Impaired IL-2 Production Research Articles

RIAM Regulate Spatio-Temporal Distribution of PLC-γ1 and Calcium Mobilization during T Cell Activation

T cell receptor (TCR) ligation induces rapid polarization of the actin cytoskeleton resulting in the formation of the immunological synapse (IS), recruitment of signaling molecules, and initiation of signaling cascades leading to T cell activation. Specific recruitment, redistribution and organization of signaling molecules in the IS is facilitated by lipid raft microdomains, which provide a scaffold for focal protein assembly. Fyn and ZAP-70 are the most proximal TCR signaling molecules that localize in the IS and are redistributed in the lipid rafts during T cell activation. Currently, it is poorly understood how signals originating from the TCR are linked to specific mechanisms that regulate T cell activation. We have identified RIAM, an adaptor molecule that contains a RA (Ras Association) domain, a PH (Plekstrin Homology) domain and proline-rich motifs. RIAM interacts with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. RIAM also interacts with ADAP/SKAP-55 and, thereby, is recruited to the plasma membrane during T cell activation. We have previously determined that, during TCR ligation by antigen, RIAM localizes at the IS and the lipid rafts and serves as a substrate for Fyn and ZAP-70. Because of these properties, we examined whether RIAM might be involved in regulating the molecular and functional outcome of T cell activation. Using RIAM-knock down (KD) T cells in which endogenous RIAM was depleted by siRNA, we determined that RIAM was necessary for IL-2 transcription and RIAM-KD cells had impaired capacity for IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking. However, despite the impaired IL-2 production, analysis of TCR-proximal signaling events did not show impairment of ZAP-70 phosphorylation or formation of the LAT signalosome comprised of phosphorylated PLC-γ1, SLP-76 and Vav1. TCR triggering of both control and RIAM-KD cells also resulted in similar phosphorylation of PLC-γ1. Activation of PLC-γ1 leads to the generation of InsP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PtdIns (4,5)P2). InsP3 binds to InsP3 receptors and triggers Ca2+ release from intracellular stores. Strikingly, TCR triggering of RIAM-KD cells resulted in markedly reduced upregulation of InsP3 compared to that in control T cells. Consistent with the defective upregulation of InsP3, calcium flux of RIAM-KD cells was dramatically impaired. This event was due to the impaired InsP3-mediated calcium release from the endoplasmic reticulum and not due to impaired store content or impaired calcium release-activated calcium (CRAC) channel entry as determined by using the Ca2+ ATPase blocker thapsigargin, which resulted in abundant calcium release in RIAM-KD cells. To analyze the consequences of deregulated InsP3 production and to investigate whether RIAM is specifically involved in PLC-γ1-mediated processes we evaluated activation of several signaling events on which PLC-γ1 activation has distinct effects. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 that are PLC-γ1 and Ca2+-dependent, was impaired in the absence of RIAM, activation of p38 and IKK was unaltered compared to control T cells. These results are consistent with a specific role of RIAM in InsP3-mediated Ca2+ release and indicate that deletion of RIAM does not result in a generalized defect in TCR-mediated signaling. Activation of PLC-γ1 at the cell membrane for proper generation of InsP3 requires appropriate docking and positioning of PLC-γ1. For this reason, we examined whether RIAM interacted with PLC-γ1 and regulated its subcellular localization after T cell activation. Detailed analysis by in vivo co-precipitation experiments in cell lysates and by in vitro association assays of purified proteins revealed a direct RIAM-PLC-γ1 interaction that was mediated via the SH3 domain of PLC-γ1. Furthermore, subcellular fractionation into cytoplasmic and cytoskeletal fractions revealed that PLC-γ1 translocated to the cytoskeleton upon T cell activation and this event was abrogated in RIAM-KD cells. These results indicate a novel and unexpected role of RIAM in T cell responses that involves regulation of spatio-temporal distribution and activation of PLC-γ1, leading to generation of InsP3 and Ca2+ mobilization after T cell receptor triggering.

Low serum levels of dehydroepiandrosterone may cause deficient IL-2 production by lymphocytes in patients with systemic lupus erythematosus (SLE).

