Interleukin-4 regulates the interleukin-2 receptors on human peripheral blood B lymphocytes.

The high-affinity interleukin-2 (IL-2) receptor (IL-2R) consists of the non-covalent association of at least two subunits, p55 and p70-75, capable of binding IL-2 with low and intermediate affinity, respectively. We studied the effects of cytokines on the IL-2R expressed on human peripheral blood B lymphocytes using monoclonal antibodies specific for IL-2R p55 and IL-2R p70-75, by means of two-colour flow cytometric analysis. In freshly isolated peripheral blood B lymphocytes, the p55 subunit was expressed only in a small population (7.0% of CD20+ cells), whereas the p70-75 subunit was expressed in a large population (89.0% of CD20+ cells). Of the cytokines studied, interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) were involved in the regulation of IL-2R on B cells. After a 2-day incubation with IL-4, expression of IL-2R p55 was markedly induced, but expression of IL2-R p70-75 was profoundly suppressed in a dose-dependent manner. These abilities of IL-4 to promote IL-2R p55 expression and suppress IL-2R p70-75 expression were inhibited by the presence of IFN-gamma. Other cytokines, including IL-1, IL-2, IL-5, and IL-6, had little effect on the expression of these two subunits. These findings suggest that IL-4 is a cytokine modulating B cell response through the regulation of IL-2R.     

Increased interleukin-2 production by human peripheral blood lymphocytes treated with low doses of dexamethasone

Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 116, N ^o 7, pp. 68–70, July, 1993.     

High recombinant interleukin-2 sensitivity of peripheral blood lymphocytes from patients with myasthenia gravis: correlations with clinical parameters

Myasthenia Gravis (MG) is an autoimmune disorder of neuromuscular transmission associated with antibodies (Ab) against acetylcholine receptor (AChR). Autoantibody production is a T-cell-dependent phenomenon perhaps caused by aberrant immunoregulation. So far, a possible role for immunoregulatory molecules has not been investigated in the pathogenesis of MG. Since interleukin-2 (IL-2) is able to induce peripheral blood mononuclear cell (PBMC) proliferation without a previous activating signal and to upregulate IL-2-receptor expression, we have evaluated the activation state of PBMC in patients with MG, by cytofluorographic analysis of CD25 expression and by testing their sensitivity to recombinant IL-2 (rIL-2) without any known previous stimulation. We found no significant difference in CD25 expression in a large group of patients compared to controls. However, proliferative responses to rIL-2 were significantly higher in MG patients than in controls. In MG, as in controls, this response was time- and dose-dependent, was inhibited by an anti-IL-2 receptor Ab and correlated with an increased percentage of CD25+ T cells after rIL-2 exposure. The response was greater in patients with a high anti-AChR Ab titer and a severe form of the disease, and in patients tested before thymectomy. Thus blood T cells in MG showed functional signs of preactivation (high sensitivity to rIL-2 alone) without detectable CD25 expression on fresh cells, raising the possibility of aberrant IL-2 receptor regulation and/or expression in MG T cells. Decreased sensitivity to rIL-2 after thymectomy, associated with general clinical improvement, suggests a role for activated cells originating from the thymus in the pathogenesis of MG, and is of clinical relevance in patient follow-up. Our findings also provide a new approach in the study of MG pathogenesis: the search for aberrant immunoregulation mechanisms linked to defects in lymphokine circuits.     

Activation of human peripheral blood lymphocytes by concanavalin A dependence of monocytes.

The activation of human peripheral blood lymphocytes or isolated T lymphocytes by concanavalin A (Con A) is hightly potentiated by the presence of autologous, mitomycin C-treated monocytes. The optimal lymphocyte: monocyte ratio within a broad dose range is 1:1 when the incorporation of [14C]thymidine is expressed as total incorporation per culture tube and 1:10 when expressed per lymphocyte. A five-to-ten-fold increase of total DNA synthesis is noted in the presence of 10-90% monocytes. The data may help to explain the wide variations in Con A responsiveness of human peripheral lymphocytes which may be partly related to differences in purification which give rise to cell preparations containing varying amounts of monocytes.     

