In vitro activity of labdane diterpene from Alomia myriadenia (Asteraceae): immunosuppression via induction of apoptosis in monocytes

A screening program in Brazilian flora was carried out to detect the presence of immunosuppressive compounds by using the in vitro phytohemagglutinin A (PHA)-induced human peripheral blood mononuclear cell (PBMC) proliferation assay. In this screening, we isolated from Alomia myriadenia Schultz-Bip. ex. Baker (Asteraceae), a labdane-type diterpene named myriadenolide. Incubation of human PBMC with this compound reduced significantly the percentage of CD14(+) cells, but it has no effect on the relative amount of CD3(+)CD4(-)CD8(+) and CD3(+)CD4(+)CD8(-) T lymphocyte subpopulations. Neither viability nor proliferative competence of T lymphocytes was significantly affected by myriadenolide. The toxic effect on monocytes (CD14(+) cells) may explain the inhibitory effect observed on PHA-induced lymphocyte proliferation. The cytotoxic effect of myriadenolide on monocytes was determined by measuring the percentage of hypodiploid nuclei content by propidium iodide staining, electron microscopy and simultaneous detection of CD14 and annexin V binding by flow cytometry. The results showed that myriadenolide induces a dose-dependent apoptosis in monocytes and thus explain the immunosuppressive effect observed.     

Interleukin-7 promotes lung-resident CD14+ monocytes activity in patients with lung squamous carcinoma

Interleukin (IL)-7 enhances cytokines secretion by CD14⁺ monocytes, and induces recruitment of monocytes to endothelium. As an important regulator to different types of immune cells, the role of IL-7 in modulation of CD14⁺ monocytes is still not fully elucidated. Thus, the aim of current study was to investigate the immunoregulatory activity of IL-7 to peripheral and lung-resident CD14⁺ monocytes in lung squamous carcinoma patients. Thirty-seven lung squamous carcinoma patients and eighteen healthy individuals were enrolled. CD14⁺ monocytes and CD4⁺ T cells were purified from both peripheral bloods and bronchoalveolar lavage fluids (BALF). IL-7 expression in plasma and BALF was measured by ELISA, and CD127 expression in peripheral and lung-resident CD14⁺ monocytes was investigated by real-time PCR and flow cytometry, respectively. Cellular proliferation, cytokine production, and molecules in IL-7 signaling pathway was assessed in CD14⁺ monocytes in response to IL-7 stimulation. IL-7-induced CD14⁺ monocytes activity to CD4⁺ T cells was also assessed in direct and indirect contact co-culture system. There were no remarkable differences of plasma IL-7 concentration or CD127 level between healthy individuals and lung squamous carcinoma patients. However, IL-7 expression was significantly reduced in BALF from tumor site in squamous carcinoma patients, especially in stage III and IV. IL-7 stimulation not only promoted proliferation, cytokines secretion, and STAT-5 phosphorylation in lung-resident CD14⁺ monocytes, but also enhanced CD14⁺ monocytes-induced Th1 and T follicular helper cells activation, which presented as elevated interferon-γ and IL-21 secretion by CD4⁺ T cells. This process required direct cell-to-cell contact, and was dependent on IL-6 secretion. The current data revealed a potential immunopromotive property of IL-7 to lung-resident CD14⁺ monocytes in lung squamous carcinoma.     

Effect of L-cycloserine on cellular responses mediated by macrophages and T cells

In this study, we examined the immunoregulatory roles of L-cycloserine (L-CS), a sphingolipid metabolism regulator with inhibitory activity of serine palmitoyltransferase (SPT), on immune responses mediated by monocytes/macrophages and T cells. Mitogenic responses of splenic lymphocytes induced by LPS, PHA, and Con A were very strongly suppressed by L-CS with IC(50) values ranging from 0.5 to 1 muM. In contrast, this compound less strongly blocked IL-2-induced CD8+ CTLL-2 cell proliferation with an IC(50) value of 540 muM. Interestingly, L-CS enhanced the number of IL-4-producing helper T cells, indicating the favored induction of Th2 condition. Although tumor necrosis factor (TNF)-alpha and nitric oxide (NO) production was not altered under 10% FCS condition, U937 cell-cell adhesion as well as the surface level of adhesion molecules (CD29 and CD98) were significantly suppressed by L-CS. In particular, reduced serum level (5%) under L-CS treatment strongly enhanced the production of TNF-alpha and the inhibitory potency of NO production and cell adhesion. Finally, sphingolipids (D-sphingosine and DL-dihydrosphingosine) did not remarkably abrogate L-CS-mediated T cell proliferation. Therefore our data suggest that de novo sphingolipid metabolism may represent an important aspect of immunomodulatory activities mediated by T cells and macrophages/monocytes, depending on serum level.     

