Immunostimulatory CpG-oligonucleotides induce functional high affinity IL-2 receptors on B-CLL cells: costimulation with IL-2 results in a highly immunogenic phenotype

OBJECTIVE: CpG-oligodeoxynucleotides (CpG-ODN) have been shown to induce proliferation, cytokine production, and surface molecule regulation in normal and malignant human B cells. In the present study, we investigated the potential of CpG-ODN to induce functional high-affinity receptors in leukemic and normal B cells and the effects of costimulation with IL-2 on proliferation, cytokine secretion, and surface molecule regulation. METHODS: Highly purified B cells from B-CLL patients and normal controls were stimulated with CpG-ODN with or without IL-2. Expression of CD25 was determined using FACS, and the presence of high-affinity IL-2 receptors was determined by scatchard analysis. Costimulatory effects of IL-2 and CpG-ODN were investigated using proliferation assays, ELISA (IL-6, TNF-alpha), and FACS analysis (CD80, CD86 expression). Reactivity of autologous and allogeneic T cells toward activated B-CLL cells was determined in mixed lymphocyte reactions and Interferon-gamma Elispot assays. RESULTS: The CpG-ODN DSP30 caused a significantly stronger induction of the IL-2 receptor alpha chain in malignant as compared with normal B cells (p = 0.03). This resulted in the expression of functional high-affinity IL-2 receptors in B-CLL cells, but fewer numbers of receptors with less affinity were expressed in normal B cells. Although addition of IL-2 to CpG-ODN-stimulated cells augmented proliferation in both normal B cells and B-CLL cells, no costimulatory effect on cytokine production or surface molecule expression could be observed in normal B cells. In contrast, TNF-alpha and IL-6 production was increased in B-CLL cells, and the expression of CD80 and CD86 was further enhanced when IL-2 was used as a costimulus. Autologous and allogeneic immune recognition of B-CLL cells stimulated with CpG-ODN and IL-2 was increased compared with B-CLL cells stimulated with CpG-ODN alone. CONCLUSION: Stimulation of B-CLL cells with CpG-ODN and IL-2 might be an attractive strategy for potential immunotherapies for B-CLL patients.     

Chemoattractive effect on the effector cells of the supernatants from melanoma cells transfected with the interleukin-2 (IL-2), IL-4 or IL-6 gene

By using the Boyden chamber system, the chemoattractive activity of the supernatants from the B16 melanoma cells transfected with the interleukin-2 (IL-2), IL-4 or IL-6 gene was investigated. We found that supernatants from IL-2- or IL-6-gene-transfected B16 melanoma cells showed chemoattractive activity on natural killer (NK) cells, CD4⁺ T cells and CD8⁺ T cells, while the supernatants from IL-4-gene-transfected B16 melanoma cells showed chemotactic activity on macrophages but not on NK cells and T cells. Further, the chemoattractive activity of the supernatants from cytokine-gene-transfected B16 cells might be the direct effects of the cytokine that they have been engineered to secrete. These results indicate that reduced tumorigenicity and increased immunogenicity of cytokine-gene-transfected B16 cells may be attributed to the direct recruitment of immune effector cells by the secreted cytokine, which plays an important role in the induction of the local antitumor immunity. Received: 26 June 1997  / Accepted: 16 October 1997     

Determination of interleukin-2 (IL-2) and soluble IL-2 receptors (S-IL-2R) in serum and cerebrospinal fluid does not discriminate purulent and aseptic meningitis

Elevated levels of soluble interleukin-2 receptors (S-IL-2R) but not interleukin-2 (IL-2) activity were found in sera from patients with aseptic meningitis, purulent meningitis, and meningism. Elevated levels of S-IL-2R in serum was also observed in 4/4 patients with bacterial pneumonia and 2/2 patients with infectious mononucleosis. The inflammation of the meninges was only reflected by an increase in S-IL-2R in cerebrospinal fluid (CSF) in 1/14 patients with aseptic meningitis and 3/10 patients with purulent meningitis. Further, IL-2 activity was only demonstrated in CSF from 2 patients with aseptic meningitis and 3 patients with purulent meningitis. In conclusion, neither S-IL-2R nor IL-2 in serum or CSF seem to have any value in the diagnosis of or discrimination between purulent meningitis and aseptic meningitis. Further, the elevation of S-IL-2R in serum is not specific for infections primarily fought by cytotoxic T-lymphocytes such as viral infections, but seems merely to reflect an unspecific activation of the immune system.     