Human recombinant interleukin-2 is mitogenic to human lymphocytes

Reported herein are the results of studies demonstrating that purified recombinant human interleukin-2 (hrlL-2) is a potent mitogen for lymphocytes of healthy human donors. The specificity of the hrlL-2-induced response was defined in experiments in which mitogenicity of this T cell growth-promoting lymphokine was completely abrogated by blocking the T cell membrane receptor for IL-2 with the anti-Tac monoclonal antibody. Depletion of adherent mononuclear leukocytes markedly reduced lymphocyte reactivity to hrlL-2, but the response could be fully recovered by the addition of interleukin-1 (IL-1). Increased proliferative responses were observed using a combination of hrlL-2 and a monoclonal antibody OKT3 that defines a T cell membrane antigen. These studies demonstrate that hrlL-2, as with antigens and phytomitogens, may serve as the first signal of T cell proliferation.     

Identification of interleukin-2 in human peripheral blood eosinophils.

Interleukin-2 (IL-2) is an essential growth factor for T cells. Previous studies have shown that human peripheral eosinophils respond to IL-2 in chemotaxis and express the IL-2 receptor (CD25). In addition, eosinophils have been shown to transcribe messenger RNA for IL-2. The aim of the present study was to determine whether eosinophils translate mRNA for IL-2 and to determine the site of intracellular localization. By immunocytochemistry, an average of 9% of cells showed cytoplasmic staining for IL-2 in freshly isolated unstimulated blood eosinophils obtained from asthmatic subjects who were not receiving oral corticosteroid treatment (n = 5). Freshly isolated, disrupted, highly purified eosinophils (> 99%, by CD16- immunomagnetic selection) contained an average of 6 pg/10(6) cells of IL-2 measured by a specific enzyme linked immunosorbent assay (ELISA) (n = 7). Purified eosinophil incubated with serum-coated Sephadex beads showed an increase in the amount of intracellularly-retained IL-2 (26.2 +/- 7.2 pg/10(6) cells) with some evidence for release of this cytokine but only in three out of six eosinophil preparations (range 1.3-5.8 pg/10(6) cells). The intracellular localization of IL-2 was determined by fractionation of the cells on a linear (0-45%) Nycodenz gradient in sucrose buffer followed by detection of IL-2 in the fractions using an IL-2-specific ELISA and dot blotting. The majority of the IL-2 detected co-eluted with known eosinophil granule markers (i.e. major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO) and beta-hexosaminidase) but small quantities were also detected in the cytosolic (lactate dehydrogenase-(LDH) associated) and membrane (CD9+) fractions. Immunogold labelling of intact eosinophils using an anti-IL-2 monoclonal antibody confirmed IL-2 immunoreactivity in association with the eosinophil crystalline granule cores. These data are consistent with the hypothesis that eosinophils synthesize, release and store IL-2 largely within cystalloid granules. This stored IL-2 may serve as a reservoir for rapid release of IL-2 in inflammatory reactions associated with eosinophilia.     

Development and maintenance of bovine cytotoxic lymphocytes with recombinant human interleukin-2

Long-term bovine lymphocyte cultures were initiated by stimulation with alloantigens and maintained in continuous culture using medium containing recombinant human interleukin-2 (rh IL-2). The development of specific and lectin-dependent killing was monitored following primary alloantigen challenge. Cytolytic activity was barely detectable after 7 days of culture, but gradually increased with peak activity occurring after 21 days of culture. A panel of monoclonal antibodies (MoAb) was used to determine whether a shift in the antigen phenotype of the cell population occurred during culture. The primary cell type that grew in culture was of the T-cell lineage with minimal or no expression of class II antigens. The activities of adenosine deaminase (ADA), purine nucleotide phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by microassay in resting peripheral blood lymphocytes (PBL) and in cells from long-term cultures. Large increases in the activities of PNP and HPRT with a decrease in the activity of ADA were observed. The data show that long-term cultures of lymphocytes can be readily generated, and that sequential changes in antigenic phenotype and function can be monitored and correlated with quantitative changes in enzyme activity.     

Differentiation of human peripheral blood B lymphocytes.