Rottlerin inhibits human T cell responses

Rottlerin is a pharmacological inhibitor of protein kinase C (PKC) theta, a novel PKC selectively expressed in T lymphocytes. PKC theta is known to regulate T cell receptor (TCR)/CD28 signalling pathways in T lymphocytes, but the impact of PKC theta inhibition on human T cell responses remains undefined. In this work, we describe the effects of rottlerin on the responses of CD4+ and CD8+ human T lymphocytes upon polyclonal activation. We observed a dose-dependent inhibition of CD4+ and CD8+ T cell proliferation in response to anti-CD3/anti-CD28 antibodies stimulation in the presence of rottlerin. This inhibition was associated with impaired CD25 expression and decreased interleukin (IL)-2 production in activated T cells. In contrast, rottlerin did not alter IL-2-induced T cell proliferation. Furthermore, we demonstrated that rottlerin blocked interferon (IFN) gamma, IL-10 and IL-13 mRNA expression in TCR/CD28 activated CD4+ T cells. These findings place rottlerin as a potent immunosuppressive agent for the development of novel therapies in T cell mediated immune disorders.     

An activated set point of T-cell and monocyte inflammatory networks in recent-onset schizophrenia patients involves both pro- and anti-inflammatory forces

We recently described a pro-inflammatory gene expression signature in the monocytes of 60% of patients with recent-onset schizophrenia (SCZ). Here we investigated whether the T-cell system is also in a pro-inflammatory state. A detailed fluorescence-activated cell sorting (FACS) analysis, e.g. of CD3+CD25+ T cells, IFN-γ+, IL-4+, IL-17A+ (CD4+) lymphocytes and CD4+CD25highFoxP3+ regulatory T cells, was performed on peripheral blood of 26 patients with recent-onset SCZ (in 19 of whom the inflammatory gene expression signature of the monocyte had been determined) and in age-/gender-matched healthy controls. Various relevant T-cell cytokines, e.g. sCD25, IFN-γ, IL-17A and IL-4, were measured in serum by a multiplex assay. We detected: (a) not only higher percentages of pro-inflammatory-prone monocytes, activated CD3+CD25+ T cells and pro-inflammatory Th17 cells in patients, but also higher percentages of anti-inflammatory CD4+CD25highFoxP3+ regulatory T cells and IL-4+ lymphocytes; (b) that this activated T-cell set point was reflected in significantly raised serum levels of sCD25; (c) that the up-regulation of IL-4+-containing lymphocytes was predominantly found in patients characterized by a monocyte pro-inflammatory set point; and (d) that regulatory T-cell and Th17-cell numbers were higher in patients irrespective of the pro-inflammatory state of the monocytes. Our data do not support the concept that the T-cell system is in a simple pro-inflammatory state in recent-onset SCZ, but do show that the monocyte and T-cell networks are activated and involve both pro- and anti-inflammatory forces. This suggests control within an activated inflammatory system.     

Type of lymphocytes affected by the islet-activating protein (IAP)

By flow cytometric analysis, we identified the subclass of lymphocytes that proliferates in response to islet-activating protein (IAP), both in vitro (human peripheral blood mononuclear cells, MNC, cultured with IAP) and in vivo (peripheral blood MNC derived from A/J mice treated with IAP). IAP caused a preferential proliferation of CD8+ T cells. These cells expressed the IL-2 receptors on their surface. CD4+ CD8+ T cells could also be detected in these cultures, IAP caused human MNC to produce IL-1 and to induce expression of HLA-DR antigen. These effects may play an important role in the T-cell proliferation induced by IAP, although IAP by itself suppressed the proliferative action of IL-1 in mouse thymocytes. IAP induced proliferation of the purified CD4+ cells but had a smaller effect on the purified CD8+ cells. This suggests that the proliferation of CD8+ cells in IAP-treated MNC depends on the function of other types of cell, e.g. CD4+ cell and macrophage.     

