The effects of interleukin-2 on immune response regulation.

The immune system has many adaptive and dynamic components that are regulated to ensure appropriate, precise and rapid response to a foreign pathogen. A delayed or inadequate immune response can lead to prolonged disease, while an excessive or under-regulated response can lead to autoimmunity. The cytokine, interleukin-2 (IL-2) and its receptor IL-2R play an important role in maintaining this balance.The IL-2 receptor transduces pSTAT5 signal through both the intermediate and high affinity receptors, which differ from each other by the presence of CD25 chain in IL-2 receptor. We present experimental data on the kinetics of pSTAT5 signalling through both of the receptors and develop a model that captures this kinetics. We then use this model to parameterize key aspects of two additional models in which we propose and study two different mechanisms by which IL-2 receptor can transduce distinct signals leading to either an activated or a non-activated cell state. We speculate that this initial state differentiation, perhaps enhanced by downstream feedbacks, may eventually lead to differential cell fates.Our result shows that non-linear dynamical models can suggest resolution of a puzzling array of seemingly contradictory experimental results on IL-2 effect on proliferation and differentiation of T-cells.

Six new C-21 steroidal glycosides from Dregea sinensis Hemsl.

Six new C-21 steroidal glycosides (1-6) were separated from the root of Dregea sinensis Hemsl. and their structures were elucidated using extensive nuclear magnetic resonance, mass spectrometry, and infrared spectral analyses. Isolated compounds were evaluated for antitumor activity, which showed that compound 3 had moderate activity in Jurkat cells (IC50 19.54 ± 0.91 µM), and compounds 1-4 had significant effects against IL-2R and TNFR2 (IC50 1.518 ± 0.06 µM to 5.9 ± 0.07 µM).

Population Pharmacokinetics of Daclizumab High-Yield Process in Healthy Volunteers and Subjects with Multiple Sclerosis: Analysis of Phase I-III Clinical Trials.

BACKGROUND AND OBJECTIVES: Daclizumab high-yield process (HYP) is a humanized IgG1 monoclonal antibody that binds to the α-subunit (CD25) of the interleukin-2 receptor. The present work characterized the population pharmacokinetics of daclizumab HYP in healthy volunteers (HVs) and subjects with relapsing-remitting multiple sclerosis (RRMS) and evaluated the effects of covariates on daclizumab HYP exposure. METHODS: Measurable serum concentrations (n = 17,139) from 1670 subjects in seven clinical studies (three phase I, one immunogenicity, one phase II with extension, and one phase III) were included in this pharmacokinetic analysis using non-linear mixed-effects modeling. The three phase I studies evaluated single or multiple doses that ranged from 50 to 400 mg with either intravenous or subcutaneous (SC) administration in HVs (n = 71). The phase II with extension studies evaluated doses of 150 or 300 mg SC every 4 weeks (n = 567), and the immunogenicity (n = 113) and the phase III (n = 919) studies evaluated 150 mg SC every 4 weeks, all in RRMS patients. RESULTS: A two-compartment model with first-order absorption and elimination adequately described daclizumab HYP pharmacokinetics. Clearance (CL) was 0.212 L/day and the central volume of distribution was 3.92 L, scaled by [body weight (kg)/68] with exponents of 0.87 and 1.12, respectively. The peripheral volume of distribution was 2.42 L. Absorption lag time, mean absorption time, and absolute bioavailability (100-300 mg SC) were 1.61 h, 7.2 days, and 88 %, respectively. The daclizumab HYP terminal half-life was 21 days. Baseline CD25, age, and sex did not influence daclizumab HYP pharmacokinetics. Body weight explained 37 and 27 % of the inter-individual variability for CL and central volume of distribution, respectively. Neutralizing antibody (NAb)-positive status (included as a time-varying covariate) increased daclizumab HYP CL by 19 %. CONCLUSIONS: Consistent with previous findings in HVs, this analysis including extensive data from RRMS patients demonstrates that daclizumab HYP is characterized by slow CL, linear pharmacokinetics at doses above 100 mg, and high SC bioavailability. The pharmacokinetics of daclizumab HYP were not influenced by age (range 18-66 years), the sex of adult subjects, or the baseline CD4+CD25+ T cells (target level). The impact of covariates (body weight, NAb) on daclizumab HYP pharmacokinetics is unlikely to be clinically relevant.

