Proinflammatory and Immunoregulatory Functions of Interleukin-12

Interleukin-12 (IL-12) is a cytokine composed of two chains, a heavy chain or p40, and a light chain or p35, forming a disulfite-linked heterodimer, or p70. IL-12 was originally discovered as a product of human B lymphoblastoid cell lines; however, the most important physiological producers of IL-12 in vitro are phagocytic cells and antigen-presenting cells rather than B cells. The major target cells of IL-12 action are natural killer and T cells, on which IL-12 induce: (1) production of cytokine, particularly interferon-γ (IFN-γ); (2) proliferation, in synergy with other mitogenic or costimulatory signals; (3) enhancement of cytotoxic activity. In addition, IL-12 has been described to have stimulatory effects on hematopoietic precursor cells and on B lymphocytes. In vivo, IL-12 is produced very early during infections or immune response, and exerts important proinflammatory functions and enhancement of innate resistance by activating natural killer cells and, through IFN-γ induction, phagocytic cells. The IL-12 produced during this inflammatory phase, both by direct action and, indirectly, by determining the composition of the cytokine milieu at the site of the murine response, induces differentiation of T helper type 1 (Th1) cells while inhibiting the generation of Th2 cells. Thus, because of its double function of a proinflammatory cytokine and an immunoregulatory factor, IL-12 plays a key role in the resistance to infections, particularly those mediated by bacteria or intracellular parasites, against which phagocytic cell activation and Th1-mediated responses are particularly effective. However, because of the same activities, IL-12 also plays a role in pathological situations, such as septic shock, tissue damage during inflammation and organ-specific autoimmune diseases.     

Human Th17 cells: Are they different from murine Th17 cells?

Type 17 Th (Th17) cells have been identified as a distinct population of CD4⁺ effector T cells different from Th1 and Th2 cells. While the pre‐eminent cytokine of Th1 cells is IFN‐γ and that of Th2 cells is IL‐4, the distinctive cytokine of Th17 cells is IL‐17A. However, although murine and human Th1 and Th2 cells exhibit strong similarities, human and murine Th17 cells seem to differ in several aspects.     

Regulation by cytokines (IL‐12, IL‐15, IL‐4 and IL‐10) of the Vγ9Vδ2 T cell response to mycobacterial phosphoantigens in responder and anergic HIV‐infected persons

Human Vγ9Vδ2 T cells contribute to immunity against intracellular pathogens and recognize nonpeptidic antigens, such as the mycobacterial phosphoantigen TUBAg. HIV infection is associated with a polyclonal decrease of peripheral Vγ9Vδ2 T cells and we previously reported that the remaining cells show a proliferative anergy to stimulation with Mycobacterium tuberculosis in 60 % of patients. Because of alterations in the Th1/Th2 cytokine balance reported in HIV infection, we analyzed, at the single‐cell level, the influence of exogenous IL‐4, IL‐10, IL‐12 and IL‐15 on the response to mycobacterial phosphoantigens of γ δ T cells from HIV‐infected patients and healthy donors. We report that the strong γ δ T cell response to TUBAg is characterized by the rapid and selective production of the Th1/proinflammatory cytokines IFN‐γ and TNF‐α in responder HIV‐infected donors. In addition, a positive regulation by IL‐12 and IL‐15 of the production of these cytokines by Vγ9Vδ2 T cells in response to nonpeptidic ligands was observed, whereas IL‐4 and IL‐10 had no effect. In contrast, Vγ9Vδ2 T cells from the anergic HIV‐infected donors had lost the ability to produce Th1 cytokines and were not shifted towards a Th2 profile. Furthermore, neither IL‐12 nor IL‐15 could reverse this functional anergy. The consequences of these observations are discussed in the context of HIV pathogenesis.     

IL‐12‐polarized Th1 cells produce GM‐CSF and induce EAE independent of IL‐23

CD4⁺ T‐helper (Th) cells reactive against myelin antigens mediate the mouse model experimental autoimmune encephalomyelitis (EAE) and have been implicated in the pathogenesis of multiple sclerosis (MS). It is currently debated whether encephalitogenic Th cells are heterogeneous or arise from a single lineage. In the current study, we challenge the dogma that stimulation with the monokine IL‐23 is universally required for the acquisition of pathogenic properties by myelin‐reactive T cells. We show that IL‐12‐modulated Th1 cells readily produce IFN‐γ and GM‐CSF in the CNS of mice and induce a severe form of EAE via an IL‐23‐independent pathway. Th1‐mediated EAE is characterized by monocyte‐rich CNS infiltrates, elicits a strong proinflammatory cytokine response in the CNS, and is partially CCR2 dependent. Conversely, IL‐23‐modulated, stable Th17 cells induce EAE with a relatively mild course via an IL‐12‐independent pathway. These data provide definitive evidence that autoimmune disease can be driven by distinct CD4⁺ T‐helper‐cell subsets and polarizing factors.     