Pertussis toxin-induced mitogenesis in human T lymphocytes

Pertussis toxin (PT) has previously been shown to affect a wide variety of immune responses and to cause lymphocyte proliferation. We have investigated the biochemical basis for the mitogenic activity of PT by using human peripheral blood lymphocytes. PT was found to induce a rapid rise in cytosolic free calcium concentration and an alkalinization of the cytosol through the Na+/H+ antiporter. The toxin was also found to induce expression of IL-2-receptor on CD3+ cells and to stimulate IL-2 production. PT induced proliferation of both CD4+ and CD8+ T cells in the presence (but not in the absence) of accessory cells. PT also stimulated IL-1 production by monocytes but neither IL-1, IL-6 alone nor a combination of the two lymphokines could replace accessory cells suggesting that cell:cell contact is required. Low doses of PT induced ADP-ribosylation of G proteins but this treatment did not affect significantly PHA-induced [Ca2+]i increase and IL-2-induced DNA synthesis suggesting that the substrates of the ADP-ribosyltransferase activity of PT are not involved in the signalling pathways leading to DNA replication.     

Partial review of immunotherapeutic pharmacology in stem cell transplantation

In two separate lymphoma populations, we examined immune reconstitution following high dose chemotherapy (HDT) and bone marrow transplantation (BMT). In the first study we followed immune reconstitution for one year after HDT and BMT. In the second study we examined the ability of the orally active immunomodulator, Bestatin to augment immune reconstitution following HDT and BMT. The studies on immune reconstitution following HDT and BMT were undertaken in a cohort of non-Hodgkin's lymphoma (NHL) patients (n = 35) and examined the peripheral blood (PB) leukocyte subsets and their in vitro functions. Our results demonstrate that monocyte and natural killer (NK) cell engraftment occurred more rapidly then did T cell reconstitution. We also observed a significant decrease in the CD4:CD8 ratio post-transplantation as compared to normal PB donors due to a decrease in CD4+ cells. In addition, following HDT and BMT, measures of T cell function (phytohemagglutinin [PHA] mitogenesis) and T helper cell activity (pokeweed mitogen [PWM] mitogenesis) were consistently depressed as compared to cells from normal PB. Further, we demonstrate a correlation between the loss of T cell function and the frequency of circulating monocytes, suggesting a cause-effect relationship. Despite the dysfunction in T cells following HDT and BMT, immune-modulating agents can still augment the immune function. One such drug is Bestatin (ubenimex), an inhibitor of aminopeptidase (AP) that binds to CD13 on macrophage/monocytes. To examine its immune modulatory activity after HDT and BMT, a dose finding (10, 30, 90 and 180 mg/day) phase Ib trial was conducted with 30 Hodgkin's disease (HD) and NHL patients who received no drug (control), or Bestatin daily for 60 days following BMT. In these studies, Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on two consecutive days. These studies revealed that Bestatin significantly increased the PHA and PWM responses in a dose-dependent manner. Flow cytometric analysis revealed a significant increase in NK cells (CD56+), B cells (CD19+), as well as the CD4:CD8 cell ratio. The latter observation was associated largely with a depression in the percent of CD8+ T cells as opposed to an increase in CD4+ T cells. We conclude that despite the peripheral tolerance observed following HDT and BMT, Bestatin could significantly increase some, but not all, immune surrogates.     