The principal cause of IL-2 deficiency, a common feature of both murine lupus and human SLE, remains obscure. Recent studies of our own as well as others have shown that dehydroepiandrosterone (DHEA), an intermediate compound in testosterone synthesis, significantly up-regulates IL-2 production of T cells, and that administration of exogenous DHEA or IL-2 via a vaccinia construct to murine lupus dramatically reverses their clinical autoimmune diseases. Thus, we have examined serum levels of DHEA in patients with SLE to test whether abnormal DHEA activity is associated with IL-2 deficiency of the patients. We found that nearly all of the patients examined have very low levels of serum DHEA. The decreased DHEA levels were not simply a reflection of a long term corticosteroid treatment which may cause adrenal atrophy, since serum samples drawn at the onset of disease, which are devoid of corticosteroid treatment, also contained low levels of DHEA. In addition, exogenous DHEA restored impaired IL-2 production of T cells from patients with SLE in vitro. These results indicate that defects of IL-2 synthesis of patients with SLE are at least in part due to the low DHEA activity in the serum.

Down-regulation of dendritic cell signaling pathways by Leishmania amazonensis amastigotes

We have previously reported a link between a deficient Th1 response to Leishmania amazonensis ( La) parasites and profound impairments in the cytokine/chemokine network at early stages of the infection. To define the molecular basis of these deficiencies, we focused on early and intracellular events in La-infected dendritic cells (DCs) in this study. La amastigote-infected DCs were less mature and less potent antigen-presenting cells (APC) than their promastigote-infected counterparts, as judged by the lower expression of CD40 and CD83, suppressed cytokine expression (IL-12p40 and IL-10), reduced effectiveness for priming CD4 + T cells from naïve or infected mice. Infection with La promastigotes, but not amastigotes, triggered transient expression of IL-12p40 by DC. Both forms of parasites markedly suppressed IL-12p40, IL-12p70, and IL-6 production and increased IL-10 production when DCs were treated with LPS, IFN-γ/LPS or IFN-α/LPS as positive stimuli. Of note, pre-infection of DCs with live amastigotes resulted in multiple alterations in innate signaling pathways, including degradation of STAT2, decreased phosphorylation of STAT1, 2, 3 and ERK1/2, and markedly reduced expression of interferon regulatory factor-1 (IRF-1) and IRF-8, some of which were partially reversed by pretreatment of parasites with proteasome or protease inhibitors. The impaired IL-12 production in infected DCs was not attributed to increased IL-10 production. Together, our data suggest that La parasites, especially in their intracellular forms, have evolved unique strategies to actively down-regulate early innate signaling events, resulting in impaired DC function and Th1 activation.

Hyaluronan Synthesis Is Required for IL-2-Mediated T Cell Proliferation

Hyaluronan (HA) is a glycosaminoglycan composed of N-acetylglucosamine and glucuronic acid subunits. Previous studies have suggested that CD44 expressed by T cells bind exogenous HA for their proliferation. However, HA endogenously synthesized by T cells may participate in their autocrine proliferation. In this study, we examined the role of endogenous HA in T cell proliferation using the highly specific HA synthase inhibitor, 4-methylumbelliferone (4-MU). We found that 4-MU inhibited the mitogen-induced synthesis of HA by T cells. Moreover, 4-MU inhibited T cell proliferation in a dose-dependent manner when cells were cultured with different stimuli, including Con A, PMA/ionomycin, and allogeneic spleen cells. Furthermore, 4-MU inhibited mitogen-stimulated IL-2 secretion, suggesting that HA may play a role in the production of this cytokine. Addition of IL-2 to T cells treated with 4-MU and Con A reversed the block in cell proliferation, showing that impaired IL-2 production is a likely mechanism for the inhibited division of T cells. Surprisingly, an anti-CD44 Ab antagonistic for HA binding did not reduce IL-2 secretion or T cell proliferation. Importantly, 4-MU did not alter the surface expression of CD44 or the ability of CD44 to bind to HA. Thus, HA-mediated IL-2 production and T cell proliferation are CD44 independent. Our results strongly suggest that HA synthesized by T cells themselves is critical for their IL-2-mediated proliferation and have revealed a previously unrecognized role for endogenous HA in T cell biology.

