Lack of correlation between defective cell-mediated-immunity and levels of secreted or circulating cytokines in a study of 90 cancer-patients

In this study we examined the levels of IL-1 alpha, IL-1 beta, IL-2, IL-6, TNF-alpha and sIL-2R in the sera and culture supernatants of PHA-stimulated lymphocytes from a series of 90 cancer patients. The expression of the IL-2R p55 chain (alpha subunit) on PHA-stimulated lymphocytes was also evaluated together with the blastogenic response of peripheral blood mononuclear cells (PBMC) to PHA, PHA plus rIL-2 and rIL-2 alone. Ninety cancer patients (70 men and 20 women; mean age 57.8 years, range 27-80) with advanced solid malignancies at different sites were studied. The lymphocyte blastogenic response to PHA was significantly lower in cancer patients than in normal individuals. The proliferative response to rIL-2 alone was also significantly depressed in cancer patients. The frequency of CD25(+) PHA-stimulated lymphocytes from cancer patients was not significantly different from that of the control group. The serum values for IL-1 alpha, IL-1 beta, IL-6 and sIL-2R were significantly higher in cancer patients than in controls, while the serum level of IL-2 was within the normal range. The levels of sIL-2R released in the supernatant of PHA-stimulated PBMC of cancer patients were significantly lower than those of the control group. However, the levels of IL-1 alpha, IL-1 beta, IL-2, IL-6 and TNF-alpha, in the supernatants of PHA-stimulated PBMC of cancer patients were in the same range as those of the control group. These results suggest that the observed immune-deficiency in cancer patients cannot be explained on the basis of a defective production of key immunoregulatory cytokines since the lymphocytes from cancer patients produced physiological amounts of cytokines. We suggest that the observed defective cell-mediated immunity may be due to a defect in transmembrane signalling by the cytokines.     

Origin of the soluble interleukin-2 receptor in the serum of patients with hairy cell leukemia

High levels of soluble IL-2 receptor (sIL-2R) are detectable in the serum of HCL patients. To determine the cell source of this molecule, we evaluated the presence of sIL-2R in the supernatants obtained from in vitro cultures of leukemic (hairy cell, HC) and non-leukemic lymphocytes from six untreated HCL patients and from an additional four patients under therapy with rIFN-alpha 2. We demonstrated that cultured HCs at resting conditions were able to spontaneously release the sIL-2R, whereas control enriched B cells did not. This phenomenon was present only when culturing HCs recovered from patients observed at the time of diagnosis but was not observed during treatment with rIFN-alpha 2. Following activation in vitro with a series of different stimulatory agents including BCGF, phorbol myristate acetate, and anti-human IgM antibody, cultured HCs increased their capability to shed the IL-2R molecules. On the other hand, the release of sIL-2R from enriched T cell populations from HCL patients did not significantly differ from the value obtained in controls. Taken together, these findings provide evidence that leukemic B cells represent the main source of sIL-2R in HCL patients and further emphasize the importance of evaluating this parameter as a relevant marker for monitoring the effectiveness of rIFN-alpha 2 therapy.     

Serum cytokine levels correlate with clinical parameters in Hodgkin's disease

Background: It has been suggested that cytokines are involvedin the pathogenesis of Hodgkin's disease. Enhanced expressionof various cytokines has been demonstrated in cell lines andbiopsy specimens from patients with Hodgkin's disease (HD).Patients and methods: In this investigation 14 cytokines wereanalysed by ELISA in sera of a large panel of patients withHD and compared with clinical and serological parameters. Results:Increased levels of soluble IL-2 receptors (sIL-2R), IL-6, IL-7,IL-8 and G-CSF, were found in many patients with HD as opposedto healthy individuals. In contrast, IL-1     

Loss of tumoral expression of soluble IL-6 receptor is associated with disease progression in colorectal cancer

Background:

Interleukin-6 (IL-6) binds both the membrane and soluble forms of the IL-6 receptor (sIL-6R), which induces a complex with gp130, and proliferation of tumour cells. The aim of this study is to clarify the relationship between tumoral sIL-6R expression and disease progression in colorectal cancer patients.

Methods:

We measured tissue concentrations of sIL-6R in tumour and normal mucosa from 161 colorectal cancer patients undergoing surgery, and in supernatants from colon cancer cell lines. The expression of IL-6, IL-6R and gp130 was evaluated by immunohistochemical analysis.