Immune reconstitution after autologous hematopoietic transplantation with Lin-, CD34+, Thy-1lo selected or intact stem cell products

In sequential studies, we compared immune reconstitution following high-dose chemotherapy (HDT) and stem cell transplantation (SCT) using intact mobilized peripheral blood stem cell (PSC) in intermediate grade non-Hodgkin's lymphoma (NHL) patients and CD34(+), lineage-negative (Lin(-)), Thy-1(lo) (CD34(+)Lin(-)Thy-1(lo)) stem cells in low-grade NHL patients. Cytokine expression and cellular phenotype and function were used as the basis of comparison. Despite differences in cellular composition of the stem cell grafts, immune reconstitution in both groups was similar. Significantly higher levels of type 1- and 2-associated cytokine messenger ribonucleic acid (mRNA) were observed both prior to and following transplant in the peripheral blood (PB) of both cohorts as compared to normal individuals. Similar levels of interleukin (IL)-4, IL-10, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha) messenger ribonucleic acid (mRNA) were seen in PB mononuclear cells following transplant with either product. In contrast, patients receiving isolated CD34(+)Lin(-)Thy-1(lo) cells expressed significantly higher IL-2 levels at all times examined post-transplant. Despite the high levels of cytokine gene expression and rapid restoration to pretransplant levels of CD3 cell number by day 30, T cell function and CD4:CD8 and CD4(+)CD45RA:CD4(+)CD45RO(+) ratios were significantly depressed in both cohorts compared to normal donors, and significantly lower in patients transplanted with CD34(+)Lin(-)Thy-1(lo) compared to patients receiving an intact PSC product. These data suggest that the peripheral tolerance in patients receiving HDT and an autologous SCT occurs independent of graft composition, although immune function and CD4 recovery are better facilitated by transplantation of an intact product.     

Immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the<Emphasis Type="Italic">In Vitro</Emphasis> immune response in pristane-induced lupus mice

Systemic lupus erythematosus (SLE) is characterized by overactive B cells that differentiate into autoantibody-forming cells, aberrant T cell function that provides helping B cells produce autoantibodies, and overproduction of proinflammatory cytokines. However, immunodysregulation in lupus pathogenensis remains incomplete. We examined mitogen-stimulated production of proinflammatory cytokines, cell proliferation, T cell activation, and T cell apoptosis in vitro in pristane-induced lupus BALB/c mice compared to normal mice. LPS-stimulated production of IL-6 and IL-10 by splenocytes and macrophages from pristane-induced lupus mice were remarkably up-regulated compared to normal mice, whereas production of macrophage TNF-alpha was significantly down-regulated. Moreover, in vitro production of IL-2, IL-6, IL-10 and IFN-gamma by Con A-stimulated splenocytes, cell proliferation in LPS- or Con A-stimulated- thymocytes and splenocytes, and expression of CD69+CD4+ T cells in Con A-stimulated splenocytes were greatly increased in cells derived from pristane-induced lupus mice compared to normal mice. In addition, splenic T cells and CD4+ T cells in thymocytes from pristane-induced lupus mice were more resistant than nonautoimmune normal cells to Con A-induced apoptosis. Our findings indicate that immunoregulatory abnormalities of T cells and hyperreactivity of B cells in the in vitro immune responses in pristane-induced lupus mice may explain some of lupus pathogenesis.     

Effect of Salmonella-infected human monocytes on natural killer cell cytotoxicity. In vitro studies.