Human B-lymphocyte differentiation was studied by measuring the capacity of such cells, isolated from peripheral blood, to synthesize and secrete Ig after pokeweed stimulation. Results show that a maximum incorporation of [3H]-thymidine took place 2 days before the appearance of detectable Ig-secreting cells. On the 7th day after pokeweed stimulation, when Ig synthesis and secretion are at a maximum, [3H]-thymidine uptake was low. Since inhibition of DNA synthesis 3 days after pokeweed stimulation completely prevents the generation of Ig-secreting plasma cells, initial DNA synthesis is apparently essential before Ig-secreting plasma cells can develop in response to pokeweed stimulation.     

Activation of human peripheral blood mononuclear cells by interleukin-2 and granulocyte-macrophage colony-stimulating factor to inhibit Cryptococcus neoformans.

The abilities of selected cytokines to activate human peripheral blood mononuclear cells (PBMC) to inhibit and kill the opportunistic fungus Cryptococcus neoformans were studied. PBMC were cultured for 7 days in cell wells containing no cytokines, tumor necrosis factor (TNF), gamma interferon (IFN-gamma), 1,25-dihydroxycholecalciferol (vitamin D3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or interleukin-2 (IL-2) and were then challenged for 24 h with a fixed number of CFU of C. neoformans. The number of CFU increased in wells containing no cytokines, TNF, IFN-gamma, or vitamin D3 and remained about the same in wells containing GM-CSF. In contrast, the number of CFU in wells containing IL-2-stimulated PBMC decreased, suggesting fungicidal activity. Optimal conditions for IL-2 stimulation included a minimum of 5 days of incubation of PBMC with IL-2, a concentration of 100 U of IL-2 per ml, and a high ratio of effectors to fungi. Separation of IL-2-stimulated PBMC based upon their adherence to plastic revealed that antifungal activity resided in the nonadherent fraction. These data demonstrate that IL-2 and GM-CSF are capable of stimulating PBMC-mediated antifungal activity and suggest that these cytokines may play physiological or pharmacological roles in host defenses against cryptococcosis.     

Interaction of influenza A virus with human peripheral blood lymphocytes.

Peripheral blood lymphocytes cultured in the presence of phytohemagglutinin, concanavalin A, or pokeweed mitogen were exposed in vitro to influenza A virus. The synthesis of several virus-specific proteins, including the nucleoprotein, membrane protein, and nonstructural 1 protein were detected, although no infectious virus was produced by the lymphocyte cultures. Evidence was obtained that only a subpopulation of mitogen-transformed cells would support virus protein synthesis. A comparison of the interactions of influenza A virus with lymphocytes from normal individuals and from rheumatoid arthritis patients showed that the same range of virus-specific proteins were made, in similar quantities, regardless of the source of lymphocytes.     

Long-term cultures of human peripheral blood lymphocytes with recombinant human interleukin-2 generate a population of virtually pure CD3+ CD16- CD56- large granular lymphocyte LAK cells.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.     

Stimulation of resting normal human peripheral blood mononuclear cells by fetal calf sera. Activation to an interleukin-2 responsive state

Normal adult human peripheral blood mononuclear cells which are negative for interleukin-2 (IL-2) receptors as assessed by flow cytofluorometry, acquire IL-2 receptors and IL-2 responsiveness after culture in media supplemented with fetal calf sera. Thus, in the absence of any known external stimuli, fetal calf sera used to supplement culture media can induce the transformation of resting (G0) peripheral blood mononuclear cells to an activated (G1) state. The activated (G1) cells are able to progress through the rest of the cell cycle (S, G2, M) in the presence of IL-2. As a result, studies of human peripheral blood mononuclear cells in fetal calf serum-supplemented culture media should be interpreted with appropriate caution.     

Retinoids synergize with interleukin-2 to augment IFN-gamma and interleukin-12 production by human peripheral blood mononuclear cells.

