IFN-gamma Induction Research Articles

Impact of interleukin-12, oxidative burst, and iNOS on the survival of murine fecal peritonitis

Abdominal sepsis due to secondary fecal peritonitis following anastomosis insufficiency is a rare but life threatening complication of colorectal surgery. The induction of IFN-gamma by IL-12 is believed to play a key role in sepsis as it promotes antibacterial effector mechanisms such as oxidative burst or nitric oxide induction. The impact of gene deficiency for IL-12 (IL-12p40 KO), oxidative burst (p47(phox) KO), or NO induction (iNOS KO) on the outcome of fecal peritonitis was characterized using the murine Colon Ascendens Stent Peritonitis model (CASP). In the IL-12p40 KO model, 3 and 12 h after surgery, serum cytokine levels of IL-1beta, TNF, IL-18, and IL-10 were analyzed. Expression of IL-1beta, IL-10, IP-10, and MIP-1alpha was measured in lung and liver by RNAse Protection Assay. IL-12p40 and iNOS-deficient mice exhibited a significantly higher susceptibility to CASP as compared to the controls, whereas no significant difference was observed in p47(phox) KO mice. Absence of IL-12 resulted in delayed expression of proinflammatory cytokines and chemokines in both the liver and the lung, and was associated with significant reduction of IL-1beta levels in the serum 12 h after CASP. IL-12 and iNOS possess protective functions in fecal murine peritonitis. Surprisingly, no significant contribution of oxidative burst to the immune response was observed. Overall, these findings suggest that IL-12 deficiency causes a profound delay of the immune response after polymicrobial challenge resulting in significantly increased susceptibility in the CASP model.

Combination Nonviral Interleukin-2 Gene Immunotherapy For Head and Neck Cancer: From Bench Top to Bedside

Intralesional delivery of cytokine genes has emerged as a promising therapeutic strategy for the treatment of cancer. In addition to the therapeutic effect of the delivered cytokine gene, the components of the gene delivery system also have been shown to induce beneficial immune responses. On the basis of these principles, we hypothesized that a molecular therapy could be developed that would provide synergistic antitumor activity by way of intralesional expression of interleukin (IL)-2 from a recombinant plasmid combined with induction of endogenous interferon (IFN)-gamma and IL-12 cytokines by immunostimulatory DNA. Our objective in these studies was to create and optimize a novel formulation of cationic lipid and DNA that generates local production of IL-2 protein within a targeted tumor environment with concomitant induction of the antitumor cytokines IFN-gamma and IL-12. Prospective laboratory drug development plan that would produce human clinical trials. Engineered bacterial plasmids containing a cytomegalovirus promoter (CMV)-IL-2 expression cassette were specifically formulated with cationic lipids and optimized for antitumor effect in a floor of mouth murine tumor model. The treated tumors were assayed for local expression of IL-2 and concurrent expression of secondary cytokines IFN-gamma and IL-12. Established tumors in C3H/HeJ mice were treated with various IL-2 gene formulations, and clinical and immunologic responses were evaluated. Immunologic studies were performed and included cytolytic T-cell assays and cytokine expression profiles. For human clinical trials, a phase I 10 patient formulated IL-2 gene therapy study was completed. Subsequently, two large scale, phase II multi-institutional and multi-international studies were initiated comparing non-viral IL-2 gene therapy to palliative methotrexate chemotherapy or in combination with cisplatin. In the preclinical stage, maximum tumor inhibition in animal models was obtained using IL-2 plasmid formulated with 1,2-dioleyloxypropyl-3-trimethyl ammonium chloride (DOTMA):cholesterol (1:1 mol:mol) at a plasmid:lipid charge ratio of 1:0.5 (-/+). Cationic lipid formulated IL-2 plasmid significantly inhibited tumor growth compared with formulated control plasmid (P < .01) or vehicle (lactose; P < .01). Consistent with previously reported studies of the immunostimulatory activity of DNA of bacterial origin, treatment of tumors with control plasmid in cationic lipid formulation induced production of endogenous IFN-gamma and IL-12 but not IL-2. Treatment of tumors with formulated IL-2 plasmid produced IL-2 protein levels that were 5-fold over background and increased IFN-gamma by 32-fold (P < .001) and IL-12 by 5.5-fold (P < .001) compared with control plasmid formulations. The phase I human trial demonstrated dose escalation safety, which was its primary objective, and there was one anecdotal reduction in tumor size. The phase II studies have been initiated and focus on either comparing the novel nonviral IL-2 gene immunotherapy formulation alone to methotrexate or comparing IL-2 gene therapy in combination with cisplatin in recurrent or unresectable patients with head and neck squamous cell carcinoma. The preclinical data provided proof of principle for matching a delivered IL-2 transgene with an immunostimulatory nonviral formulation to enhance intralesional production of therapeutic cytokines for the maximization of antitumor response. Human clinical trials have demonstrated this novel therapy to be safe in the human clinical setting. Phase II trials have been initiated to assess efficacy and feasibility as a single or combination therapy for head and neck cancer.