Various chemicals, including some bacteria-derived components, modulate natural killer cell (NKC) activity. We have analyzed the effect of wild-type Ty2 and of mutant strain TYT1231 Salmonella typhi-infected monocytes (U937 cells and human autologous monocytes) on NKC cytotoxicity of peripheral blood mononuclear cell (PBMC) and highly purified NKC (HPNKC; CD16+/56+ > 95%; the rest corresponding to CD3+ T-cells). PBMC's co-culture with either S. typhi strain infected U937 cells (medium or non-infected U937 cells as controls) resulted in the induction of lymphocyte activated killer (LAK) cell activity showing cytotoxicity against target human NKC-resistant lymphoblastoid Daudi cell line. Comparable experiments using autologous monocytes gave similar results. Co-culture of HPNKC preparations with either S. typhi strain infected U937 cells resulted in increased LAK cell activity against target Daudi cells in each and everyone of the five samples tested; paired Student's t-test p < 0.01 for both times (20 and 40 h) tested. Similar to the results observed in the experiments using PBMC, we did not find significant differences in the ability between medium and non-infected cells, or between wild-type S. typhi Ty2 and mutant strain TYT1231 infected U937 cells, to induce LAK activity in HPNKC preparations. PBMC co-incubation with either S. typhi strain infected U937 cells or autologous monocytes resulted in significant increases in IL-12, TNF-alpha, and IFN-gamma secretion. In similar experiments using HPNKC samples instead, infected U937 cells significantly increased IL-12 and IFN-gamma, but not TNF-alpha secretion. PBMC co-incubation with non-infected U937 cells, but not with non-infected monocytes, significantly increased supernatant IL-12 and TNF-alpha levels (no significant changes in IFN-gamma were recorded). Secreted cytokines remained essentially unchanged after co-incubating HPNKC preparation with non-infected U-937 cells. Incubation of PBMC or HPNKC preparations with either S. typhi strain infected U937 cells failed to produce significant changes in the expression of NKC lineage (CD16+/56+) or activation (CD28+, CD69+ and CD95+) markers. The ability of infected monocytes to induce LAK activity, release NKC cytokines and upmodulate NKC's CD95+ marker expression was essentially the same for both infecting Salmonella strains used. These results suggest a role for NKC in the physiological defensive response against intracellularly infected monocytes representing, perhaps one of the earliest antimicrobial mechanisms of the innate immune system.     

Effect of coronary percutaneous revascularization on interferon-gamma and interleukin-10 producing CD4+ T cells during acute myocardial infarction

T lymphocytes play an important role in the induction and progression of acute coronary syndromes (ACS). To gain insight into how different T cell subsets can influence ACS, we analyzed the frequencies of circulating CD4+ T cells producing either pro-inflammatory interferon(IFN)-gamma or anti-inflammatory interleukin (IL)-10 in subjects presenting with ST-elevation myocardial infarction (STEMI). The effect of coronary bare metal (BS) and paclitaxel-eluting stent (PES) on the balance between CD4+IFN-gamma+ and CD4+IL-10+ lymphocytes was also investigated. Peripheral blood mononuclear cells (PBMC) were isolated from 38 consecutive patients with STEMI before and 48 hrs or 6 days after implantation of either BS or PES. Twenty patients with no history of coronary artery disease were included as basal controls. PBMC were stimulated in vitro with anti-CD3/anti-CD28 monoclonal antibodies, and CD4+IFN- gamma+ or CD4+IL-10+ T cells were detected by flow cytometry intracellular staining. The frequency of peripheral CD4+IL-10+ T cells was significantly higher in STEMI patients as compared with controls. Conversely, the frequency of CD4+IFN-gamma+ T lymphocytes did not differ between STEMI and subjects without history of coronary artery disease. Six days after the revascularization procedure, the percentage of CD4+IL-10+ T cells was significantly decreased in BS but not in the PES group, whereas the relative percentage of CD4+IFN-gamma+ T lymphocytes were diminished in both groups as compared with baseline levels. Our data indicate that STEMI is associated with a peripheral expansion of CD4+IL-10+ T lymphocytes, and that primary coronary revascularization with implantation of either BS or PES is followed by a reduction in circulating CD4+IFN-gamma+ T lymphocytes. PES implantation, however, appears to inhibit the relative decrease of the IL-10 producing lymphocyte as observed in BS implanted patients, shifting the balance between pro-inflammatory and anti-inflammatory T cell populations in favor of the latter.     