Results:

Loss of tumour expression of sIL-6R as defined by sIL-6R Ca/N ratio <1.0 was significantly associated with factors reflecting disease progression, and was an independent prognostic factor not only in all the patients in this study, but also in the patients with curative intent. Colon cancer cell lines produced sIL-6R in vitro, and the production of sIL-6R in cancer cell lines was stimulated by cytokine stimulation. Immunohistochemistry revealed that loss of tumour expression of sIL-6R was significantly inversely correlated with intense IL-6 expression in the cytoplasm of cancer cells. In addition, tumoral IL-1β expression was significantly correlated with sIL-6R expression.

Conclusion:

Loss of tumour expression of sIL-6R is associated with colorectal cancer disease progression.     

氟达拉滨对多发性骨髓瘤细胞系KM3细胞分泌IL-6的影响

目的：研究氟达拉滨（fludarabine）在体外对多发性骨髓瘤细胞KM3细胞生长的影响及对细胞上清液IL-6、sIL-6R的影响。方法：采用MTT法及细胞计数法分别观察不同浓度氟达拉滨对KM3细胞生长的影响；用双抗夹心法检测不同浓度氟达拉滨作用KM3细胞后上清液白细胞介素6（interleukin-6，IL-6）和可溶性白细胞介素6受体（soluble interleukin-6 receptor，sIL-6R）的表达水平。结果：MTT法及细胞计数法均显示氟达拉滨对KM3细胞有明显的抑制作用，氟达拉滨作用KM3细胞72h后，25、50、100、200、400nmol／L组的增殖抑制率均高于对照组（P〈0．01），且与浓度成正比。400nmol/L组对KM3细胞增殖抑制率随时间的延长而增加。双抗夹心法检测结果显示KM3细胞经氟达拉滨作用96h后，其IL-6水平先升高后明显下降，而sIL-6R水平随药物浓度的升高而逐渐下降。结论：氟达拉滨能明显抑制KM3细胞的增殖，同时能抑制KM3细胞自分泌IL-6和sIL-6R。     

The relationship between soluble receptor of interleukin-6 with angiogenic cytokines and proliferation markers in multiple myeloma

Soluble interleukin-6 receptor (sIL-6R) is part of IL-6 receptor that may stimulate cells that do not express the whole molecule. It may enhance myeloma cell proliferation and furthermore angiogenesis. The aim of the study was to evaluate the clinical significance and the relationship between serum levels of sIL-6R, with various stimulators of angiogenesis, such as hepatocyte growth factor (HGF) and interleukin-18 (IL-18) and with markers of proliferation, such as beta-2 microglobulin (B2M) levels and plasma cell Ki-67 proliferation index in the bone marrow, in patients with multiple myeloma (MM). We studied 45 newly diagnosed MM patients. Serum levels of sIL-6R, HGF, IL-18, and B2M and Ki-67 proliferation index (Ki-67 PI) in bone marrow’s plasma cells were determined. The mean concentrations of sIL-6R, HGF, IL-18, and B2M and the value of Ki-67 were significantly higher in the patients compared to controls and with increasing disease stage. sIL-6R was strongly positively correlated with HGF, IL-18, B2M, and Ki-67 PI. There is a positive correlation between plasma cell growth, as determined by Ki-67 PI, and different angiogenic cytokines, such as HGF and IL-18, with sIL-6R. This relationship suggests the significant role of these cytokines in the proliferation and disease activity in MM patients.     

Levels of soluble interleukin-2 receptors are predictive of response in patients treated with interleukin-2 and lymphokine-activated killer cells