IP-10 Mediates Selective Mononuclear Cell Accumulation and Activation in Response to Intrapulmonary Transgenic Expression and During Adenovirus-Induced Pulmonary Inflammation

CXC chemokines that lack the glutamine-leucine-arginine (ELR) motif, including interferon (IFN)-inducible protein 10 (IP-10 or CXCL10), have been shown to mediate the generation of type 1 immune responses. In this study, we found that the intrapulmonary transient transgenic expression of murine IP-10 in mice using adenoviral gene transfer resulted in the early accumulation of neutrophils, natural killer (NK) cells, and NK T cells within the lung, followed by the delayed accumulation of CD4+ T cells. Adenovirus-mediated transgenic expression of IP-10 also resulted in selective activation of mononuclear cells, including gamma(delta)-T cells and NK cells, as manifest by CD69 expression or induction of cell-associated IFN-gamma. Importantly, the intratracheal (i.t.) administration of a control human type 5 adenovirus also caused significant accumulation of NK, NK T, and CD4+ T cells, which was maximal at 7 days post vector administration and was associated with the induction of IP-10. Neutralization of endogenous IP-10 in animals receiving control adenovirus resulted in decreases in the numbers of NK, CD4+, and CD8+ T cells. These results indicate that IP-10 can direct the accumulation and activation of neutrophils and selected mononuclear cells to the lung and that adenovirus-induced IP-10 contributes to lung inflammatory cell recruitment/activation observed in response to adenoviral vectors used for gene therapy.

B7-1 and B7-2 act differentially in the induction of a T cell response: Their impact for a HLA-matched and HLA-mismatched anti-tumor immunotherapy

The efficacy of T cell-based immunotherapy is primarily due to efficient cellular activation that requires the engagement of 2 separate signals, i.e., via the T cell receptor complex and via co-stimulatory molecules the prototype of which is CD28. In cellular activation, the CD28 ligands B7-1 (CD80) and B7-2 (CD86) are thought to play nearly identical roles in T cell activation. We monitored the T cell response upon co-culture with HLA Class I-matched and mismatched renal carcinoma cells, respectively, that express different levels of B7-1 and B7-2, respectively. In a HLA Class I-mismatched co-culture, T cell proliferation, IFN-gamma and GM-CSF secretion equally depend on the levels of B7-1 and B7-2 on tumor cells. In contrast, in a HLA Class I-matched situation, B7-2 is more effective in the induction of IFN-gamma and GM-CSF secretion than B7-1, but both B7 molecules induce T cell proliferation equally efficient. B7-2 is more effective than B7-1 in inducing TNF-alpha and IL-10 secretion in both HLA Class I-matched and mismatched situations. The distinct patterns of cytokine induction by B7-1 and B7-2 obviously depend on the HLA Class I compatibility. These conclusions have substantial implications for the development of B7-based vaccines used for immunotherapies.