LIGHT Is Critical for IL-12 Production by Dendritic Cells, Optimal CD4+ Th1 Cell Response, and Resistance to Leishmania major

Although studies indicate LIGHT (lymphotoxin (LT)-like, exhibits inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator (HVEM), a receptor expressed by T lymphocytes) enhances inflammation and T cell-mediated immunity, the mechanisms involved in this process remain obscure. In this study, we assessed the role of LIGHT in IL-12 production and development of CD4(+) Th cells type one (Th1) in vivo. Bone marrow-derived dendritic cells from LIGHT(-/-) mice were severely impaired in IL-12p40 production following IFN-gamma and LPS stimulation in vitro. Furthermore, blockade of LIGHT in vitro and in vivo with HVEM-Ig and LT beta receptor (LTbetaR)-Ig leads to impaired IL-12 production and defective polyclonal and Ag-specific IFN-gamma production in vivo. In an infection model, injection of HVEM-Ig or LTbetaR-Ig into the usually resistant C57BL/6 mice results in defective IL-12 and IFN-gamma production and severe susceptibility to Leishmania major that was reversed by rIL-12 treatment. This striking susceptibility to L. major in mice injected with HVEM-Ig or LTbetaR-Ig was also reproduced in LIGHT(-/-) --> RAG1(-/-) chimeric mice. In contrast, L. major-infected LTbeta(-/-) mice do not develop acute disease, suggesting that the effect of LTbetaR-Ig is not due to blockade of membrane LT (LTalpha1beta2) signaling. Collectively, our data show that LIGHT plays a critical role for optimal IL-12 production by DC and the development of IFN-gamma-producing CD4(+) Th1 cells and its blockade results in severe susceptibility to Leishmania major.

Exogenous Interferon-α and Interferon-γ Increase Lethality of Murine Inhalational Anthrax

Background Bacillus anthracis, the etiologic agent of inhalational anthrax, is a facultative intracellular pathogen. Despite appropriate antimicrobial therapy, the mortality from inhalational anthrax approaches 45%, underscoring the need for better adjuvant therapies. The variable latency between exposure and development of disease suggests an important role for the host's innate immune response. Type I and Type II Interferons (IFN) are prominent members of the host innate immune response and are required for control of intracellular pathogens. We have previously described a protective role for exogenous Type I and Type II IFNs in attenuating intracellular B.anthracis germination and macrophage cell death in vitro.Methodology and Principal FindingsWe sought to extend these findings in an in vivo model of inhalational anthrax, utilizing the Sterne strain (34F2) of B.anthracis. Mice devoid of STAT1, a component of IFN-α and IFN-γ signaling, had a trend towards increased mortality, bacterial germination and extrapulmonary spread of B.anthracis at 24 hrs. This was associated with impaired IL-6, IL-10 and IL-12 production. However, administration of exogenous IFN-γ, and to a lesser extent IFN-α, at the time of infection, markedly increased lethality. While IFNs were able to reduce the fraction of germinated spores within the lung, they increased both the local and systemic inflammatory response manifest by increases in IL-12 and reductions in IL-10. This was associated with an increase in extrapulmonary dissemination. The mechanism of IFN mediated inflammation appears to be in part due to STAT1 independent signaling.ConclusionsIn conclusion, while endogenous IFNs are essential for control of B.anthracis germination and lethality, administration of exogenous IFNs appear to increase the local inflammatory response, thereby increasing mortality.

Diacylglycerol kinase zeta regulates microbial recognition and host resistance to Toxoplasma gondii (51.16)

Abstract Mammalian Toll-like receptors (TLRs) recognize microbial pathogen-associated molecular patterns and are critical for innate immunity against microbial infection. Diacylglycerol kinases (DGKs) regulate the intracellular levels of two important second messengers involved in signaling from many surface receptors by converting diacylglycerol (DAG) to phosphatidic acid (PA). Here, we demonstrate that the ζ isoform of the DGK family (DGKζ) is expressed in macrophages (Mϕ) and dendritic cells (DC). DGKζ deficiency results in impaired IL-12 and TNFα production following TLR stimulation in vitro and in vivo, increased resistance to endotoxin shock, and enhanced susceptibility to Toxoplasma gondii infection. We further show that DGKζ negatively controls the PI3K-Akt pathway and that inhibition of PI3K activity can restore LPS-induced IL-12 production by DGKζ deficient macrophages. Collectively, our data provide the first genetic evidence that an enzyme involved in DAG/PA metabolism plays an important role in innate immunity and indicate that DGKζ promotes TLR responses via a pathway involving inhibition of PI3K.