Soluble interleukin-2 receptor (sIL-2R) α (CD25) levels were serially determined in the sera of 20 patients who had undergone adoptive immunotherapy with high-dose IL-2 and lymphokine-activated killer (LAK) cells for various types of metastatic solid tumors or Hodgkin’s disease. The treatment course consisted of 5 days of high-dose IL-2 priming followed by the collection of peripheral blood leukocytes by leukapheresis, andin vitro activation of mononuclear cells with IL-2, and the subsequent infusion of such prepared LAK-cells together with IL-2. sIL-2R levels increased in all patients following IL-2 administration, and the ratio of baseline sIL-2R levels to those measured after 5 days of IL-2 was signifıcantly correlated with pre-IL-2 levels (p=0.016) in that higher pre-IL-2 levels resulted in a larger increase upon IL-2 administration. In terms of treatment outcome, the variables analysed included sIL-2R levels, total IL-2 doses administered, the expression of membrane-bound CD25 onin vitro cultured cells (pre- and post-IL-2 exposure), the total number of LAK-cells infused andin vitro cytotoxic activity of LAK-cells against the natural killer cell-resistant cell line Daudi. In a multivariate analysis, low baseline sIL-2R levels (p=0.095) and highin vitro cytotoxic activity of LAK-cells against Daudi cells (p=0.082) were jointly associated with response. Our data suggest that serum sIL-2R levels provide a fast and non-invasive parameter for predicting the response in patients treated with IL-2 and LAK-cells.     

Epstein-Barr virus (EBV)-induced B-cell proliferative disorder after chemotherapy in a patient with hemophagocytic lymphohistiocytosis with associated EBV-induced T-cell proliferation

We report a case of Epstein-Barr virus (EBV)-associated lymphoproliferative disorder (LPD) which developed after chemotherapy for hemophagocytic lymphohistiocytosis (HLH), who had no history of immunodeficiency or familial X-linked LPD. In HLH, the presence of EBV in T-cells was confirmed by a combination of in situ hybridization (ISH) and immunostaining. Southern blot analysis using EBV-TR and immunoglobulin JH probes revealed oligoclonal proliferation of B-cells in each organ involved by abnormal B-lymphoid cells at autopsy. Combined ISH and immunostaining disclosed the presence of EBV in proliferating B-cells. Cytokine analysis during the period of T-cell activation in HLH revealed marked elevation of interferon (IFN) gamma, interleukin (IL)-10 and soluble IL-2 receptor (sIL-2R) and mild to moderate increases of tumor necrosis factor (TNF)-alpha were observed, while IFN gamma, IL-10 and sIL-2R were elevated initially during the HLH phase, which then decreased as LPD developed and B-cell proliferation predominated. Immunosuppressive chemotherapy for HLH may then have allowed latent EBV in B lymphocytes to induce transformation and oligoclonal proliferation of B-cells, finally resulting in LPD. Mechanisms of EBV-induced cell proliferation remain unclear, but alteration of various cytokines may be responsible for it.     

Monocyte disorders associated with T cell defects in patients with solid tumors.

T cells proliferate in response to autologous monocytes in the autologous mixed lymphocyte reaction (AMLR). AMLR was found to be impaired in patients with advanced cancer (stages III and IV), whereas normal values were found in the early stages of the disease (stages I and II). Peripheral T lymphocytes from patients with advanced stages also exhibited a decreased ability to produce Interleukin-2 (IL-2) during an AMLR response, whereas production of IL-2 by T cells in stages I and II was comparable to that of normal donors. The impaired IL-2 production by T lymphocytes in the AMLR was associated with high concentrations of soluble interleukin-2 receptor (sIL-2R) in culture supernatants and reduced expression of membrane-bound interleukin-2 receptors (IL-2R) on the same AMLR-activated T lymphocytes. These abnormalities in T cells from cancer patients were demonstrated to be associated with dysfunctions of autologous monocytes. Thus monocytes from patients with advanced cancer exhibited diminished expression of HLA-DR antigens and produced low levels of Interleukin-1 beta (IL-1 beta) and Tumor Necrosis Factor a (TNFa). No changes were detected in the expression of HLA-A, -B, -C antigens. The results presented here demonstrate that decreased in vitro T cell responses may be attributed to monocyte dysfunctions in these patients and provide new information for a better understanding of the impaired T cell function in cancer patients.     

