL-10 but downregulated expression of IL-2 in a dose-dependent and time-dependent manner. The levels of IL-2 produced by IL-12-treated T cells correlated inversely with the levels of IL-10. Moreover, neutralization of IL-10 activity with anti-IL-10 antibodies normalized IL-2 production by IL-12-treated T cells, confirming that the inhibition of IL-2 production by IL-12 was IL-10 mediated. Thus, IL-12 amplified expression of IFN-gamma and IL-10 and, via its ability to upregulate production of IL-10, inhibited expression of IL-2. These findings demonstrate that IL-12 differentially regulates expression of the Th1-type lymphokines, IFN-gamma and IL-2, in T lymphoblasts.     

Interleukin-12 increases interleukin-4 production by established human ThO and Th2-like T cell clones

Interleukin (IL)-12 is a potent inducer of cell-mediated immunity: it favors the generation of interferon (IFN)-γ-producing T cells, increases IFN-γ production by T cells and natural killer cells and prevents the generation of a Th2 response in murine in vivo models. Nevertheless, the effects of IL-12 on an established Th2 response remain poorly documented. In the present paper, we analyzed the effect of IL-12 on the profile of lymphokines produced by established IL-4-producing Th0 and Th2-like human T cell clones (TCC) and by polyclonal T cells. We found that IL-12 (i) enhances, as previously reported, IFN-γ production by Th0 TCC and, to a smaller extent, by Th2-like TCC, (ii) increases the proliferation of Th0 and Th2-like TCC and (iii) unexpectedly, synergizes with T cell receptor-associated or nonspecific stimuli in increasing IL-4 production by these TCC. Thus, IL-12 potentiates the production of IFN-γ and also of IL-4 by established IL-4-producing TCC. Although IL-12 has been widely reported to induce a Th1 response and to prevent the development of a Th2 response in vivo, IL-12 may on the contrary potentiate an established Th2 response in humans.     

Contribution of mast cells to the T helper 2 response induced by simultaneous subcutaneous and oral immunization

This work examines the contribution of mast cells to the synergistic enhancement of the T helper 2 (Th2) immune response elicited following simultaneous oral and subcutaneous (s.c.) immunization. The s.c. route induced a Th1-biased immune response, characterized by increased interferon-gamma (IFN-gamma) and immunoglobulin G2a (IgG2a) antibody production. In contrast, oral immunization stimulated a primarily Th2-type response in which interleukin-4 (IL-4) and IgG1 antibody production were dominant. Simultaneous immunization also triggered a Th2-biased response, the magnitude of which exceeded the additive effects of s.c. and oral immunization alone by greater than threefold. To analyse whether mast cells in gut-associated lymphoid tissue contributed to this synergistic response, mast cell-deficient mice WBB6F1-w/wv were studied. Whereas the primary response following simultaneously antigen administration was reduced only twofold in these animals compared with wild type controls WBB6F1-+/+ (suggesting that mast cells were not needed to initiate Th2 immunity), reconstitution with bone-marrow-derived mast cells from WBB6F1-+/+ mice resulted in a superoptimal response (suggesting that mast cells contribute to the magnitude and perpetuation of these Th2-biased responses).     

IL-12 plays a pivotal role in LFA-1-mediated T cell adhesiveness by up-regulation of CCR5 expression

The chemokine receptor CCR5 has been implicated in the recruitment of T cells to inflammatory sites. However, the regulation of CCR5 induction on T cells and its contribution to T cell adhesiveness are poorly understood. Using a Th1 clone, 2D6, that can be maintained with interleukin (IL)-12 or IL-2 alone (designated 2D6(IL-12) or 2D6(IL-2), respectively), we investigated how CCR5 is induced on T cells and whether CCR5 is responsible for up-regulating the function of adhesion molecules. 2D6(IL-12) grew, forming cell aggregates, in culture containing IL-12. This was due to lymphocyte function-associated antigen (LFA)-1-intercellular adhesion molecule (ICAM)-1 interaction, because 2D6(IL-12) expressed both LFA-1 and ICAM-1 and cell aggregation was inhibited by anti-ICAM-1 monoclonal antibody. Despite comparable levels of LFA-1 and ICAM-1 expression, 2D6(IL-2) cells did not aggregate in culture with IL-2. It is important that there was a critical difference in CCR5 expression between 2D6(IL-12) and 2D6(IL-2); the former expressed high levels of CCR5, and the latter expressed only marginal levels. Both types of cells expressed detectable albeit low levels of RANTES (regulated on activation, normal T expressed and secreted) mRNA. Unlike IL-12 or IL-2, IL-18 induced high levels of RANTES mRNA expression without modulating CCR5 expression. Therefore, combined stimulation with IL-12 and IL-18 strikingly up-regulated 2D6 cell aggregation. Notably, LFA-1-mediated aggregation of 2D6(IL-12) cells was suppressed by anti-CCR5 antibody. These results indicate that IL-12 plays a critical role in CCR5 expression on Th1 cells and consequently contributes to CCR5-mediated activation of LFA-1 molecules.