Intestinal VIP receptors: differential effect of trypsin on the high and low affinity binding sites

The effects of trypsin treatment on VIP binding to rat intestinal epithelial cell membranes were examined. The decrease in specific binding of [125I]VIP is dependent on the amount of trypsin used and digestion time. Specific binding decreases by 50% after 8 min with 20 micrograms/ml trypsin. Trypsin is active in the 1-100 micrograms/ml concentration range (ED50 approximately equal to 5 micrograms/ml). Non-specific binding is unaltered by the enzyme. The effect of trypsin is abolished by trypsin inhibitor. Scatchard analysis of VIP binding reveals two types of binding sites: sites I characterized by a high affinity, a low capacity and a high sensitivity to low trypsin levels (1-5 micrograms/ml); sites II characterized by a low affinity, a high capacity, resistant to low trypsin levels (1-5 micrograms/ml) but sensitive to a high trypsin level (20 micrograms/ml). Trypsin decreases the binding capacity by lowering the site number without altering their affinity. Sites not destroyed by trypsin retain their functional characteristics: KD, sensitivity to GTP and coupling with adenylate cyclase. It is concluded that sites I and II are proteins with different structures and/or differently localized in the membrane.     

Serum interleukin-6 (IL-6) and interleukin-4 (IL-4) in patients with multiple myeloma (MM)

In this study we determined, in patients with multiple myeloma (MM), serum levels of IL-4 and IL-6 at diagnosis and during the course of the disease, seeking a correlation with disease activity and prognosis. We studied 54 MM patients, 41 of whom responded to chemotherapy whilst 11 were resistant. At diagnosis, IL-6 was increased in 66% of patients (median 35.5 pg/ml) whereas IL-4 was low (median 4 pg/ml) in 75% of patients. In responding patients, IL-4 increased in remission (median 25 pg/ml), whereas IL-6 decreased (median 4 pg/ml). In chemotherapy-resistant patients, IL-6 and IL-4 values remained stable during the course of the disease.     

The prognostic value of serum albumin in healthy older persons with low and high serum interleukin-6 (IL-6) levels

OBJECTIVE: We sought to determine the prognostic value of serum albumin for 4-year mortality among high-functioning persons who did or did not have evidence of inflammation as indicated by high interleukin-6 (IL-6) levels. DESIGN: We used a case-cohort design of healthy, nondisabled older persons who had serum albumin and plasma IL-6 measured at baseline. Crude and multiply adjusted (for sociodemographics and chronic diseases) proportional hazards models were used to identify the effect of baseline levels of serum albumin level on 4-year mortality among those with higher and lower levels of IL-6. RESULTS: Among subjects without evidence of IL-6-mediated inflammation (IL-6 < 3.20 pg/mL), having a lower (< or = 4.4 g/dL) albumin level was associated with a multiply adjusted relative risk of 2.1 for 4-year mortality compared with those with higher albumin. In the presence of inflammation (IL-6 > or = 3.20 pg/mL), higher and lower serum albumin levels had similar risks (adjusted relative risks 4.0 and 3.8, respectively) compared with the referent group (higher albumin and low IL-6). CONCLUSIONS: High serum albumin has a protective effect in healthy older persons who do not have evidence of cytokine-mediated inflammation. This protective effect is not conferred in presence of inflammation. The mechanisms by which inflammation eliminates the protective effect of high albumin remain to be determined.     

Opposite effects of high and low doses of interleukin-2 on T cell-mediated hepatitis in mice (interleukin-2 on hepatitis)

Purpose  

Concanavalin A (Con A)-induced hepatitis is an extensively used animal model of T cell-mediated acute hepatitis. A variety of cytokines, including interleukin 4 (IL-4), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), have been shown to play important roles in Con A-induced liver injury. However, the role of IL-2, a critical cytokine in the development and function of T cells and a clinical therapeutics for virus infection and tumor, has not been carefully examined in this model.     