Activation of colo-rectal high-threshold afferent nerves by Interleukin-2 is tetrodotoxin-sensitive and upregulated in a mouse model of chronic visceral hypersensitivity.

BACKGROUND: Chronic visceral pain is a defining feature of irritable bowel syndrome (IBS). IBS patients often show alterations in innate and adaptive immune function which may contribute to symptoms. Immune mediators are known to modulate the activity of viscero-sensory afferent nerves, but the focus has been on the innate immune system. Interleukin-2 (IL-2) is primarily associated with adaptive immune responses but its effects on colo-rectal afferent function in health or disease are unknown. METHODS: Myeloperoxidase (MPO) activity determined the extent of inflammation in health, acute trinitrobenzene-sulfonic acid (TNBS) colitis, and in our post-TNBS colitis model of chronic visceral hypersensitivity (CVH). The functional effects of IL-2 on high-threshold colo-rectal afferents and the expression of IL-2R and NaV 1.7 mRNA in colo-rectal dorsal root ganglia (DRG) neurons were compared between healthy and CVH mice. KEY RESULTS: MPO activity was increased during acute colitis, but subsided to levels comparable to health in CVH mice. IL-2 caused direct excitation of colo-rectal afferents that was blocked by tetrodotoxin. IL-2 did not affect afferent mechanosensitivity in health or CVH. However, an increased proportion of afferents responded directly to IL-2 in CVH mice compared with controls (73% vs 33%; p < 0.05), and the abundance of IL-2R and NaV 1.7 mRNA was increased 3.5- and 2-fold (p < 0.001 for both) in colo-rectal DRG neurons. CONCLUSIONS & INFERENCES: IL-2, an immune mediator from the adaptive arm of the immune response, affects colo-rectal afferent function, indicating these effects are not restricted to innate immune mediators. Colo-rectal afferent sensitivity to IL-2 is increased long after healing from inflammation.

C-21 steroidal glycosides from Dregea sinensis.

Two new C-21 steroidal glycosides, dregeosides D (1) and E (2), were isolated from the roots of Dregea sinensis. The structures of the isolated compounds were elucidated on the basis of 1D and 2D NMR spectra and HR-ESI-MS analysis. Finally, the inhibited effects of the isolated compounds on interleukin 2 receptor were evaluated by enzyme-linked immunosorbent assay.

IL-2 enhances cervical cancer cells proliferation and JAK3/STAT5 phosphorylation at low doses, while at high doses IL-2 has opposite effects.

The IL-2R signaling is critical for normal lymphocyte proliferation. However, the role of the IL-2 signaling in cervical cancer is not yet fully understood. We show that in IL-2R-expressing cervical cancer cells, JAK1 molecules are not phosphorylated. At low doses of IL-2, the constitutive phosphorylation of JAK3 and STAT5 increases in the tumor cells and decreases in lymphocytes, whereas the opposite occurs at high doses of IL-2. Using AG-490, the activation of JAK3 and the proliferation of cervical cancer cells were inhibited. We describe differences in the response of molecules downstream the IL-2R in lymphocytes and tumor cells.

Zinc signals promote IL-2-dependent proliferation of T cells.