Mesothelin overexpression promotes autocrine IL-6/sIL-6R trans-signaling to stimulate pancreatic cancer cell proliferation

Mesothelin (MSLN) overexpression in pancreatic cancer (PC) leads to enhanced cell survival/proliferation and tumor progression. After screening for a number of growth factors/cytokines, we found that the MSLN expression correlated closely with interleukin (IL)-6 in human PC specimens and cell lines. Stably overexpressing MSLN in different PC cell lines (MIA-MSLN and Panc1-MSLN) led to higher IL-6 production. Silencing MSLN by small interfering RNA (siRNA) significantly reduced IL-6 levels. Blocking the observed constitutive activation of nuclear factor-kappaB (NF-κB) with IKK inhibitor wedelolactone in MIA-MSLN cells also reduced IL-6. Silencing IL-6 by siRNA reduced cell proliferation, cell cycle progression and induced apoptosis with significant decrease of c-myc/bcl-2. Interestingly, recombinant IL-6-induced proliferation of MIA-MSLN cells but not MIA-V cells. Although messenger RNA/protein levels of IL-6R did not vary, soluble IL-6R (sIL-6R) was significantly elevated in MIA-MSLN and was reduced by treatment with the TACE/ADAM17 inhibitor TAPI-1, indicating intramembrane IL-6R cleavage and IL-6 trans-signaling may be operative in MIA-MSLN cells. Blocking the IL-6/sIL-6R axis using sIL-6R antibody abrogated basal proliferation/survival as well as recombinant human IL-6-induced cell proliferation. Our data suggest that MSLN-activated NF-κB induces elevated IL-6 expression, which acts as a growth factor to support PC cell survival/proliferation through a novel auto/paracrine IL-6/sIL-6R trans-signaling. In addition, using a panel of PC cells with varying MSLN/IL-6 expressions, we showed that MSLN/IL-6 axis is a major survival axis in PC supporting tumor cell growth under anchorage-dependent and independent conditions. The close correlation between MSLN and IL-6 provides a new rationale for combination therapy for effective control of MSLN-overexpressing PCs.     

Macrophage-mediated immunostimulation modulates therapeutic efficacy of interleukin-2 based chemoimmunotherapy in advanced metastatic melanoma patients

The biological mechanisms of chemoimmunotherapy efficacy in vivo have not been fully clarified; furthermore, few data are available to predict its efficacy on the basis of clinical and immunological pretreatment factors. In this paper, pre- and post-treatment serum levels of cytokines (interleukin [IL]-6, IL-10, IL-12 and neopterin) and soluble IL-2 receptors (sIL-2R), as well as circulating levels of T-cell and NK subpopulations, were analysed according to clinical outcome in 66 advanced metastatic melanoma (MM) patients treated with subcutaneous IL-2 in association with interferon-alpha, cisplatin and tamoxifen. Our purpose was to correlate the immune modifications during treatment with the clinical response and to define pretreatment factors with predictive value for clinical outcome. The overall response rate was 35%, with a median overall survival of 11.3 months. During treatment, responding patients showed a common marked increase in IL-12 (mainly released by activated macrophages), sIL-2R and neopterin serum levels, associated with high levels of total lymphocytes and circulating natural killer lymphocytes; progressing patients were characterized by an increase in IL-6 serum levels (directly related to the increase in tumour burden). Multivariate analysis showed that high pretreatment IL-12 levels (P = 0.05) and, to a lesser extent, lactate dehydrogenase levels in the normal range (< or = 450 U/I; P = 0.061) are independent favourable prognostic factors for survival. Our results show that macrophage activation in an immunostimulating way either before or during treatment is associated with a better clinical response and improved survival in advanced MM patients treated with IL-2-based chemoimmunotherapy.     

Immune regulation within human tumors - contribution of different components of the tumor microenvironment

We have examined the ability of various components of breast, colorectal and ovarian tumours to regulate the activation and function of LAK cells and TILs, immune effectors which have been used as anti-tumour therapies. Tumour cell or supernatants derived from their short-term in vitro culture, inhibited the activation of PBMC by IL-2 but supported the continued proliferation of LAK or TIL cells which had already been activated. Despite being able to enhance or suppress growth of a range of cell lines, a cell-free, soluble preparation (TDS) from primary tumours was uniformly inhibitory to IL-2 activated cells, suggesting that it reflected the immunoregulatory nature of human tumours more accurately than cell-cultures or their supernatants.     