Administration of interleukin-2 (IL-2) results in increased plasma concentrations of IL-5 and eosinophilia in patients with cancer

Peripheral eosinophilia is almost invariably observed during the course of interleukin-2 (IL-2) therapy and is frequently accompanied by the development of a capillary leak syndrome characterized by edema, weight gain, and oliguria. We studied five patients with advanced malignancy treated with IL-2. Eosinophilia was not present initially but developed in all patients late in the course of therapy, with counts ranging from 2,328/mm3 to 15,958/mm3. In all patients, there was a temporal relationship between the infusion of IL-2 and the appearance of elevated plasma concentrations of IL-5, a growth factor for eosinophils. Granulocyte-macrophage colony-stimulating factor was not detectable in plasma. IL-4 and gamma-interferon plasma levels were variably elevated. Plasma concentrations of major basic protein, a toxic eosinophil granule protein, began increasing before eosinophil counts increased. By the time of the third IL-2 infusion, high concentrations of major basic protein were present in all five patients (up to 5,600 ng/mL) and skin biopsies showed major basic protein deposition in the dermis. Four patients developed significant capillary leak syndrome and all of these patients showed markedly elevated major basic protein levels. The lowest peak plasma concentration of major basic protein (1,751 ng/mL) was observed in the one patient who did not develop edema and weight gain. These results suggest that IL-2 induces IL-5 leading to marked peripheral eosinophilia and extravascular eosinophil degranulation. The release of toxic eosinophil products at extravascular sites and in the circulation may contribute to the pathogenesis of the capillary leak syndrome complicating IL-2 therapy.     

变应性鼻炎、哮喘患者T细胞亚群、白介素水平及肺功能测定

目的评价变应性鼻炎、哮喘患者血清中白细胞介素IL-4、IL-5、IL-6检测指标的方法。方法变应性鼻炎、哮喘及鼻炎并哮喘患者外周血CD3、CD4、CD8较正常对照组均有明显下降,经t检验有显著性差异(P<0.01),鼻炎组、哮喘组及鼻炎并哮喘组血清IL-4、IL-5、IL-6水平明显高于对照组。其气道反应性明显高于对照组。结论IL-4、IL-5、IL-6参与了变应性鼻炎、哮喘的发生和促进了变态反应性疾病的发展,有必要开展更广泛研究和更深层的原因探索。     

Lymphokine-activated killer (LAK) cells and interleukin-2 (IL-2) therapy that improved lymphadenopathy in a patient with B cell non-Hodgkin's lymphoma]

A 70-year-old man was admitted to our hospital on March 9, 1989 because of fever, superficial generalized lymphadenopathy, upper abdominal mass and right pleural effusion. The diagnosis of non-Hodgkin's lymphoma (follicular medium sized cell type, B cell) was made by a biopsy of the neck lymph node. Peripheral blood mononuclear cells were obtained from the patient by cytopheresis. The cells were cultured for 8 days with interleukin-2 (IL-2) to generate Lymphokine-activated killer (LAK) cells. The patient received a total of 7.7 x 10(9) LAK cells intravenously over a period of 3 weeks. He also received continuous intravenous infusion of IL-2 for 17 days, starting 2 days before the first infusion of LAK cells. After this therapy, although his superficial generalized lymphadenopathy disappeared or decreased in size, the size of the upper abdominal mass did not decrease. Therefore, it is suggested that adoptive immunotherapy is a beneficial treatments for B cell lymphoma. However, LAK cells should be generated in much larger quantities for a more successful therapeutic result.     

Function of the interleukin-2 (IL-2) receptor gamma-chain in biologic responses of X-linked severe combined immunodeficient B cells to IL-2, IL-4, IL-13, and IL-15

The interleukin-2 (IL-2) receptor gamma-chain is a common component of several members of the cytokine receptor superfamily including those for IL-2, IL-4, IL-7, IL-9, IL-15, and possibly IL-13, and has recently been renamed the common gamma-chain (gamma c-chain). Transfection experiments have shown that the gamma c-chain participates in signal transduction by IL-2, IL-4 and IL-7, but a functional role for the gamma c-chain in biological responses by normal T cells and B cells to these cytokines has not been established. In this study, we have used X- linked severe combined immunodeficiency (X-SCID) as a naturally occurring gamma c-chain gene disruption model to examine the role of the gamma c-chain in human B-cell responses to IL-2, IL-4, IL-13, and IL-15. Our experiments show that B cells from two X-SCID patients with characterized gamma c-chain gene mutations do not respond to IL-2 or IL- 15, but respond as well or better than normal B cells to both IL-4 and IL-13 in assays for B-cell activation, proliferation, and IgE secretion. This finding raises important questions about the function of the gamma c-chain in receptors for IL-4 and IL-13, and the nature of the immune defect in X-SCID.     