Zinc signals, i.e. a change of the intracellular concentration of free zinc ions in response to receptor stimulation, are involved in signal transduction in several immune cells. Here, the role of zinc signals in T-cell activation by IL-2 was investigated in the murine cytotoxic T-cell line CTLL-2 and in primary human T cells. Measurements with the fluorescent dyes FluoZin-3 and Zinquin showed that zinc is released from lysosomes into the cytosol in response to stimulation of the IL-2-receptor. Activation of the ERK-pathway was blocked by chelation of free zinc with N,N,N',N'-tetrakis-2(pyridyl-methyl)ethylenediamine, whereas zinc was not required for STAT5 phosphorylation. In addition, the key signaling molecules MEK and ERK were activated in response to elevated free intracellular zinc, induced by incubation with zinc and the ionophore pyrithione. Downstream of ERK activation, ERK-specific gene expression of c-fos and IL-2-induced proliferation was found to depend on zinc. Further experiments indicated that inhibition of MEK and ERK-dephosphorylating protein phosphatases is the molecular mechanism for the influence of zinc on this pathway. In conclusion, an increase of cytoplasmic free zinc is required for IL-2-induced ERK signaling and proliferation of T cells.

A novel indole-3-propanamide exerts its immunosuppressive activity by inhibiting JAK3 in T cells.

We previously identified an indole-3-propanamide derivative, 3-[1-(4-chlorobenzyl)indol-3-yl]-N-(pyridin-4-yl)propanamide (AD412), as a potential immunosuppressive agent. Here, we document that AD412 inhibited the proliferative response of CD3/CD28-stimulated human T cells without inhibiting their interleukin 2 (IL-2) production and also inhibited the proliferation of CTL-L2 cells in response to IL-2. These results prompted us to analyze the effect of our compound on the three main signaling pathways coupled to the IL-2 receptor. We provide evidence that AD412 inhibited the JAK1/3-dependent phosphorylations of Akt, STAT5a/b, and ERK1/2 in IL-2-stimulated CTL-L2 cells. In contrast, AD412 had little effect on the JAK1/2-dependent INF-gamma-induced phosphorylation of STAT1 in U266 cells. This suggested a preferential inhibition of JAK3 over JAK1 or JAK 2 activities by AD412 that was confirmed by in vitro kinase assays with purified JAK2 and JAK3 kinases. In addition, we provide evidence that the inhibition of IL-2 response by AD412 was not due to inhibition of IL-2Ralpha up-regulation because neither AD412 nor JAK3 inhibitors described previously [4-[(3-bromo-4-hydroxyphenyl)amino]-6,7-dimethoxyquinazoline (WHI-P154) and alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamid (AG-490)] significantly inhibited IL-2-induced IL-2Ralpha overexpression. Finally, we further document the immunosuppressive activity of AD412 in vivo by showing that its administration per os significantly prolonged heart allograft graft survival. This molecule may thus represent an interesting lead compound to develop new immunosuppressive agents in the field of transplantation and autoimmune diseases.

Fish oil n-3 polyunsaturated fatty acids selectively affect plasma cytokines and decrease illness in Thai schoolchildren: a randomized, double-blind, placebo-controlled intervention trial.

OBJECTIVE: To determine whether very long-chain n-3 polyunsaturated fatty acids (PUFAs) affect illness and selected plasma cytokines in schoolchildren. STUDY DESIGN: Thai schoolchildren aged 9 to 12 years consumed milk containing placebo (soybean) oil (n = 86) or fish oil (n = 94) on 5 days per week for 6 months; the latter provided 200 mg eicosapentaenoic acid plus 1 g docosahexaenoic acid daily. Episodes and duration of illness were recorded, and plasma interleukin (IL)-2 receptor, IL-6, IL-10, and transforming growth factor (TGF)-beta1 concentrations and the fatty acid profile of plasma phosphatidylcholine determined. RESULTS: After intervention, very long-chain n-3 PUFAs were higher in plasma phosphatidylcholine in the fish oil group than in the placebo group (P < .001). The fish oil group showed fewer episodes (P = .014) and shorter duration (P = .024) of illness (mainly upper respiratory tract) than the placebo group. Plasma IL-2 receptor, IL-10, and IL-6 were not affected by either treatment. Plasma TGF-beta1 increased in both groups, but the increase was smaller in the fish oil group, and at the end of supplementation TGF-beta1 concentration was lower in the fish oil group (P < .001). CONCLUSIONS: Very long-chain n-3 PUFAs reduce illness, mainly infections, in healthy Thai schoolchildren.