IL-4-mediated fine tuning of IL-12p70 production by human DC

IL‐4 is expressed at high levels in allergic diseases and dominates the early phases of multiple acquired immune responses. However, the precise role of IL‐4 during early inflammation and its impact on the differentiation of newly recruited DC precursors remains elusive. In order to characterize the impact of IL‐4 on the differentiation of human DC, we investigated the role of IL‐4 on the differentiation of monocytes into DC. Human DC were differentiated from peripheral blood precursors under either low or high concentrations of IL–4. We analyzed their cytokine profile and capacity to polarize T‐cell differentiation. Concentrations of 5 (low) and 50 (high) ng/mL IL‐4 induced two distinct types of DC. DC differentiated under low‐dose IL‐4 (5 ng/mL) produced almost no IL–12p70, and primed naïve CD4⁺ T cells allowing IL‐4 secretion and Th2 induction. In contrast, DC generated under high concentrations of IL‐4 (50 ng/mL) produced large amounts of IL‐12p70, low IL‐10 and primed naïve CD4⁺ T cells to become Th1 cells. Thus, we demonstrate that the Th2 cell cytokine IL‐4 decisively determines the phenotype of ongoing immune responses by orchestrating the functional phenotype of newly immigrating DC precursors.     

C5a receptor‐deficient dendritic cells promote induction of Treg and Th17 cells

C5a is a proinflammatory mediator that has recently been shown to regulate adaptive immune responses. Here we demonstrate that C5a receptor (C5aR) signaling in DC affects the development of Treg and Th17 cells. Genetic ablation or pharmacological targeting of the C5aR in spleen‐derived DC results in increased production of TGF‐β leading to de novo differentiation of Foxp3⁺ Treg within 12 h after co‐incubation with CD4⁺ T cells from DO11.10/RAG2^?/? mice. Stimulation of C5aR^?/? DC with OVA and TLR2 ligand Pam₃CSK₄ increased TGF‐β production and induced high levels of IL‐6 and IL‐23 but only minor amounts of IL‐12 leading to differentiation of Th cells producing IL‐17A and IL‐21. Th17 differentiation was also found in vivo after adoptive transfer of CD4⁺ Th cell into C5aR^?/? mice immunized with OVA and Pam₃CSK₄. The altered cytokine production of C5aR^?/? DC was associated with low steady state MHC class II expression and an impaired ability to upregulate CD86 and CD40 in response to TLR2. Our data suggest critical roles for C5aR in Treg and Th17‐cell differentiation through regulation of DC function.     

Control of Borrelia burgdorferi-specific CD4+-T-cell effector function by interleukin-12- and T-cell receptor-induced p38 mitogen-activated protein kinase activity

Infection with Borrelia burgdorferi, the causative agent of Lyme disease, results in a Th1 response and proinflammatory cytokine production. Mice deficient for MKK3, an upstream activator of p38 mitogen-activated protein (MAP) kinase, develop a lower Th1 response and exhibit an impaired ability to produce proinflammatory cytokines upon infection with the spirochete. We investigated the contribution of p38 MAP kinase activity in gamma interferon (IFN-gamma) production in CD4+ T cells in response to specific antigen through T-cell receptor (TCR)- and interleukin-12 (IL-12)-mediated signals. The specific inhibition of p38 MAP kinase in T cells and the administration of a pharmacological inhibitor of the kinase during the course of infection with the spirochete resulted in reduced levels of IFN-gamma in the sera of infected mice. Our results also demonstrate that although p38 MAP kinase activity is not required for the differentiation of B. burgdorferi-specific CD4+ T cells, the production of IFN-gamma by Th1 effector cells is regulated by the kinase. Both TCR engagement and IL-12 induced the production of the Th1 cytokine through the activation of the p38 MAP kinase pathway. Thus, the inhibition of this pathway in vitro resulted in decreased levels of IFN-gamma during restimulation of B. burgdorferi-specific T cells in response to anti-CD3 and IL-12 stimulation. These results clarify the specific contribution of the p38 MAP kinase in the overall immune response to the spirochete and its role in the effector function of B. burgdorferi-specific T cells.     