Healthy centenarians show high levels of circulating interleukin-22 (IL-22)

Aging is characterized by a progressive alteration of homeostatic mechanisms modulated by environmental and genetic factors. It is associated with a pro-inflammatory status. In centenarians, an increase of pro-inflammatory cytokine production balanced by anti-inflammatory immune response that would promote longevity is observed. Cytokine dysregulation is believed to play a key role in the proposed remodeling of the immune-inflammatory responses accompanying old age. IL-22 is a pro-inflammatory cytokine belonging to the IL-10 family and represents an important effector molecule of activated T helper (Th)-22, Th-1, and Th-17 cells. We recruited 17 healthy centenarians (4 males, 13 females, range 100-105 years). All ultralongeval subjects were living at home or in a nursing home. Sixteen healthy, sex-matched individuals (4 males, 12 females, range 60-95 years) were also recruited as controls. Centenarians displayed significantly higher circulating IL-22 levels compared to control population (45.7±66.9 pg/ml versus 11.1±6.5 pg/ml; p=0.031). It's well known that IL-22 is a pro-inflammatory cytokine produced by activated T lymphocytes and NK cells. IL-22 stimulates the production of acute phase reactants and promotes the antimicrobial defense. The results of the present study show, for the first time, that there is an increase of IL-22 in healthy centenarians. This pro-inflammatory condition probably is protective against infection, promoting the longevity of these subjects.     

Phase I trial of an interleukin-2 (IL-2) fusion toxin (DAB486IL-2) in hematologic malignancies expressing the IL-2 receptor.

DAB486IL-2 is a recombinant fusion toxin in which the native receptor binding domain of diphtheria toxin has been replaced with human interleukin-2 (IL-2). It selectively binds and intoxicates only cells that bear the high-affinity receptor for IL-2. In the first clinical trial of a genetically engineered ligand fusion-toxin, we have treated 18 patients with chemotherapy-resistant IL-2 receptor expressing hematologic malignancies with escalating doses of DAB486IL-2. The maximal tolerated dose of a daily intravenous bolus of DAB486IL-2 was 0.1 mg/kg per day for 10 doses, established by asymptomatic, reversible elevations of hepatic transaminases without changes in other tests of liver function. Other mild reversible side effects noted were rash, nausea, elevated creatinine, chest tightness, and fever. Pharmacokinetic analysis showed a monophasic clearance of 5.8 +/- 0.7 minutes with peak levels of 3,549 +/- 1,041 mg/mL at the 0.1 mg/kg dose. Approximately 50% of patients developed an antibody response to diphtheria toxin or DAB486IL-2. The presence of such antibodies did not preclude patients from experiencing an antitumor response as four of the six patients with antitumor effect had detectable antibody titers. Although this was a phase I trial designed to define the safety of DAB486IL-2, remissions were observed in three patients lasting from 5 to over 18 months. The ability to achieve significant tumor reductions in this group of heavily treated patients is encouraging and suggests additional trials are warranted in hematologic malignancies.     

Diminished production of interleukin-6 in chronic lymphocytic leukaemia (B-CLL) cells from patients at advanced stages of disease

The production of the cytokines interleukin-6 (IL-6) and tumour necrosis factor-alpha (TNF-α) in B-CLL cells from 24 patients at different stages of chronic lymphocytic B-cell leukaemia (B-CLL) was investigated in vitro . In the majority of these cases, low spontaneous IL-6 production was measured. Mitogenic stimulation with phorbol 12-myristate 13-acetate (PMA) or PMA plus interleukin-2 (IL-2) resulted in a tremendous increase in TNF-α and IL-6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B-CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA-stimulated (1 ng/ml) IL-6 production. In stimulated 72 h cultures, IL-6 concentrations were 1280 ± 1080 pg/ml for Binet A ( n  = 11), 757 ± 597 pg/ml for Binet B ( n  = 8) and 46.0 ± 84.0 pg/ml for Binet C ( n  = 5). The differences in IL-6 production between stage C v B and stage C v A were both statistically significant ( P  = 0.025). Similar effects, but to a lesser extent, were observed in TNF-α production. These results suggest that the varying capacity to produce IL-6 and TNF-α may play a role in B-CLL progression and in clinical manifestations of the disease.     