Immunological evaluation of Lactobacillus casei Zhang: a newly isolated strain from koumiss in Inner Mongolia, China.

BACKGROUND: There is increasing evidence to suggest an immunomodulation function both within the intestines and systemically upon consuming probiotic species. We recently isolated a novel LAB, Lactobacillus caseiZhang (LcZhang) from koumiss. LcZhang exhibited favorable probiotic properties, such as acid resistance, bile resistance, gastrointestinal (GI) colonization ability, etc. In order to examine the immunomodulatory qualities of LcZhang, we administered LcZhang to healthy mice with varying doses of either live or heat-killed LcZhang and measured various parameters of the host immune response. RESULTS: The study was performed in four separate experiments via oral administration of live and heat-killed LcZhang to BALB/c mice for several consecutive days. We investigated the immunomodulating capacity of LcZhang in vivo by analyzing the profile of cytokines, T cell subpopulations, and immunoglobulin concentrations induced in blood serum and intestinal fluid in BALB/c mice. Only live bacteria elicited a wide range of immune responses, which include the increased production of interferon-gamma (IFN-gamma), and depression of tumor necrosis factor-alpha (TNF-alpha) levels. In addition, interleukin-2 (IL-2) and IL-2 receptor gene transcription increased significantly, but the proportion of T cell subsets appeared to be unaffected. We also observed that LcZhang was capable of inducing gut mucosal responses by enhancing the production of secretory Immunoglobulin A (sIgA) as well influencing the systemic immunity via the cytokines released to the circulating blood. CONCLUSION: The present work shows that the dose-dependent administration of LcZhang is capable of influencing immune responses, implying that it may be a valuable strain for probiotic use in humans.

Optimizing denileukin diftitox (Ontak) therapy.

Denileukin diftitox (Ontak) is a novel recombinant fusion protein consisting of peptide sequences for the enzymatically active and membrane translocation domain of diphtheria toxin linked to human IL-2. Denileukin diftitox specifically binds to IL-2 receptors on the cell membrane, is internalized via receptor-mediated endocytosis and inhibits protein synthesis by ADP ribosylation of elongation factor 2, resulting in cell death. This article focuses on the clinical trial that led to the US FDA approval of the drug for cutaneous T-cell lymphoma in 1999, and other investigational studies for hematologic malignancies, recurrent and refractory chronic lymphocytic leukemia, non-Hodgkin B-cell lymphoma, graft-versus-host disease and autoimmune disease, demonstrating the activity and adverse effects of the drug.

Role of endotoxin in the pathogenesis of critical illness polyneuropathy.

Critical illness polyneuropathy (CIP) occurs in association with sepsis and multiple organ failure; however, little is known about the pathomechanisms of CIP and its therapy. In order to determine the parameters which interfere with development of CIP, electrophysiological investigations of peripheral nerves and biochemical measures were correlated to each other. The present study includes 20 consecutive patients in an intensive care unit developing severe sepsis or septic shock. Nerve conduction studies and electromyography were performed with occurring sepsis (day 1, 7, 14) and neurophysiological parameters were correlated with biochemical measures, especially indicators of infection and inflammation. It was found that all patients developed neurophysiological signs of axonal motor polyneuropathy. There was a significant correlation between serum concentrations of endotoxin and interleukin-2 receptors (IL2-R) and reduction of the amplitude of the compound motor action potentials. Other clinical and biochemical parameters showed no significant correlations with neurophysiological data. This finding apparently indicates that endotoxin damages nerve axons directly or indirectly, e.g. by activation of inflammatory cascades (IL2-R). Endotoxin appears to be an essential factor in the pathogenesis of CIP in sepsis, and therapeutic options neutralizing endotoxin may prevent development of CIP.

In vitro and in vivo immunomodulating and immunorestorative effects of Astragalus membranaceus.