プロラクチンはＩＤＯ（Ｉｎｄｏｌｅａｍｉｎｅ２，３‐ｄｉｏｘｙｇｅｎａｓｅ）の発現増強を介し妊娠維持に関与する

Indoleamine 2,3-dioxygenase (IDO), one of the enzymes of tryptophan catabolism, has been shown to play an essential role for successful pregnancy through the inhibition of allogenic fetus-induced T-cell proliferation, and interferon-gamma (IFN-gamma) induces the expression of IDO in CD14-positive (CD14(+)) cells. On the other hand, prolactin (PRL) is the hormone whose serum levels drastically elevate during pregnant period and is shown to play an important role in the early stages of pregnancy including implantation. However little is known about the physiological significance of the elevation of PRL from second trimester except for its fundamental role in lactation. Since receptors of PRL and IFN-gamma share their structure and the signal transduction pathway, we hypothesized the potential crosstalk between two substances. To test this idea, we examined the effect of PRL on IFN-gamma-induced IDO expression in CD14(+) cells. CD14(+) cells were prepared from peripheral blood of 12 healthy controls who had informed consent, and IDO expression and transcriptions were analyzed by flow cytometry and RT-PCR. The results showed that although PRL by itself had little effect on IDO expression, PRL significantly enhanced the IFN-gamma-induced IDO expression at comparable to those seen in a pregnant period. In contrast, no such effect was observed with PRL at lower concentrations comparable to those seen in a non-pregnant period. These findings suggest that PRL may contribute to the maintenance of pregnancy by augmenting IFN-gamma induction of IDO expression.

Clinical improvement in chronic plaque‐type psoriasis lesions after narrow‐band UVB therapy is accompanied by a decrease in the expression of IFN‐γ inducers – IL‐12, IL‐18 and IL‐23

Type-1 cytokine-producing T cells are important in the pathogenesis of psoriasis vulgaris, for which efficient therapy is provided by means of narrow-band ultraviolet-B (NB-UVB). The expression of the type-1 cytokine interferon-gamma (IFN-gamma) is regulated by interleukin-12 (IL-12), IL-15, IL-18 and IL-23; however, not much is known about the effect of this therapy on the levels of these cytokines in lesional psoriatic skin in situ. In this study, we investigated the effects of NB-UVB therapy on the expression of IFN-gamma-inducing cytokines. Ten patients with chronic plaque-type psoriasis selected to be treated with NB-UVB therapy were recruited for these experiments and the expression of cytokines IL-12, IL-15, IL-18, IL-23 and IFN-gamma in lesional psoriatic skin before, during and after therapy was determined with the help of immunohistochemistry. Double staining was performed in order to determine the cell types expressing these cytokines. The decrease in the psoriasis area and severity index was accompanied by a significant decrease in the expression of IFN-gamma, and concomitantly, significant reduction of IFN-gamma inducers -- IL-12, IL-18 and IL-23. Thus, we concluded that the decrease of IFN-gamma expression in psoriasis lesions after NB-UVB therapy could be a result of diminished expression of IL-12, IL-18 and IL-23 in lesional skin. Therapies targeting these three cytokines should, therefore, be considered in the treatment of psoriasis.

Marked induction of IL-6, haptoglobin and IFNγ following experimental BRSV infection in young calves

Bovine respiratory syncytial virus (BRSV) has been identified worldwide as an important pathogen associated with acute respiratory disease in calves. An infection model has been developed reflecting accurately the clinical course and the development of pathological signs during a natural BRSV-infection. In the experiments described in the present study, calves were infected at 13-21 weeks of age and reinfected 14 weeks later. Blood samples from the entire infection period were analysed for acute phase protein (haptoglobin) by ELISA and for expression (mRNA level in peripheral blood mononuclear cells) of the cytokines interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-6 (IL-6) and interferon-gamma (IFNgamma) by quantitative real-time reverse transcribed polymerase chain reaction (RT-PCR). IFNgamma, interleukin-6 and haptoglobin were markedly induced together with development of clinical signs in response to the first infection with BRSV. The IFNgamma response was biphasic, with an early peak at day 1-3 post infection (p.i.) and a later increase between day 5 and 8 p.i. Reinfection also resulted in an induction of IFNgamma, but without induction of clinical signs, IL-6 and haptoglobin. These results indicate that early mediators connected with the innate responses are induced on a first encounter with the pathogen, but not on a second encounter (reinfection) where the adaptive immune system may act as the first line defence.