RIAM Regulates Actin Reorganization at the Immunological Synapse and Calcium Mobilization during T Cell Activation.

Engagement of immune recognition subunits on lymphocytes results in cytoskeletal reorganization, polarization of polymerized actin, conjugate formation between T cells and APC, stabilization of the immunological synapse (IS), and initiation of signaling cascades leading to T cell activation. Actin remodeling is essential for these events and is mandatory for T cell activation. We have identified RIAM, a novel adaptor molecule that interacts specifically with active GTP-bound Rap1 and with regulators of the actin cytoskeleton Evl, VASP and Profilin. Profilin associates with G-actin and promotes actin polymerization by adding actin monomers to the barbed ends of F-actin. Ena/VASP family proteins are cytoskeletal proteins and regulate actin dynamics. Via these interactions, RIAM functions as a regulator of the actin cytoskeleton. RIAM also has a mandatory role in regulating activation of integrins downstream of Rap1. The ability of a T cell to recognize and conjugate with an APC is dependent on cytoskeletal dynamics and “inside out” signaling leading to integrin-mediated adhesion. Activation of the b2 integrin LFA-1 leads to high avidity binding to ICAM proteins, which is crucial for stable T cell-APC conjugation. Because RIAM is involved both in cytoskeletal dynamics and integrin activation, we examined whether RIAM had a role in this process. Stable GFP-transfected Jurkat T cells were incubated with Raji B cells as APC, in the presence or absence of SEE. T cell:APC conjugates and formation of IS were examined by confocal microscopy. Synapse formation between GFP-Jurkat cells and Raji B cells was detected only in the presence of SEE. Under these conditions, polymerized (F) actin was highly recruited at the IS, as determined by phalloidin staining. Staining with RIAM-specific antibody indicated that in the absence of antigen, RIAM was diffusely expressed in the cytoplasm and at the plasma membrane. Impressively, upon incubation with SEE loaded Raji cells, RIAM was redistributed at the IS where it co-localized with polymerized (F) actin and ZAP-70. Knockdown of RIAM in Jurkat T cells resulted in significant defects in conjugate formation with SEE loaded B cells. Thus RIAM is involved in actin reorganization in the IS and in the formation of T cell:APC conjugates. Because appropriate actin reorganization is required for T cell activation we next explored whether RIAM knockdown leads to defects in TCR-mediated signaling events. RIAM-knockdown Jurkat T cells displayed significantly impaired IL-2 production in response to SEE loaded Raji and in decreased IL-2 promoter driven transcriptional activity. Surprisingly, despite the significant decrease in IL-2 transcription, RIAM-knockdown Jurkat cells did not show altered TCR-induced phosphorylation of PLCg1, or activation of MAP kinases including MEK, Erk1/2 and JNK, compared to control cells. Moreover, AP-1 and NF-kB-mediated gene transcription induced by CD3-plus-CD28 was unaffected. In contrast, NFAT-driven transcription was significantly impaired. Assessment of intracellular calcium by flow cytometry indicated that RIAM-knockdown cells had significantly reduced TCR-mediated intracellular calcium signal. These results indicate that RIAM is an important component of a signaling pathway that regulates calcium mobilization and NFAT mediated gene transcription in T lymphocytes.

CD40/CD154 Interactions Are Required for the Optimal Maturation of Skin-Derived APCs and the Induction of Helminth-Specific IFN-γ but Not IL-4

The mechanisms through which Schistosoma mansoni larvae induce Th1 rather than Th2 immune responses are not well understood. In this study, using CD154-/- mice exposed to radiation-attenuated S. mansoni larvae, we demonstrate roles for CD154/CD40 in the activation of skin-derived APCs and the development of Th1 cells in the skin-draining lymph nodes (sdLN). The presence of CD154 was important for optimal IL-12p40 and essential for Ag-specific IFN-gamma, but CD154 expression by wild-type CD4- cells was insufficient to rescue recall responses of CD4+ cells from CD154-/- mice. This defect is probably due to impaired CD40-dependent IL-12 production in vivo, because administration of anti-CD40 Ab, or rIL-12, restored IFN-gamma production by sdLN cells from CD154-/- mice. CD154 ligation of CD40 was not required for the migration of skin-derived APCs, but did have a limited role in their maturation (increased MHC II and CD86). Unexpectedly, although CD4 cells from CD154-/- mice were deficient in their ability to produce IFN-gamma, they produced significant amounts of IL-4 and IL-5 in the presence of skin-derived APCs from wild-type and CD154-/- mice. Thus, in contrast to IFN-gamma, the production of Th2-associated cytokines is (in this model) independent of CD154. We conclude that whereas the priming of Th1 responses soon after exposure to schistosome larvae is completely CD40/CD154 dependent, IL-4, IL-5, and IL-13 are independent of CD154, suggesting a dichotomy in the specific mechanisms that induce these cytokines by CD4+ cells in the sdLN.