Suppression of the generation of lymphokine-activated killer (LAK) cells by serum-free supernatants of in vitro maintained tumour cell lines

Serum-free supernatants from in vitro maintained gastrointestinal cancer and melanoma cell lines inhibit the generation of lymphokine (IL-2) activated killer (LAK) cells in a time and dose-related manner. Concentrations as low as 5% can inhibit the generation of LAK cytotoxicity but inhibition of proliferation is not observed until higher concentrations are included in the culture system. Inhibition is not observed with supernatants from a breast cancer cell line nor with supernatants from normal cells. There was complete concordance between the capacity of the tumour cells themselves to inhibit LAK generation and the presence of inhibitory activity in the corresponding supernatant. The inhibitory factor(s) is stable after heating to 44 and 56 degrees C. Production of the inhibitory factor(s) is sensitive to metabolic inhibitors and has a molecular weight greater than 25 kD. The inhibition of LAK cell stimulation by tumour cells may partially explain the failure of adoptively transferred LAK cells and IL-2 therapy to cause tumour regression in man.     

CTCL患者外周血淋巴细胞亚群、血清sIL-2R及炎性因子的水平变化特点及意义

目的:探讨皮肤T细胞淋巴瘤（CTCL）患者外周血淋巴细胞亚群及血清可溶性白细胞介素2受体（sIL-2R）、炎性因子的水平变化特点及意义。方法:选取2013年1月至2018年1月在我院治疗的CTCL患者35例（CTCL组）,同时选取炎症性皮肤病患者35例作为对照组,检测两组血清sIL-2R、B淋巴细胞、T淋巴细胞和NK细胞水平以及白细胞介素-6（IL-6）、肿瘤坏死因子 （TNF-α）和C反应蛋白（CRP）。结果:CTCL组血清sIL-2R为（770.15±87.44）U/ml,明显高于对照组（P＜0.05）,而B淋巴细胞为6.10（0.15～10.57）%,明显低于对照组（P＜0.05）;CTCL组和对照组T淋巴细胞和NK细胞比较差异无统计学意义（P＞0.05）;CTCL组血清IL-6、TNF-α和CRP分别为110.02(67.39,230.03)pg/ml、（31.29±7.28）pg/ml和（36.60±6.10）mg/L,明显高于对照组（P＜0.05）;皮损改善患者治疗后sIL-2R为（230.04±78.11）U/ml,明显低于皮损未改善患者（P＜0.05）。结论:CTCL患者血清sIL-2R、IL-6、TNF-α和CRP升高,而B淋巴细胞水平降低,其中sIL-2R与治疗效果有一定关系,值得进一步研究。     

Generation of Monoclonal Antibodies to the Rabbit Interleukin-2 Receptor a Chain (CD25) and Its Distribution in HTLV-1-transformed Rabbit T Cells

Rabbits can be infected with human retroviruses such as human T-cell leukemia virus-1 (HTLV-1) and human immunodeficiency virus (HIV), and provide useful animal models to study retroviral diseases such as adult T-cell leukemia and HIV. Previously we have succeeded in generating monoclonal antibodies (mAbs) against rabbit CD4, CD5 and CD11a antigens. To make this animal species more amenable to cellular and molecular studies, we have attempted to extend the panel of mAbs against rabbit CD antigens. Here we report on the generation of three neutralizing mAbs against interleukin-2 receptor α chain (IL-2Rα) (CD25), Kei-α1 (IgG2b), Kei-α2 (IgG2a) and Kei-α3 (IgG1). They specifically recognize the rabbit Mr 55,000 IL-2 binding protein, IL-2Rα, and completely inhibit both high- and low-affinity IL-2 binding to F648b cells that express IL-2Rα as well as IL-2Rβ. The use of mAb Kei-α1 confirmed that the rabbit IL-2Rα is not only a low-affinity IL-2R on its own but also an essential component of high-affinity IL-2R as found in other animal species, and that rabbit activated T cells including HTLV-1-transformed cell lines express high levels of the IL-2Rα. Together with mAbs against various rabbit CD antigens that we reported previously, these neutralizing mAbs to IL-2Rα will be valuable for studies of human retrovirus infections, such as those induced by HTLV-1 or HIV, in rabbits.     