奥沙利铂联合Exosome诱导的效应性T细胞抑制HepG2细胞增殖的研究

目的观察奥沙利铂(OXA)联合负载HepG2细胞裂解物的Exosome诱导的效应性T细胞对肝癌HepG2细胞系增殖的抑制作用。方法自健康人外周血中提取单个核细胞和T淋巴细胞,并以GM-CSF+IL-4的培养方案获得树突状细胞(DC)。将反复冻融后产生的HepG2细胞裂解物体外致敏DC,收集培养上清后以超高速离心法分离得到Exosome,电镜下观察其形态特征。用致敏DC及其分泌的Exosome刺激T淋巴细胞的增殖和活化。以ELISA试验检测不同刺激环境下T细胞分泌IL-1β、IL-2、IL-4、IL-6、IL-10、IFN-γ和TNF-α的能力。以细胞杀伤实验(MTT法)检测不同因素刺激后的T细胞、OXA(2.5、5.0、10.0mg.L-1)以及两者联合应用对HepG2细胞生长的抑制效应。结果与未致敏DC及其分泌的Exosome相比,HepG2细胞冻融裂解物致敏的DC及其分泌的Exosome能显著促进T细胞的增殖,并使其活化和分泌大量的IL-1β、IL-2、IL-6、IFN-γ和TFN-α等Th1型细胞因子。活化后的T细胞、OXA以及两者联合应用对HepG2细胞体外增殖均有抑制作用,而活化T细胞联合10mg.L-1OXA对HepG2细胞的杀伤率最高,但与联合5 mg.L-1OXA的效应相比,差异无统计学意义(P0.05)。结论负载肿瘤细胞抗原的DC所分泌的Exosome可在体外刺激T细胞,使其活化为具有抗肿瘤效应的细胞毒性T细胞,该细胞联合低浓度OXA能显著抑制肝癌细胞的体外增殖,并降低OXA的用量。     

Differential effect of sodium arsenite during the activation of human CD4+ and CD8+ T lymphocytes

Contamination of water with arsenic is a problem affecting several regions of the world. Peripheral blood mononuclear cells (PBMC) from chronically exposed individuals show a lower replicating activity than non-exposed individuals when stimulated with phytohemagglutinin (PHA). We have previously reported that PBMC from healthy donors treated in vitro with 1 muM sodium arsenite (NaAsO2) and stimulated with PHA showed a reduction in proliferation by a delay in cell cycle entry and a decrease in the rounds of cell division. In this paper we tested the effect of 1-5 muM NaAsO2 on the proliferation, viability, blast transformation, expression of the CD4 and CD8 molecules, and during the activation and proliferation of both CD4+ and CD8+ T lymphocytes. We found a reduction in cell proliferation and an increase in non-dividing cells with higher concentrations of NaAsO2 (2-5 microM) when proliferation was studied by 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) dilution. The use of 7-aminoactinomycin D (7-AAD) in CFSE-labeled cells allowed us to detect an increase in percentage of non-dividing cells, and an increase in apoptotic/dead cells mainly in non-proliferating cells. Analysis of the expression of CD4 and CD8 molecules on these cells showed that concentrations > or = 2 microM NaAsO2 reduced the expression of the CD8 molecule and induced apoptosis/death in CD4+ cells. Analysis of blast transformation by flow cytometry showed an accumulation of CD8+ resting cells in the presence of NaAsO2. Analysis of CD25 and CD69 expression in kinetics experiments in both subtypes showed a delay in the expression of CD25 and a delay in the downregulation of the CD69 molecule, in both CD4+ and CD8+ cells. However, in the case of CD8+ cells, we detected an accumulation of a CD25- CD69- population in the presence of increasing concentrations of NaAsO2. Altogether, our results show that NaAsO2 alters the expression kinetics of the early activation molecules CD25 and CD69 similarly in both subtypes. In addition, activated and non-activated CD4+ cells die by apoptotic mechanisms and although a percentage of CD8+ cells also die by apoptosis, a subpopulation of these cells is unable to activate and thus accumulates as resting cells.     

Cardiotoxin-III selectively enhances activation-induced apoptosis of human CD8+ T lymphocytes