Possibility of inducing anterior chamber-associated immune deviation by TGF-β2 treatment of monocytes isolated from Behcet's patients

Murine macrophages treated with TGF-β2 are capable of inducing anterior chamber-associated immune deviation (ACAID), and these macrophages are characterized by impaired IL-12 production and CD40 expression, consequently failing to promote Th1 cell differentiation. In this study, we investigated whether human monocytes can also acquire the specific functions by TGF-β2 treatment, even when the monocytes are isolated from patients with Behcet's disease (BD). Adherent monocytes isolated from peripheral blood mononuclear cells (PBMC) of 16 BD patients and 16 healthy controls, were cultured overnight with or without 5 ng/ml of TGF-β2. Then, TGF-β2-treated or untreated adherent cells were co-cultured with allogeneic CD4 + T cells obtained from healthy subjects. TGF-β2 treatment inhibited the abilities of adherent monocytes obtained from BD patients to stimulate the proliferation and IFN-γ production of allogeneic CD4 + T cells. The reduced IFN-γ production was also confirmed by IFN-γ mRNA expression in the co-cultured T cells. IL-12 production and CD40 molecule expression by adherent monocytes obtained from BD patients were strikingly reduced by TGF-β2 treatment. These results suggest a possibility that adherent monocytes isolated from BD patients may acquire a property to induce ACAID by treatment with TGF-β2.

Cutting Edge: Diminished T Cell TLR Expression and Function Modulates the Immune Response in Human Filarial Infection

Patent lymphatic filariasis is characterized by profound Ag-specific T cell hyporesponsiveness with impaired IFN-gamma and IL-2 production. Because T cells have been shown to express a number of TLR and to respond to TLR ligands, we hypothesized that diminished T cell TLR function could partially account for the T cell hyporesponsiveness in filariasis. T cells expressed TLR1, TLR2, TLR4, and TLR9, and the baseline expression of TLR1, TLR2, and TLR4, but not TLR9 was significantly lower in T cells of the filarial-infected individuals compared with the uninfected individuals (both endemic and nonendemic). TLR function was significantly diminished in the T cells of filarial-infected individuals based on decreased T cell activation/cytokine production in response to TLR ligands. Thus, diminished expression and function of T cell TLR is a novel mechanism underlying T cell immune tolerance in lymphatic filariasis.

Defective nuclear translocation of nuclear factor of activated T cells and extracellular signal-regulated kinase underlies deficient IL-2 gene expression in Wiskott-Aldrich syndrome

Proliferation and IL-2 production in response to T-cell receptor ligation are impaired in patients with Wiskott-Aldrich syndrome (WAS). The transcription factors nuclear factor-kappaB (NF-kappaB), nuclear factor of activated T cells (NF-AT), and activating protein-1 (AP-1) play a critical role in IL-2 gene expression. To investigate the mechanisms of impaired IL-2 production after T-cell receptor ligation in T cells deficient in WAS protein (WASP). T cells from WASP-/- mice were stimulated with anti-CD3 and anti-CD28. Nuclear NF-kappaB, NF-AT, and AP-1 DNA-binding activity was examined by electroshift mobility assay. NF-ATp dephosphorylation and nuclear localization were examined by Western blot and indirect immunofluorescence. Phosphorylation of the mitogen-activated protein kinases Erk and Jnk, and of their nuclear substrates Elk-1 and c-Jun, was examined by Western blot. Expression of mRNA for IL-2 and the NF-kappaB-dependent gene A20 and of the AP-1 components c-fos and c-Jun was examined by quantitative RT-PCR. Nuclear translocation and activity of NF-kappaB were normal in T cells from WASP-/- mice. In contrast, NF-ATp dephosphorylation and nuclear localization, nuclear AP-1 binding activity, and expression of c-fos, but not c-Jun, were all impaired. Phosphorylation of Jnk, c-Jun, and Erk were normal. However, nuclear translocation of phosphorylated Erk and phosphorylation of its nuclear substrate Elk1, which activates the c-fos promoter, were impaired. These results suggest that WASP is essential for NF-ATp activation, and for nuclear translocation of p-Erk, Elk1 phosphorylation, and c-fos gene expression in T cells. These defects underlie defective IL-2 expression and T-cell proliferation in WAS.