TLR ligands of ryegrass pollen microbial contaminants enhance Th1 and Th2 responses and decrease induction of Foxp3hi regulatory T cells

Microbial contamination of grass pollens could affect sensitization, subsequent allergic response, and efficacy of allergen‐specific immunotherapy. We investigated whether bacterial immunomodulatory substances can direct PBMC responses of allergic and nonatopic subjects against ryegrass pollen (RGP) toward Th1, Th2, or regulatory T (Treg) cells. Aqueous extracts of RGP with high or low LPS were fractionated into large and small molecular weight (MW) components by diafiltration. CFSE‐labeled PBMCs from allergic and nonatopic subjects were stimulated with RGP extracts (RGPEs) and analyzed for cytokine secretion and T‐cell responses. High LPS RGPE increased IFN‐γ⁺ Th1 and IL‐4⁺ Th2 effector cell induction and consistently decreased CD4⁺Foxp3^hi Treg‐cell induction. IL‐10‐producing T‐cell frequency was unaltered, but IL‐10 secretion was increased by high LPS RGPE. RGPE‐stimulation of TLR‐transfected cell lines revealed that high LPS pollen also contained a TLR2‐ligand, and both batches a TLR9‐ligand. Beta‐1,3‐glucans were detected in large and small MW fractions and were also T‐cell stimulatory. In conclusion, coexposure to allergen and proinflammatory microbial stimuli does not convert an established Th2‐ into a Th1‐response. Instead, proinflammatory responses are exacerbated and Foxp3^hi Treg‐cell induction is decreased. These findings show that adjuvants for specific immunotherapy should enhance Treg cells rather than target immune deviation from Th2 to Th1.     

Progressive disease or protective immunity to Leishmania major infection: the result of a network of stimulatory and inhibitory interactions

 The study of experimental infection of inbred strains of mice with the intracellular protozoan parasite Leishmania major has contributed significantly not only to our understanding of this fascinating host/parasite relationship but also to that of many basic immunological phenomena. Much has been learned about the cognate interaction of antigen-specific T cells and antigen-presenting cells, about cytokine and T cell subset regulation, and the requirements for costimulation. Specifically, the immune response to experimental L. major infection is the paradigm for polarized T helper cell (Th) 1 and Th2 differentiation. In this model system a Th1 response characterized by interleukin (IL)-2 and interferon (IFN)-γ secretion leads to self-curing disease, whereas a Th2 response (IL-4, IL-10) leads to nonhealing disease. Numerous manipulations, including the injection of cytokines and of neutralizing anti-cytokine antibodies, cytokine transgene expression, and more recently cytokine and cytokine receptor gene knockout studies, have all provided intriguing new pieces to the still incomplete mosaic of our understanding of the immune response. Some of these findings were clearly unexpected and are still incompletely understood. For instance, based on earlier neutralizing anti-IL-4 monoclonal antibody injection studies, IL-4 gene-disrupted BALB/c mice were expected to be unable to mount the biased Th2 response typical of the IL-4^+/+ wild-type mice and to be able to control their lesions; quite unexpectedly, the BALB/c IL-4 knockout mice remain unable to heal their L. major infection. Based on these unexpected findings, we reexamine the literature in an attempt to resolve this apparent paradox and to relate the large body of experimental findings in the mouse system to that which is known about natural and experimental infections in the human. Received: 18 February 1997 / Accepted: 31 July 1997     

ATP conditions intestinal epithelial cells to an inflammatory state that promotes components of DC maturation

Intestinal epithelial cells (IECs) normally promote the development of gut resident tolerogenic dendritic cells (DCs) and regulatory T cells, but how this process is altered in inflammatory bowel disease is not well characterized. Recently, we published that the cell injury signal ATP modulates IEC chemokine responses to the TLR5 ligand flagellin and exacerbates colitis in the presence of flagellin. We hypothesized that ATP switches these IECs from tolerogenic to proinflammatory, enhancing DC activation and immune responses to commensal antigens. Here, we report that ATP enhanced murine IEC production of KC, IL‐6, TGF‐β, and thymic stromal lymphopoietin in response to TLR1/2 stimulation by Pam₃CSK₄ (PAM). Moreover, supernatants from IECs stimulated with ATP+PAM enhanced expression of CD80 on bone marrow derived dendritic cells, and increased their production of IL‐12, IL‐6, IL‐23, TGF‐β, and aldh1a2, suggesting a Th1/Th17 polarizing environment. DCs conditioned by stressed IECs stimulated an enhanced recall response to flagellin and supported the expansion of IFN‐γ⁺ and IL‐17⁺ memory T cells. Lastly, colonic administration of nonhydrolysable ATP increased production of IL‐6 and Cxcl1 (KC) by IECs. These findings indicate that ATP influences the response of IECs to TLR ligands and biases the maturation of DCs to become inflammatory.     