We have demonstrated previously that cells from both the skin and peripheral blood from patients with cutaneous T cell lymphoma (CTCL) have elevated levels of protein and mRNA for Th2 cytokines, interleukin-4 (IL-4) and IL-5, and depressed levels of Thl cytokines, IL-2 and interferon-gamma (IFN-gamma). Furthermore, IL-12 in vitro can restore IFN-gamma production by these patients' cells to near normal levels. Because retinoids exert therapeutic activity in CTCL and are potent modulators of growth and differentiation of hematopoietic cells, we investigated the role of retinoids in modulating Thl cytokine production. Peripheral blood mononuclear cells (PBMC) from normal donors and patients with CTCL were cultured with medium, IL-2, 13-cis-retinoic acid, all-trans-retinoic acid, acetretin or etretinate alone, or IL-2 plus the retinoids for 24 h, and levels of IFN-gamma were determined using ELISA. IL-2 or retinoids alone could induce low but significant levels of IFN-gamma. However, when IL-2 was cultured with each retinoid, a synergistic augmentation of IFN-gamma levels (4-fold to 90-fold) was observed except in the case of etretinate. All-trans-retinoic acid (ATRA) was the most potent IFN-y inducer. Similar studies performed using PBMC from CTCL patients indicated the IFN-gamma augmentation occurred but in a blunted manner. The IFN-y-inducing effect of ATRA and 13-cis-retinoic acid could be abrogated by addition of anti-IL-12 antibodies, suggesting that IL-12 plays a role in the synergistic upregulation of IFN-gamma. Using an IL-12 p40-specific radioimmunoassay (RIA), we confirmed the presence of IL-12 in IL-2 plus retinoid-treated culture supernatants. Purified monocytes cultured with IL-2 plus ATRA did not secrete IL-12. Only when monocytes were cocultured with lymphocytes was there an increase in IL-12 production, suggesting the involvement of a paracrine feedback loop requiring both monocytes and lymphocytes. These data suggest that retinoids can induce Th1 cytokines from normal and CTCL PBMC and that this induction may be mediated through IL-12 production.     

重组白介素—4促进外周血淋巴细胞增殖和抗辐射的作用

目的 研究重组人白介素－4(rhIL-4)对正常人外周血淋巴细胞亚群的促增殖和对离体照射淋巴细胞的保护作用。方法 用MTT、碱性磷酸酶免疫组化和TUNEL方法,检测淋巴细胞的增殖,T、B淋巴细胞亚群和淋巴细胞凋亡。结果（1）在适当浓度PWM存在下,一定浓度的rhIL-4能明显促进正常人外周血淋巴细胞增殖,其中以无血清体系和培养后24h的作用较强;（2）rhIL－4的促增殖作用主要表现在,促进TH、Ts细胞亚群和B细胞的增殖;（3）一定浓度的rhIL-4能明显抑制6Gy γ-线照射引起的淋巴细胞总数、TH,Ts细胞亚群和B细胞数量的减少;（4）rhIL-4能明显抑制急性照射引起的大量淋巴细胞凋亡,可能是减轻淋巴细胞辐射损伤和改善机体免疫功能的重要原因。结论rhIL-4能使正常人外周血T、B淋巴细胞数量增加,对致死剂量照射引起的淋巴细胞减少也具有明显的抑制作用。     

Surface properties of LDL-binding lymphocytes in human peripheral blood.

Surface properties of low density lipoprotein (LDL)-binding lymphocytes were evaluated to determine whether LDL binds with a subpopulation of human peripheral blood lymphocytes (PBL). B- and T-cell rich fractions were prepared from PBL using E-rosette formation or nylon reticulum columns. Binding of FITC-labelled LDL with these cell fractions was determined with a fluorescent microscope and a fluorescence-activated cell sorter (FACS II). The specificity of the binding was evaluated by a dose-dependent inhibition of LDL binding with the addition of unlabelled lipoproteins. In parallel studies, surface properties including E-rosette formation, surface immunoglobulins, and receptors for IgG-Fc, as well as human and mouse C3 were examined. LDL binding lymphocytes were enriched in the B-cell rich fraction, and depleted in the T-cell rich fraction. In addition, FITC-LDL binding lymphocytes were selectively collected by the FACS II. These LDL binding cells restored surface immunoglobulins after incubation in serum-free medium following trypsinization. The majority of lymphocytes stimulated by PHA and PWM in vitro bound with LDL. It is concluded that LDL binds with B cells in fresh human PBL, while it binds with B and T cells in mitogen-stimulated lymphocytes. It is suggested that the selective collection of LDL binding lymphocytes by the FACS II can be applied to the evaluation of cellular interaction of these cells in various immunological reactions.     