CCR8 Is Expressed by Antigen-Elicited, IL-10-Producing CD4+CD25+ T Cells, Which Regulate Th2-Mediated Granuloma Formation in Mice

CCR8 was initially described as a Th2 cell-restricted receptor, but this has not been fully tested in vivo. The present study used ex vivo and in vivo approaches to examine the distribution and functional significance of CCR8 among CD4+ T cells. Populations of cytokine-secreting CD4+ T cells were generated in primed mice with Th1 or Th2 cell-mediated pulmonary granulomas, respectively elicited by i.v. challenge with either Mycobacteria bovis purified protein derivative- or Schistosoma mansoni egg Ag (SEA)-coated beads. Cytokine-producing CD4+ T cells were isolated from Ag-stimulated draining lymph node cultures by positive selection. Quantitative analysis of cytokine mRNA indicated enriched populations of IFN-gamma-, IL-4-, and IL-10-producing cells. Analysis of chemokine receptor mRNA indicated that IL-10+ cells selectively expressed CCR8 in the SEA bead-elicited type 2 response. The IL-10+CCR8+ populations were CD25+ and CD44+ but lacked enhanced Foxp3 expression. Adoptive transfer to naive recipients indicated that IL-10+ T cells alone could not transfer type 2 inflammation. Analysis of SEA bead-challenged CCR8-/- mice indicated significantly impaired IL-10 production as well as reductions in granuloma eosinophils. Adoptive transfer of CD4+CCR8+/+ T cells corrected cytokine and inflammation defects, but the granuloma eosinophil recruitment defect persisted when donor cells were depleted of IL-10+ cells. Accordingly, local IL-10 production correlated with CCR8 ligand (CCL1) expression and the appearance of CCR8+ cells in granulomatous lungs. Thus, IL-10-producing, CCR8+CD4+CD25+CD44+ T cells are generated during SEA challenge, which augment the Th2-mediated eosinophil-rich response to the parasite Ags.

Defective production of soluble HLA-G molecules by peripheral blood monocytes in patients with asthma

HLA-G, a human nonclassic MHC class I molecule, is responsible for complex immunoinhibitory functions. HLA-G is expressed as membrane-bound and is secreted as soluble molecules by the peripheral blood CD14+ monocytes activated by IL-10. It has been reported that LPS stimulation induces IL-10 production by PBMCs and that IL-10 levels are reduced in patients with severe asthma compared with patients with mild asthma and healthy subjects. The study was designed to investigate whether this impaired IL-10 production can affect the expression and the secretion of soluble HLA-G (sHLA-G)-1/HLA-G5 molecules. We investigated the production of sHLA-G1/HLA-G5 and IL-10 by specific ELISAs in the culture supernatants of LPS-activated PBMCs from 24 healthy subjects and 20 patients with moderate to severe persistent asthma. LPS stimulation induced the secretion of IL-10 and sHLA-G1/HLA-G5 molecules in all healthy subjects, whereas in patients with asthma, the levels of IL-10 were significantly lower (P < .001) and the number of cultures exhibiting detectable sHLA-G1/HLA-G5 was reduced (7/20; P < .001). The addition of exogenous IL-10 to LPS-stimulated PBMCs from patients with asthma restored normal sHLA-G1/HLA-G5 production. Our data suggest that a specific deficit of IL-10 secretion in patients with asthma could prevent the normal production of sHLA-G1/HLA-G5 molecules. The reduction of immunosuppressive activity mediated by HLA-G could in turn contribute to the persistence of chronic airway inflammation in asthma.