Astragalus membranaceus is a common traditional Chinese medicinal plant widely used as a tonic to enhance the body's natural defense mechanisms. In this study, bioactive fractions were isolated from the roots of Astragalus membranaceus. One of these fractions, designated as AI, was found to be the most potent with respect to its mitogenicity on murine splenocytes. Effects of AI on both specific and nonspecific immunity in mouse models were examined. Results showed that AI could exhibit mitogenic and co-mitogenic activities on mouse splenocytes, both in vitro and in vivo. Experiments in human cell culture demonstrated that AI was also active on human lymphocytes. It was found that AI was mitogenic to T cell depleted population but virtually inactive on B cell depleted population. Intraperitoneal injection of AI into mice markedly augmented the antibody response to sheep red blood cells. Besides, both the influx of macrophages into the peritoneal cavity and the phagocytic activity of macrophages were found to be enhanced by AI in vivo. On the other hand, AI could significantly increase the interleukin-2 receptor expression on mouse splenocytes in vitro. In terms of immunorestorative activity, it was found that AI could restore the lymphocyte blastogenic response of the older mice to values that are normally found in the younger mice. Moreover, administration of AI in vivo could partially restore the depressed immune functions in tumour-bearing mice and cyclophosphamide-treated mice. Collectively, the results clearly showed that AI could exhibit immunomodulating and immunorestorative effects, both in vitro and in vivo.

Blockade of IL-2 receptor suppresses HTLV-I and IFN-gamma expression in patients with HTLV-I-associated myelopathy/tropical spastic paraparesis.

OBJECTIVE: Th1 activation based on a high HTLV-I proviral load is one of the characteristic immunological abnormalities in the peripheral blood lymphocytes of patients with HTLV-I-associated myelopathy/tropical spastic paraparesis (HAM/TSP). To clarify the cause of this abnormality with the potential to be one of the therapeutic targets, we analyzed the involvement of interleukin-2 (IL-2)/IL-2 receptor (IL-2R) signaling in HTLV-I and interferon-gamma (IFN-gamma), which is a representative Th1 cytokine, expression in peripheral blood CD4(+) T cells from HAM/TSP patients. PATIENTS AND METHODS: Twelve patients with HAM/TSP were included in the study. After the peripheral blood CD4(+) T cells were treated in cultures under the presence of each anti-IL-2Ralpha, beta,and gamma blocking antiboby for 48 hours, both HTLV-I p19 antigen and IFN-gamma levels in the culture supernatants were measured using ELISA methods. To check the influence on cell proliferation under these culture conditions, the numbers of viable cells were simultaneously determined by MTS assay. RESULTS: Treatment with anti-IL-2Ralpha blocking antibody, but not anti-IL-2Rbeta or anti-IL-2Rgamma blocking antibody, suppressed HTLV-I p19 antigen expression levels. In addition, treatment with all types of anti-IL-2R blocking antibodies also suppressed IFN-gamma expression levels. All of the types of anti-IL-2R blocking antibodies did not inhibit the proliferation. CONCLUSION: These results indicate that IL-2/IL-2R signaling is involved in HTLV-I and IFN-gamma expression on peripheral blood CD4(+) T cells from HAM/TSP patients, suggesting that the interruption of this signaling has therapeutic potential against HAM/TSP in patients with the focus on the down-regulation of Th1 activation based on a high HTLV-I proviral load in the peripheral blood.

Anti-Tac (daclizumab, Zenapax) in the treatment of leukemia, autoimmune diseases, and in the prevention of allograft rejection: a 25-year personal odyssey.

Twenty-five years ago, we reported the production of the monoclonal antibody, anti-Tac that identifies the IL-2 receptor alpha subunit and blocks the interaction of IL-2 with this growth factor receptor. In 1997, daclizumab (Zenapax), the humanized form of this antibody, was approved by the FDA for use in the prevention of renal allograft rejection. In addition, we demonstrated that daclizumab is of value in the treatment of patients with noninfectious uveitis, multiple sclerosis, and the neurological disease human T-cell lymphotropic virus I associated myelopathy/tropical spastic paraparesis (HAM/TSP). Others demonstrated therapeutic efficacy with daclizumab in patients with pure red cell aplasia, aplastic anemia, and psoriasis. Thus, translation of basic insights concerning the IL-2/IL-2 receptor system obtained using the monoclonal antibody daclizumab provided a useful strategy for the prevention of organ allograft rejection and the treatment of patients with select autoimmune diseases or T-cell leukemia/lymphoma.