人外周血T淋巴细胞胀亡现象的观察

目的观察人外周血T淋巴细胞的胀亡现象,探讨建立T细胞胀亡检测方法.方法密度梯度离心法及尼龙棉柱法分离健康成年人外周血T淋巴细胞,分空白组及地塞米松组,培养后观察细胞光镜、荧光镜及电镜形态学,并用流式细胞仪检测胀亡细胞比例变化.结果①人外周血T淋巴细胞经96小时体外培养,可自然出现典型细胞胀亡形态学改变.②经72小时培养,不同浓度地塞米松组(1×10-6、1×10-5、1×10-4、1×10-3 mol/L)T细胞的胀亡率分别为(3.49±0.42)%、(5.17±0.48)%、(8.44±0.72)%、17.93±1.50)%.③在1×10-5mol/L地塞米松作用下,不同培养时间(48、72、96、120小时)T淋巴细胞的胀亡率分别为(0.53±0.10)%、(6.36±0.80)%、(20.60±1.59)%、25.56±1.76)%.结论人外周血T淋巴细胞存在胀亡现象,地塞米松可诱导人外周血T淋巴细胞胀亡.     

Enhancement of human T lymphocyte function by prothymosin alpha: increased production of interleukin-2 and expression of interleukin-2 receptors in normal human peripheral blood T lymphocytes

The in vitro incubation of phytohemagglutinin (PHA)- or alloantigen-stimulated peripheral blood T cells with prothymosin alpha (ProT alpha) resulted in a marked and reproducible increase in the production of interleukin-2 (IL-2). Incubation of T cells with ProT alpha, in the absence of PHA or alloantigen, failed to induce any production of IL-2. ProT alpha by itself did not exert any IL-2 activity. Finally, ProT alpha was shown to increase the expression of IL-2 receptors on phytohemagglutinin- or alloantigen-activated T cells. These data provide the basis for understanding the in vitro immunoenhancing effects of ProT alpha in cellular immune systems.     

Carbohydrate metabolism in PHA stimulated human peripheral blood lymphocytes.

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-14C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can be easily quantitated by an enzymatic assay.     

Regulation of interleukin-2 induced interleukin-5 and interleukin-13 production in human peripheral blood mononuclear cells

Adjuvant interleukin (IL)-2 immunotherapy has been used for many years for a variety of malignant and nonmalignant entities. In many cases, a dose escalation might have seemed desirable, but was prevented by the rather severe adverse effects of systemic IL-2 application. Only recently has the regulation of IL-2 induced cytotoxicity been understood better, so that now efforts can be aimed at the design of cytokine cocktails that would selectively induce cytotoxicity but result in as few adverse effects as possible. Previously, induction of IL-5 under systemic IL-2 therapy has been described, and a number of the side-effects have been attributed to this event. We therefore investigated the regulation of IL-2 induced production of IL-5 and IL-13 (which, similarly to IL-5, is a mediator of allergy-like symptoms). At the same time, the effects of regulatory cytokines, such as IL-4, IL-10 and IL-12, on interferon-gamma (IFN-gamma), the major cytotoxic mediator of IL-2 therapy, were studied. All three have been discussed as antitumour immunotherapeutics, either alone or in combination with IL-2. In anti-CD3-treated peripheral blood mononuclear cells, IL-2 induced IL-5 and IL-13 alongside IFN-gamma, IL-10 and IL-12. In the presence of IL-2, inhibition of endogenous IL-12 production further enhanced the IL-5 and IL-13 responses, while IFN-gamma and IL-10 were markedly suppressed. Co-incubation with IL-2 and IL-12 suppressed IL-5/IL-13 below, but enhanced IFN-gamma and IL-10 above, levels induced by IL-2 alone. IL-10 was suppressive on all the investigated cytokines, while IL-4 interfered with IL-2 induced IFN-gamma and IL-12 production, but was additive to IL-2 in its effect on IL-5 and IL-13. These data suggest that the combination of IL-12 with IL-2 would enhance the cytotoxic activity of this regimen, but might reduce its adverse effects.