Issues in T‐helper 1 development – resolved and unresolved

T-helper 1 cell (Th1) development participates in immunity to many pathogens in part by providing a source of interferon (IFN)-gamma that contributes numerous protective effects. The process of Th1 development involves signals provided by antigen-presenting cells and cytokines produced in response to pathogens, with IFN-gamma itself, interleukin (IL)-12, and IL-18 each promoting the process in some way. Despite the rapid progress into mechanisms of Th1 development in recent years, there are still a number of important unresolved issues in this area. The precise sequence of effector and cellular mechanisms represents a relatively recent avenue of research but is still the subject of current debate, as is the basis of mechanisms that may stabilize a Th1 response. Another unresolved issue is the role of type I IFNs in substituting for IL-12-mediated activation of signal transducer and activator of transcription 4 (Stat4) and induction of IFN-gamma in either murine or human T cells. It is now clear that Th1 cells acquire the property of being capable of nonantigen-dependent activation through the coordinate signaling of IL-12 and IL-18, but the precise order of intracellular signaling events and the uniqueness of this pathway's reliance on the p38 mitogen-activated protein kinase (MAPK) pathway are still issues in need of resolution. Finally, the process of verifying the effects of Stat4 mutations on functional responses has led to the recognition of an unexpected action of the STAT N-domain that may apply generally to other STAT proteins as well. None of these areas is static or resolved fully, and they likely will remain topics of rapid progress.

Bioactivity and secretion of interleukin-18 (IL-18) generated by equine and feline IL-18 expression constructs

Interleukin 18 (IL-18) is a cytokine capable of induction of IFNgamma, granulocyte monocyte-colony stimulating factor (GM-CSF), TNFalpha and IL-1 in immunocompetent cells. Equine and feline plasmid vectors expressing pro-IL-18, mature IL-18 and IL-18 fused to a synthetic signal sequence from human IL-1beta receptor antagonist protein (ILRAP), ILRAP-IL-18, have been generated. In vitro protein expression of these constructs was compared by Western blot analysis. These data demonstrated that ILRAP-IL-18 protein was secreted readily from transfected chinese hamster ovary (CHO) cells. A simple bioassay for human IL-18 was recently described using human myelomonocytic KG-1 cells, which produce human IFNgamma in response to human IL-18 in a dose dependent manner (Konishi et al., 1997). We demonstrated bioactivity of equine and feline IL-18 protein in transfection products of CHO cells using this assay. Bioactivity of ILRAP-IL-18 protein was demonstrated in the culture medium of transfected CHO cells. These data imply that the ILRAP-IL-18 construct shows potential for use in vivo, where cell secretion of protein is crucial.

Frontline: absence of functional STAT4 activation despite detectable tyrosine phosphorylation induced by murine IFN-alpha.

We previously reported that IL-12, but not IFN-alphaA/D, induces T helper type (Th) 1 development and STAT4 phosphorylation in murine CD4+ T cells. However, a recent study reported that IFN-alphaA/D and recombinant murine IFN-alphaA can induce STAT4 phosphorylation, although more weakly than IL-12, largely in CD8+ rather than CD4+ T cells. That report did not examine Th1 development or directly demonstrate induction of IFN-gamma by IFN-alpha. To address these differences, we compared IFN-alphaA/D, murine IFN-alphaA, and IL-12 for STAT4 phosphorylation, formation of active nuclear DNA binding complexes, induction of Th1 development, and production of IFN-gamma in murine CD4+ T cells. IFN-alphaA induced detectable STAT4 phosphorylation, although at significantly lower levels than induced by IL-12. Furthermore, in contrast to IL-12, IFN-alphaA failed to induce Th1 development or the formation of DNA binding complexes or to synergize with IL-18 for induction of IFN-gamma production. STAT1-deficient CD4+ T cells showed increased IFN-alphaA-induced STAT4 phosphorylation but still exhibited significantly lower amounts of cytokine-induced IFN-gamma compared to IL-12. In summary, these results suggest that in contrast to IL-12, IFN-alphaA does not play a functionally significant role in meditating the STAT4-dependent induction of Th1 development or IFN-gamma production in CD4+ T cells.

Protective role of arginase in a mouse model of colitis.

Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg). Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines. We investigated the role of these enzymes in the Citrobacter rodentium model of colitis. Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated. l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement. There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion. Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice. Polyamine levels were increased in colitis tissues, and were further increased by l-Arg. In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis. Taken together, these data indicate that arginase is protective in C. rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS. Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Cutaneous T-cell lymphoma (CTCL) responses to a TLR9 agonist CPG immunomodulator (CPG 7909), a phase I study

6600 Background: Available systemic therapies for CTCL produce significant response rates and may relieve skin symptoms or complications, but there is a lack of well-tolerated systemic therapies with reliable and durable responses in patients with recurrent or advanced disease. A new class of chemically defined CpG immunomodulators target dendritic cell TLR9 receptors with induction of IL-12, IFN-gamma, and NK cell function. In several early trials, CPG 7909 has been well tolerated by weekly s.c. dosing and has clinical anti-tumor activity. Methods: Cohorts of 3 patients with recurrent/refractory CTCL were enrolled to receive weekly s.c. doses of 0.08, 0.16, 0.24, or 0.28 mg/kg of CPG 7909. Clinical response was monitored using the Composite Assessment of Index Lesion Disease Severity Index (CA) and the Physician's Global Assessment of Clinical Condition (PGA). Results: Of 12 patients treated to date (8 females, 10 Caucasians, 1 Hispanic and 1 African-American), 2 at the lowest dose had progressive disease (PD), 5 have stable disease, and 3 have had partial or total clearing of disease (see table below). Stable disease and response was maintained for up to 6 months of treatment. Doses were generally well tolerated; dose related injection site reactions (erythema, induration, redness and pain) and mild or moderate flu-like symptoms were common. Conclusions: CPG 7909 has significant antitumor activity in CTCL. Responses will be correlated with immunomodulator biologic responses and PK. Enrollment is ongoing with additional dose escalations to optimize response at tolerable doses. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group

Cutaneous T-cell lymphoma (CTCL) responses to a TLR9 agonist CPG immunomodulator (CPG 7909), a phase I study

6600 Background: Available systemic therapies for CTCL produce significant response rates and may relieve skin symptoms or complications, but there is a lack of well-tolerated systemic therapies with reliable and durable responses in patients with recurrent or advanced disease. A new class of chemically defined CpG immunomodulators target dendritic cell TLR9 receptors with induction of IL-12, IFN-gamma, and NK cell function. In several early trials, CPG 7909 has been well tolerated by weekly s.c. dosing and has clinical anti-tumor activity. Methods: Cohorts of 3 patients with recurrent/refractory CTCL were enrolled to receive weekly s.c. doses of 0.08, 0.16, 0.24, or 0.28 mg/kg of CPG 7909. Clinical response was monitored using the Composite Assessment of Index Lesion Disease Severity Index (CA) and the Physician's Global Assessment of Clinical Condition (PGA). Results: Of 12 patients treated to date (8 females, 10 Caucasians, 1 Hispanic and 1 African-American), 2 at the lowest dose had progressive disease (PD), 5 have stable disease, and 3 have had partial or total clearing of disease (see table below). Stable disease and response was maintained for up to 6 months of treatment. Doses were generally well tolerated; dose related injection site reactions (erythema, induration, redness and pain) and mild or moderate flu-like symptoms were common. Conclusions: CPG 7909 has significant antitumor activity in CTCL. Responses will be correlated with immunomodulator biologic responses and PK. Enrollment is ongoing with additional dose escalations to optimize response at tolerable doses. Author Disclosure Employment or Leadership Consultant or Advisory Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Coley Pharmaceutical Group Coley Pharmaceutical Group Coley Pharmaceutical Group

The role of Tyk2, Stat1 and Stat4 in LPS-induced endotoxin signals.

Mice lacking Tyk2, Stat1 or Stat4, which are members of the Jak-Stat signaling cascade, were resistant to LPS-induced endotoxin shock. Interestingly, Tyk2-deficient mice had higher resistance to LPS challenge than mice lacking either Stat1 or Stat4. The activation of MAPK and NF-kappaB by LPS, and the production of TNF-alpha and IL-12 after LPS injection, were not abrogated by the absence of Tyk2, Stat1 or Stat4. In Stat1-deficient mice, the induction of IFN-beta by LPS in macrophages was severely reduced, although the serum level of IFN-gamma was elevated after LPS injection. In contrast, in Stat-4 deficient mice, the induction of IFN-beta by LPS was normal, but the serum level of IFN-gamma remained low after LPS injection. Interestingly, the induction of both IFN-beta and IFN-gamma by LPS was severely reduced in Tyk2-deficient mice. Therefore, Stat1 and Stat4 independently play substantial roles in the susceptibility to LPS. Tyk2 is essential for LPS-induced endotoxin shock, and this signaling pathway is transduced by the activation of Stat1 and Stat4.