Interaction between prostaglandin D2 and chemoattractant receptor‐homologous molecule expressed on Th2 cells mediates cytokine production by Th2 lymphocytes in response to activated mast cells

The mechanisms by which immunologically activated mast cells stimulate the production of proinflammatory cytokines by T helper type 2 (Th2) lymphocytes were investigated in a human cell culture system. Supernatants collected from cord blood‐derived mast cells after treatment with immunoglobulin E (IgE)/anti‐IgE contained an activity that stimulated the production of interleukin (IL)‐4, IL‐5 and IL‐13 (both mRNA and protein) by Th2 lymphocytes. This activity was not detected in supernatants from unactivated mast cells and its production was inhibited by treatment of activated mast cells with the cyclo‐oxygenase inhibitor diclofenac. The concentration of diclofenac used inhibited completely the production of prostaglandin D₂ (PGD₂) but did not inhibit the release of histamine or leukotriene C₄. The effect of supernatants from activated mast cells was mimicked by exogenous PGD₂ at concentrations similar to those detected in the cultures of activated mast cells, and addition of exogenous PGD₂ to supernatants from diclofenac‐treated mast cells restored their ability to stimulate Th2 cytokine production. The ability of the mast cell supernatants to stimulate production of Th2 cytokines was not affected by addition of diclofenac to the Th2 cells directly, indicating that the production, but not the action, of the factor was sensitive to diclofenac treatment. Inhibition of chemoattractant receptor‐homologous molecule expressed on Th2 cells (CRTH2) abolished the effect of the mast cell supernatants on Th2 cytokine production. These data indicate that mast cells have the ability to stimulate Th2 cells to elaborate cytokines independently of T cell receptor activation or co‐stimulation and this response is mediated by PGD₂ acting upon CRTH2 expressed by Th2 cells.     

Cytokine responses in acute and persistent human parvovirus B19 infection

The aim of this study was to characterize the proinflammatory and T helper (Th)1/Th2 cytokine responses during acute parvovirus B19 (B19) infection and determine whether an imbalance of the Th1/Th2 cytokine pattern is related to persistent B19 infection. Cytokines were quantified by multiplex beads immunoassay in serum from B19-infected patients and controls. The cytokine responses were correlated with B19 serology, quantitative B19 DNA levels and clinical symptoms. In addition to a proinflammatory response, elevated levels of the Th1 type of cytokines interleukin (IL)-2, IL-12 and IL-15 were evident at time of the initial peak of B19 viral load in a few patients during acute infection. This pattern was seen in the absence of an interferon (IFN)-gamma response. During follow-up (20-130 weeks post-acute infection) some of these patients had a sustained Th1 cytokine response. The Th1 cytokine response correlated with the previously identified sustained CD8+ T cell response and viraemia. A cross-sectional study on patients with persistent B19 infection showed no apparent imbalance of their cytokine pattern, except for an elevated level of IFN-gamma response. No general immunodeficiency was diagnosed as an explanation for the viral persistence in this later group. Neither the acutely infected nor the persistently infected patients demonstrated a Th2 cytokine response. In conclusion, the acutely infected patients demonstrated a sustained Th1 cytokine response whereas the persistently infected patients did not exhibit an apparent imbalance of their cytokine pattern except for an elevated IFN-gamma response.     

Effect of Dehydroepiandrosterone Sulfate on Maturation and Functional Properties of Interferon-α-Induced Dendritic Cells

We studied the effect of adrenal cortex hormone dehydroepiandrosterone sulfate on maturation and functional activity of interferon-α-induced dendritic cells. Dehydroepiandrosterone sulfate stimulated differentiation and maturation of interferon-α-induced dendritic cell, which manifested in a decrease in the number of CD14⁺ cells and increase in the ratio of mature CD83⁺ dendritic cells expressing costimulatory molecules (CD80 and CD86). The induction of dendritic cell differentiation after treatment with dehydroepiandrosterone sulfate was accompanied by an increase in the production of interferon-γ. At the stage of dendritic cell maturation, the effect of dehydroepiandrosterone sulfate manifested in a 4-fold increase in tumor necrosis factor-α production. Dehydroepiandrosterone sulfate had little effect on the production of Th2/antiinfl ammatory cytokines at the stages of differentiation and maturation of interferon-α-induced dendritic cells. Dehydroepiandrosterone sulfate increased the ability of dendritic cells to stimulate Th1 cytokine production by T cells (interferon-γ). This hormone had no effect on the ability of interferon-α-induced dendritic cells to activate CD3⁺IL-4⁺T cells in mixed lymphocyte culture.