Unexpected effects of viral interleukin-10–secreting dendritic cells in vivo: Preferential inhibition of TH2 responses

Viral interleukin (IL)-10 (vIL-10) has been widely described as an immunoregulatory cytokine that does not possess the T-cell costimulatory activities of cellular IL-10; it was therefore believed to be a more potent tolerogenic mediator. The immunosuppressive properties of this cytokine are partly attributed to its capacity to render dendritic cells (DCs) unable to undergo full maturation and to activate T cells. We reported here that myeloid DCs retrovirally transduced with vIL-10 had an impaired production of IL-12 and a decreased expression of MHC class II molecules but had minor defects in costimulatory molecule expression and no alteration on CCR5 and CCR7 expression. In mixed leukocyte reaction, vIL-10–transduced C57BL/6 bm12 (MHC class II mismatch) DCs had a reduced capacity to stimulate C57BL/6 wild-type CD4 + T-cell proliferation. We show that bm12 vIL-10–transduced DC administration in CD8 −/− C57BL/6 mice promoted IFN-γ production, down-regulated TH2-type cytokine production, and did not induce skin graft tolerance. These findings suggest that vIL-10–transduced DC may surprisingly facilitate Th1-type inflammatory responses in vivo.

Side-by-Side Comparison of Lentivirally Transduced and mRNA-Electroporated Dendritic Cells: Implications for Cancer Immunotherapy Protocols

The use of tumor antigen-loaded dendritic cells (DC) is one of the most promising approaches to inducing a tumor-specific immune response. We compared electroporation of mRNA to lentiviral transduction for the delivery of tumor antigens to human monocyte-derived and murine bone marrow-derived DC. Both lentiviral transduction and mRNA electroporation induced eGFP expression in on average 81% of human DC. For murine DC, eGFP mRNA electroporation (62%) proved to be more efficient than lentiviral transduction (47%). When we used tNGFR as a transgene we observed lentiviral pseudotransduction that overestimated lentiviral efficiency. Neither gene transfer method had an adverse effect on viability, phenotype, or allostimulatory capacity of either human or murine DC. Yet, the mRNA-electroporated DC showed a reduced production of IL-12p70 compared to their lentivirally transduced and unmodified counterparts. Human Ii80MAGE-A3-modified DC and murine Ii80tOVA-modified DC were able to present antigenic epitopes in the context of MHC class I and class II. Both types of modified murine DC were able to induce OVA-specific cytotoxic T cells in vivo; however, the mRNA-electroporated DC were less potent. Our data indicate that this may be related to their impaired IL-12 production.

Graves' hyperthyroidism and the hygiene hypothesis in a mouse model.

Graves' hyperthyroidism is an organ-specific autoimmune disease mediated by stimulatory autoantibodies against the TSH receptor (TSHR; thyroid-stimulating antibodies), causing thyroid hyperplasia and hyperthyroidism. Development of this ailment is well known to be under polygenic and environmental control. For example, we recently demonstrated that parasite helminth Schistosoma mansoni infection suppressed a T helper cell type 1 (Th1)-type anti-TSHR immune response and prevented disease development in our mouse model of Graves' disease using adenovirus coding for the TSHR. In the present study we examined the outcome of infection with Mycobacterium bovis bacillus Calmette-Guerin (BCG), a Th1-promoting infectious pathogen, on Graves' disease. Our results show that prior infection with M. bovis BCG differentiates the TSHR-specific immune response toward a Th1 phenotype, as demonstrated by enhanced secretion of a Th1 cytokine interferon-gamma and impaired production of a Th2 cytokine IL-10 from splenocytes stimulated in vitro with TSHR antigen. M. bovis BCG also significantly suppressed disease induction. These data together with our recent report that coinjection of adenovirus expressing the Th1 cytokine IL-12 induced a Th1-polarized, TSHR-specific immune response without affecting disease development support the hygiene hypothesis, rather than Th1-mediated disease suppression. Thus, some infectious pathogens may influence the development of Graves' disease regardless of their ability to modify the Th1/Th2 balance.

In vitro treatment of dendritic cells with tacrolimus: impaired T-cell activation and IP-10 expression.