Prevention of acute and chronic allograft rejection by a novel retinoic acid receptor-alpha-selective agonist.

To investigate the involvement of retinoic acid receptor (RAR)-alpha in allograft rejection, we investigated the effect of a novel selective agonist to the receptor, ER-38925, in a mouse cardiac allograft model. Prophylactic treatment with ER-38925 inhibited the acute rejection of the mouse cardiac allograft (BALB/c --> C3H/HeN) at 0.3 and 3 mg/kg, and its effect was enhanced in combination with tacrolimus. In this model, ER-38925 remarkably inhibited cytotoxic T lymphocyte induction and alloantigen-stimulated production of cytokines, i.e. IL-2, IL-12 and IFN-gamma. In the chronic rejection model, combined treatment with tacrolimus and ER-38925 reduced the grade and incidence of arteriosclerosis in the cardiac allografts significantly more potently than tacrolimus monotherapy. ER-38925 inhibited the proliferation of rat aortic smooth muscle cells stimulated in vitro, presumably through the induction of a cyclin-dependent kinase inhibitor, p27(kip-1). Those results provide a rationale for using RAR-alpha agonists as immunosuppressants in human organ transplantation.

Plasmodium vivax and Plasmodium falciparum gametocyte stages are neglected in vaccine development

Plasmodium gametocytes are responsible for transmission from the vertebrate host to the mosquito. Plasmodium gametocytes undergo a complex cycle from asexual stages, through a poorly understood process characterized by expression of stage-specific proteins and adhesion molecules. Gametocytes are capable of inducing specific humoral IgG, and cellular responses, which include induction of TNFalpha, IFNgamma and gammadelta+ lymphocyte proliferation, in addition to immune responses to other stages of the parasite (sporozoite, exo-erythrocytic stages, erythrocytic stages). Although transmission-blocking vaccines against Plasmodium do not currently include components against the gametocytes (rather they focus on gametes, zygotes or ookinetes, stages which occur in the mosquito), further understanding of the mechanisms underlying gametocytogenesis and immune responses against these stages may provide additional strategies for more effective transmission inhibition.

The alcohol-induced conformational changes in casein micelles: a new challenge for the purification of colostrinin.

Recent advances in protein separation technology have allowed for the isolation of whey proteins and peptides of significant biological importance. In this study, we report a novel method for isolation and purification of the neuroprotective proline-rich polypeptides, also known as Colostrinin (CLN). Although CLN was first isolated from ovine colostrum and characterized as a complex of small molecular peptides, its constituents are present also in other mammal colostrums. The previous purification protocols are very tedious, time consuming, and, due to the diverse characteristics of colostrum, also very difficult to validate. Thus, the aim of this study was to develop a simple protocol with a maximum recovery rate for CLN peptides. Here we demonstrate the two-step extraction/purification method that consists of methanol extraction and ammonium sulfate precipitation as the general principles. When compared with the original material, CLN obtained by this method shows (1) similar pattern of peptides in SDS PAGE, (2) identical amino acid analysis, characterized by high content of proline (22%), a high proportion of nonpolar amino acids, a low percentage of glycine, alanine, arginine, histidine, and no tryptophan, methionine, and cysteine residues, (3) similar pattern of HPLC profiles, and (4) its ability to induce IFN gamma and TNF alpha. More importantly, the protocol for the production of high-quality CLN can be accomplished in less than a 48 h timeframe. In addition, avoidance of excessively harsh conditions preserves the structure and biological activity of the peptides.

A mechanism underlying STAT4-mediated up-regulation of IFN-gamma induction inTCR-triggered T cells.