Immunomodulatory effects of rexinoids.

Rexinoids have shown clinical activity in hematologic malignancies by mediating genes associated with both growth and differentiation. Consequently, these compounds are increasingly being investigated for the treatment of cutaneous T-cell lymphomas. Combining rexinoids with interleukin-2 receptor-targeted therapies, such as denileukin diftitox, would appear to be a rational therapy option in the treatment of lymphoid malignancies. This article discusses the use of rexinoids in combination with these pharmacotherapeutic agents, together with their use in combination with extracorporeal photophoresis and explores practical clinical approaches that may help to evoke immunomodulatory effects in targeted tumor cells, and ultimately lead to improved clinical outcome.

DNA and adenoviral vectors encoding carcinoembryonic antigen fused to immunoenhancing sequences augment antigen-specific immune response and confer tumor protection.

A panel of vectors was constructed to encode carcinoembryonic antigen (CEA) fused at its C-terminal end to various polypeptides, so as to compare their immunogenicity by plasmid DNA immunization and adenovirus injection in wild-type and CEA transgenic (CEA.tg) mice. Fusions between CEA and the minimized domain of tetanus toxin fragment C (CEA-DOM) or the Fc portion of IgG1 (CEA-FcIgG) were identified as highly immunogenic and elicited significant CEA-specific antibody and CD8+ T cell responses. CEA.tg mice were protected from tumor growth on challenge with MC38-CEA tumor cells only when immunized with repeated injections of plasmid pV1J/CEA-DOM followed by Ad/CEA-DOM. Depletion of T-regulatory cells resulted in an increased immune response and antitumor effect with DNA plus adenovirus immunization. In addition, this protective effect was abrogated if the NK, CD4+, or CD8+ cell population from immunized mice was depleted before tumor challenge. Passive transfer studies demonstrated that CD4+ and CD8+ T cells and antibodies contributed to the antitumor effect, thus suggesting that a genetic vaccine based on the use of plasmid DNA and adenoviral vectors encoding CEA fused to immunoenhancing sequences augments CEA-specific immune responses and effectively protects from tumor development.

Cytomodulation of interleukin-2 effect by L-2-oxothiazolidine-4-carboxylate on human malignant melanoma.

Glutathione (GSH), the most prevalent intracellular non-protein thiol, plays an important role in the interleukin-2 (IL-2)-induced proliferative activity of normal and tumour cells expressing IL-2 receptor (IL-2R). In the present study, we investigate the effect of IL-2 on proliferation of the human melanoma A375 cell line, and the possible selective cytomodulation effect of this cytokine by L-2-oxothiazolidine-4-carboxylate (OTZ) on these melanoma cells and on human peripheral blood mononuclear cells (PBMCs). We found that recombinant IL-2 (rIL-2) significantly increased the proliferation rate of A375 melanoma cells, which was associated with an increase in GSH levels, the enhancement of IL-2Ralpha expression and the endogenous production of IL-2 in these tumour cells. In contrast, OTZ decreased GSH content and the proliferation rate of A375 cells, and abrogated the growth-promoting effects of rIL-2. Thus, compared to cells treated with rIL-2, pre-treatment with OTZ reduced IL-2Ralpha expression, and also decreased the consumption of rIL-2 and the endogenous secretion of IL-2 by these tumour cells. With regard to PBMCs, the combination of OTZ plus rIL-2 resulted in a more rapid and greater increase of IL-2Ralpha expression than rIL-2 alone, with the proliferation rate being similar in the first 24 h, but with a lower PBMC' count found thereafter compared to rIL-2 treatment alone. These results suggest that OTZ plays a crucial role in obtaining a selective cytomodulation of rIL-2, enabling it to exert its growth-promoting effect on normal cells, but not on melanoma cells, thereby possibly improving biochemotherapy with rIL-2.