Functional interleukin-7 receptors (IL-7Rs) are expressed by marrow stromal cells: binding of IL-7 increases levels of IL-6 mRNA and secreted protein

DNA spotted microarrays were used to compare gene expression profiles from 2 functionally distinct human marrow stromal cell lines: HS-27a, which supports cobblestone area formation by early hematopoietic progenitors, and HS-5, which secretes multiple cytokines that support the proliferation of committed progenitors. One unexpected result was the high level of interleukin-7 receptor (IL-7R) gene expression in HS-27a stromal cells. Northern blot analysis confirmed the IL-7R RNA expression, and Western blots for the IL-7R protein detected both a full-length (90-kd) IL-7R and a smaller 30-kd fragment in both HS-27a cells and primary stromal cell cultures, whereas only the 90-kd receptor protein was detected in peripheral blood mononuclear cells. Biotinylated IL-7 was shown to bind to HS-27a cells under physiologic conditions, and this binding was inhibited by blocking anti-IL-7 antibodies. Tyrosine phosphorylation of several proteins (55 kd, 30 kd, and 24 kd) in HS-27a cells was rapidly increased after incubation with recombinant IL-7. One of the phosphorylated proteins proved to be the 30-kd IL-7R fragment. Exposure of HS-27a cells to IL-7 resulted in a 10-fold increase in secretion of IL-6 into culture supernatants but no increase in the cytokines stromal cell-derived factor 1, macrophage inflammatory protein 1 alpha, or IL-1 beta. The up-regulation of IL-6 secretion is associated with a rapid but transient increase in detectable levels of IL-6 messenger RNA. These data suggest that IL-7 may function to regulate the milieu of the microenvironment by modulating IL-6 secretion by the IL-7R-expressing stromal elements.     

Partial impairment of interleukin-12 (IL-12) and IL-18 signaling in Tyk2-deficient mice

Tyk2 is activated in response to interleukin-12 (IL-12) and is essential for IL-12-induced T-cell function, including interferon-gamma (IFN-gamma) production and Th1 cell differentiation. Because IL-12 is a stimulatory factor for natural killer (NK) cell-mediated cytotoxicity, we examined whether tyk2 is required for IL-12-induced NK cell activity. IL-12-induced NK cell activity in cells from tyk2-deficient mice was drastically reduced compared to that in cells from wild-type mice. IL-18 shares its biologic functions with IL-12. However, the molecular mechanism of IL-18 signaling, which activates an IL-1 receptor-associated kinase and nuclear translocation of nuclear factor-kappaB, is different from that of IL-12. We next examined whether biologic functions induced by IL-18 are affected by the absence of tyk2. NK cell activity and IFN-gamma production induced by IL-18 were reduced by the absence of tyk2. Moreover, the synergistic effect of IL-12 and IL-18 for the production of IFN-gamma was also abrogated by the absence of tyk2. This was partially due to the absence of any up-regulation of the IL-18 receptor treated with IL-12, and it might suggest the presence of the cross-talk between Jak-Stat and mitogen-activated protein kinase pathways in cytokine signaling.     

The expression of NK cell inhibitory receptors on cytotoxic T cells in B-cell chronic lymphocytic leukaemia (B-CLL)

Immune surveillance of tumours is mediated by cytotoxic T cells (CTL) that recognise tumour antigen. Reduced reactivity of CTL towards tumour cells could thus lead to disease progression and loss of tumour control. In B-cell chronic lymphocytic leukaemia (B-CLL), the function of tumour-reactive CTL seems to correlate inversely to disease stage. Inhibitory NK cell receptors are known to suppress the CTL response upon interaction with major histocompatibility complex (MHC) class I and increased expression of such receptors on CTL may inhibit the anti-tumour response. So, the aim of this study was to investigate the expression of NK cell inhibitory receptors on CTL in B-CLL patients and if such expression correlated to disease stage. CD8+ T cells from B-CLL patients in Binet stage A (n = 26) and stage C (n = 14) and healthy controls (n = 14) were analysed for the expression of killer immunoglobulin-like receptors (KIR) CD158a (KIR2DL1), CD158b (KIR2DL2), CD158e (KIR3DL1) and the C-type lectin receptor CD94, by flow cytometry analysis. Patients with advanced disease (Binet stage C) had a significantly greater percentage of CTL expressing CD158b, CD158e and CD94 than patients with non-progressive disease (Binet stage A) and healthy controls. Stage C patients also had a significantly higher percentage of CTL expressing CD158a than stage A patients. No statistically significant differences were found between Binet A patients and healthy controls. Our results suggest that increased expression of KIR and CD94 on CTL in advanced stage B-CLL may potentially contribute to the impaired anti-tumour immune response in these patients.