IL-12 promotes T(h)1 development/IFN-gamma expression by activating STAT4. However, it is still unclear how STAT4 elicits IFN-gamma promoter activation. Here, we investigated the mechanism by which IL-12-activated STAT4 functions for IFN-gamma induction in TCR-triggered T cells. TCR stimulation induced high levels of IFN-gamma production depending on co-stimulation with IL-12. IL-12 stimulation greatly enhanced the promoter-binding activity of c-Jun/AP-1, a critical transcription factor for IFN-gamma gene expression in wild-type T cells, but not in STAT4-deficient (STAT4(-/-)) T cells. Comparable amounts of c-Jun were induced by TCR stimulation in both wild-type and STAT4(-/-) T cells irrespective of IL-12 co-stimulation. However, c-Jun bound to STAT4 in IL-12-co-stimulated wild-type T cells. c-Jun forming a complex with STAT4 efficiently interacted with the AP-1-related sequence of the IFN-gamma promoter. Such an enhanced c-Jun binding did not occur in STAT4(-/-) T cells. These results show that STAT4 contributes to enhancing IFN-gamma expression by up-regulating the binding of TCR signal-induced AP-1 to the relevant promoter sequence.

Macrophage-stimulating protein, the ligand for the stem cell-derived tyrosine kinase/RON receptor tyrosine kinase, inhibits IL-12 production by primary peritoneal macrophages stimulated with IFN-gamma and lipopolysaccharide.

IL-12, produced by APCs during the initial stages of an immune response, plays a pivotal role in the induction of IFN-gamma by NK and gammadeltaT cells and in driving the differentiation of Th1 cells, thus providing a critical link between innate and acquired immunity. Due to the unique position occupied by IL-12 in the regulation of immunity, many mechanisms have evolved to modulate IL-12 production. We have shown previously that macrophage-stimulating protein (MSP), the ligand for the stem cell-derived tyrosine kinase/recepteur d'origine nantais (RON) receptor, inhibits NO production by macrophages in response to IFN-gamma and enhances the expression of arginase. Mice lacking RON exhibit increased inflammation in a delayed-type hypersensitivity reaction and increased susceptibility to endotoxic shock. In this study we demonstrate that pretreatment of macrophages with MSP before IFN-gamma and LPS results in the complete inhibition of IL-12 production due to suppression of p40 expression. This response is mediated by the RON receptor, and splenocytes from RON(-/-) animals produce increased levels of IFN-gamma. MSP pretreatment of macrophages resulted in decreased tyrosine phosphorylation of Stat-1 and decreased expression of IFN consensus sequence binding protein in response to inflammatory cytokines. In addition to IL-12, the expression of IL-15 and IL-18, cytokines that are also dependent on IFN consensus sequence binding protein activation, is inhibited by pretreatment with MSP before IFN-gamma and LPS. We also show that the ability of MSP to inhibit IL-12 production is independent of IL-10. Taken together, these results suggest that MSP may actively suppress cell-mediated immune responses through its ability to down-regulate IL-12 production and thus inhibit classical activation of macrophages.

IFN-gamma gene expression in epithelial trophectoderm cells is linked to downregulation of the p44/p42 MAP kinase pathway.

We previously stated that interferon-gamma (IFN-gamma) is highly expressed in a transient and developmentally regulated manner by the trophectoderm of the pig blastocyst, which represents a monolayer of polarized epithelial cells, during early pregnancy. In order to study the molecular mechanisms of this atypical IFN-gamma gene expression, we established the pig trophectoderm cell line TBA B4-3. These cells develop a polarized phenotype with high transepithelial electrical resistance (TER) when grown on a microporous membrane. We previously showed that treatment of polarized TBA B4-3 cells with the strong protein kinase C (PKC) agonist phorbol 12-myristate-13-acetate (PMA) induced 3-4 days later a transient IFN-gamma mRNA expression and apical IFN-gamma protein secretion. In the present paper, we report that after PMA removal, a transient phase of p44/p42 mitogen-activated protein (MAP) kinase activation occurs, followed by a strong downregulation preceding the phase of IFN-gamma expression. Surprisingly, we found that inhibition of this surge of p44/p42 MAP kinase activation with MEK inhibitors (U0126 and PD98059) triggers earlier IFN-gamma mRNA and protein expression, correlated with earlier TER rising and restoration of epithelial phenotype. These results indicate that in the TBA B4-3 cell system, activation of this signaling pathway has a negative effect on IFN-gamma gene expression. These observations reinforce the hypothesis of a link between establishment of cell polarity and induction of IFN-gamma that could be mediated by signaling from intercellular junctions.