High doses (10(-6)-10(-8) M) of tacrolimus (FK506) were reported to induce a type-2 T-helper cell (Th2)-promoting function in developing dendritic cells (DC). We used a therapeutic dose (2.4 x 10(-9 )M) of tacrolimus to investigate its effect on human monocyte-derived DC. Using untreated and treated immature and mature DC we compared T cell-activating capacity, surface marker expression, T cell and DC cytokine profile and transcription of genes coding for a panel of DC function-related molecules. Tacrolimus-treated mature DC had reduced T-cell stimulatory capacity. Although interleukin (IL)-12 production of DC was impaired, they did not promote Th2 development as T cells activated by tacrolimus-treated DC produced less interferon (IFN)-gamma, IL-4 and IL-10. The up-regulation of the T-cell activation marker CD69 and the production of IL-2 were impaired. In addition, tacrolimus-treated DC produced less IP-10 (CXCL10), which is known to be involved in allograft rejection. Other molecules related to DC function remained unchanged. Tacrolimus treatment reduces the ability of DC to stimulate T cells and the impaired production of DC-derived IP-10 (CXCL10) and IL-12 might play a role in the immunosuppressive action of tacrolimus.

Deregulated production of protective cytokines in response to Candida albicans infection in patients with chronic mucocutaneous candidiasis.

Patients with chronic mucocutaneous candidiasis (CMC) are selectively unable to clear the yeast Candida, which results in persistent debilitating infections affecting the skin, nails, and mucous membranes. The underlying defect is unknown. Recent animal studies highlighted the importance of type 1 cytokines in protection against Candida, and previous work suggested that CMC patients may exhibit altered cytokine production in response to Candida. Based on these findings, in this study we investigated cytokine production in CMC patients by assessing a range of inflammatory, anti-inflammatory, type 1, and type 2 cytokines (interleukin-2 [IL-2], IL-4, IL-5, IL-6, IL-10, IL-12, gamma interferon [IFN-gamma], tumor necrosis factor alpha [TNF-alpha]) in whole-blood cultures in response to five different fractions of Candida albicans (carbohydrate, purified mannan, and protein-rich fractions, etc.), as well as non-Candida antigens. Our results demonstrate that cytokine production is deregulated in a Candida-specific way for some cytokines (IL-2, IL-10), is deregulated more generally for other cytokines (IL-12, IL-6, IFN-gamma), and is not markedly altered for still other cytokines (TNF-alpha, IL-4, IL-5). The most notable finding in CMC patients was the markedly impaired production of IL-12 in parallel with dramatically increased levels of IL-6 and IL-10 that occurred selectively in response to Candida. These results suggest that patients with CMC have impaired production of type 1-inducing cytokines (possibly a macrophage or dendritic cell defect?), which could result in an inability to mount protective cell-mediated responses and a failure to clear Candida. Continued tissue damage and inflammation may trigger production of high levels of inhibitory cytokines, such as the IL-10 production seen in our study, which would further reduce production of type 1-inducing cytokines in a positive feedback loop leading to persistent infection.

IL-11-induced reduction of C/EBP transcription factor binding may contribute to the IL-12 downregulation in tumor-bearing mice

Using a mammary tumor model syngeneic to BALB/c mice, we have characterized several tumor-derived factors. We now report that the DA-3 cell line derived from this tumor, as well as the in vivo tumor itself, express IL-11. The expression of IL-11 in the tumor is detectable at the transcriptional and translational levels, as evidenced by RT-PCR and Western blots. Using a murine IL-11 ELISA, we observed no differences in IL-11 production between normal and tumor-bearer's macrophages or T cells, with or without activation. Interestingly, elevated levels of IL-11 were found in the sera of tumor-bearers, when compared to normal animals and even higher levels of IL-11 were detected in the tumor cystic fluid. Macrophages from mice bearing large mammary tumors show an impaired production of IL-12 and NO, whereas T cells from the same animals display a deficient production of IFN-gamma. Pretreatment of normal macrophages with IL-11 resulted in no decrease in NO production, nor an impaired production of IFN-gamma was observed in normal T cells upon pretreatment with IL-11. However, pretreatment of normal macrophages with IL-11 resulted in a decreased production of IL-12, as revealed by ELISA and RT-PCR. Electromobility shift assays showed decreased binding of the transcription factor C/EBP to the IL-12p40 promoter of LPS-activated macrophages from normal animals, upon pretreatment with IL-11. In contrast, no differences were observed in the levels of NFkappaB binding under the same experimental conditions. Our results suggest that tumor-derived IL-11 may play a role in the depressed IL-12 production by macrophages, leading to the impaired immune functions observed during mammary tumorigenesis.