Differential effects on expression of IL-2 receptors (p55 and p70) by the HTLV-I pX DNA

Abnormal expression of the low-affinity receptor for interleukin-2 (IL-2R) is a characteristic of the HTLV-I (+) leukemic T cells in adult T-cell leukemia (ATL). Despite the expression of IL-2R bearing Tac antigen (IL-2R/p55), leukemic cells of the majority of ATL patients do not proliferate in response to IL-2. In the human NK cell line, YT, as well as in ATL-derived T cells, the co-expression of IL-2R/p55 and the second IL-2R without the Tac epitope (IL-2R/p70) is required to produce high-affinity IL-2R. To study the effect of HTLV-I on both of the IL-2Rs, we transfected a fragment of HTLV-I containing the p40X gene into YT cells. One of the 2 transfected YT clones (YT/pX-5.1) had an increased level of expression of IL-2R/p55. In contrast, expression of IL-2R/p70 was unaffected, as determined by Scatchard analysis and the cross-linking study using 125I-IL-2. Our results show that the T-cell phenotype is not required for induction of IL-2R/p55 by p40X. We suggest that HTLV-I infection induces a disproportionate induction of IL-2R/p55 without significant enhancement of IL-2R/p70 expression, resulting in the predominant expression of low-affinity IL-2R in ATL. IL-2R/p70 may be a critical parameter determining the IL-2 reactivity of HTLV-I-infected T cells as well as of normal lymphocytes.     

Down-regulation of expression of interleukin-6 and its receptor results in growth inhibition of MCF-7 breast cancer cells

Interleukin-6 (IL-6) plays an important role in the neoplastic process through its action on cancer cell adhesion, motility, proliferation, tumor-specific antigen expression, and thrombopoiesis. IL-6 exerts its activity by binding to a high affinity receptor complex consisting of two membrane glycoproteins: the 80 kDa IL-6 a-receptor subunit (IL-6R) and the 130 kDa signal-transducing protein (GP130). In the present study, MCF-7 breast cancer cells were cultured with human IL-6 and IL-6 soluble receptor (sIL-6R). MCF-7 cells were also treated with either antibodies specific to human IL-6 and IL-6R, or synthetic antisense oligodeoxynucleotides (ODNs) targeted to IL-6 and IL-6R genes. Cell growth was measured, and it was found that human IL-6 and sIL-6R did not significantly increase the proliferation of MCF-7 cells. When IL-6 produced by the MCF-7 cells was bound by rabbit anti-human IL-6 antibody, there was a significant dose-dependent inhibition of cell proliferation. IL-6 and IL-6R antisense ODNs caused a marked and specific decrease in IL-6 and IL-6R mRNA and proteins, respectively. Both IL-6 and IL-6R antisense ODNs significantly inhibited the proliferation of MCF-7 cells, but the inhibitory effect of IL-6R antisense ODN was greater than that of IL-6 antisense ODN (IC??: IL-6R: 1 μM; IL-6: 5 μM, 72-hour incubation). Addition of exogenous IL-6 partially reversed the growth inhibition caused by IL-6 antisense ODN but not the growth inhibition caused by IL-6R antisense ODN. In conclusion, IL-6 plays an important role in maintaining the growth of MCF-7 breast cancer cells. These results suggest careful modulation of IL-6 and IL-6R expression of cells as a potential approach for breast cancer therapy.     

Prognostic significance of serum soluble interleukin-2 receptor level in non-Hodgkin's lymphoma: a single center study in Japan

Interleukin 2 receptor is expressed not only on the surface of activated T or B lymphocytes, but also on certain lymphoid malignancies. The receptor is released from the cell membrane as soluble form (sIL-2R). Serum sIL-2R level is a sensitive and quantitative marker of circulating peripheral blood mononuclear cell activation or specific tumor cell growth including non-Hodgkin's lymphoma (NHL). However, the relevance of serum sIL-2R levels relating to clinical outcome in adult patients with NHL remains uncertain. Therefore, we investigated the serial serum sIL-2R levels in 28 untreated patients with NHL to evaluate its correlation with clinical characteristics. High serum sIL-2R level (>1000 U/ml) at diagnosis was associated with a high incidence of treatment failure (p=0.03) and poor overall survival (p=0.057). The serum sIL-2R levels decreased significantly after achieving complete remission (p=0.003). Further larger studies are required to evaluate whether serum sIL-2R level is an independent prognostic factor or not. However, adding this parameter to those already employed in the International Prognostic Index would perhaps provide a better prognostic index for adult patients with NHL.