Cardiotoxin-III (CTX-III), a major cardiotoxin isolated from the venom of the Taiwan cobra (Naja naja atra), is a highly basic, hydrophobic, toxic protein, which can induce lysis of mononuclear cells by an unknown mechanism. This study was undertaken to investigate the effects of CTX-III on untreated and PHA-activated peripheral blood mononuclear cells (PBMCs) in vitro. The results show that treatment of PHA-activated lymphocytes with CTX-III (10 microg/ml) induced apoptosis and depletion of the CD8(+) population. In both untreated and PHA-treated lymphocytes, interferon-gamma production was dramatically reduced and interleukin-2 (IL-2) production was moderately reduced by CTX-III treatment. In PHA-activated lymphocytes, CD4 expression was increased, whereas CD8 and IL-2R beta chain (CD25) expression were decreased. In contrast, CTX-III had no effect on the viability of PHA-activated monocytes but significantly enhanced their tumor necrosis factor-alpha production. These results show that CTX-III selectively enhanced activation-induced apoptosis in CD8(+) T cells. CTX-III was found to bind to the cell membrane of PHA-stimulated PBMCs, and three CTX-III-binding proteins, with molecular weights of 92, 77, and 68 kDa, were identified. We therefore propose that CTX-III interacts with one or more cell surface proteins and initiates a signal pathway causing functional changes. These findings provide an insight into the immunomodulatory properties of CTX-III and suggest a novel method for the selective induction of apoptosis in CD8(+) T lymphocytes.     

Administration of macrophage colony-stimulating factor mobilized both CD11b+CD11c+ cells and NK1.1+ cells into peripheral blood

We attempted the phenotypic characterization of peripheral blood (PB) cells after daily administration of macrophage colony-stimulating factor (M-CSF) in mice. The number of CD11b+ cells was increased by M-CSF treatment (2- and 5-day injections). Notably, CD11bbrightCD11cdim, CD11b+CD11c+ and CD11b+CD80+ cells were significantly increased by 2-day treatment of M-CSF. On the other hand, the number of NK1.1+ cells was not changed by the 2-day treatment, but it was significantly increased by the 5-day treatment. However, the numbers of CD3+ and NK1.1+CD3+ cells were not changed by M-CSF treatment. Then, mononuclear cells (MNCs) were separated from the PB of mice treated with saline or M-CSF, and they were incubated with GM-CSF + IL-4 or IL-2. Compared with the saline-treated one (S-MNCs), the MNCs of M-CSF-treated mice (M-MNCs) showed strong proliferation by the GM-CSF + IL-4 stimulation. The MNCs could stimulate proliferation of allo-T cells in the mixed lymphocyte reaction (MLR), especially the M-MNCs showed strong reaction. On the other hand, the stimulation by IL-2 induced strong cell growth of MNCs. And M-CSF treatment enhanced this response. Furthermore, the M-MNCs (stimulated by IL-2 in vitro) exhibited greater cytotoxicity against Yac-1 cells than the S-MNCs. In conclusion, we found that administration of M-CSF mobilized CD11b+, CD11b+CD11c+, CD11b+CD80+, and NK1.1+cells into PB. And the injection of M-CSF facilitates the generation of dendritic and natural killer cells from PB cells in vitro. These results suggest that the mobilized cells may provide for application of immunotherapy.     

Measurement of circulating T cell subsets positive for Fas antigen (CD95) in patients with alcoholic hepatitis.

T cell subsets positive for Fas antigen in peripheral blood of patients with alcoholic hepatitis were measured, using monoclonal antibodies in two colour immunofluorescence assay with flowcytometry. 1) In the patients with alcoholic hepatitis, the ratio of mean +/- standard deviation (M +/- SD) of CD4+ cells positive for CD95 (Fas antigen) in peripheral blood lymphocytes of patients with alcoholic hepatitis tended to increase, but the ratio of CD95-positive cells in CD4+ cells of peripheral blood was almost the same, compared with those of healthy controls. In the alcoholics (overdrink) who did not show alcoholic hepatitis with or without apparent alcoholic damage, the ratio of CD95-positive CD4+ cells in peripheral blood lymphocytes was within normal range, while CD95-positive cells in CD4+ cells of peripheral blood tended to decrease. 2) In the alcoholic hepatitis, the ratios of CD8+ cells positive for CD95 and CD95-positive cells in CD8+ cells of peripheral blood decreased significantly, and in the alcoholics (overdrink) they also tended to decrease. 3) The fluorescence intensity of CD95 on CD4+ cells in peripheral blood of the alcoholics (overdrink) decreased apparently, although the one on CD8+ cells did not. 4) The ratios of T cell subsets, that is, CD4+ and CD8+ cells, positive for HLADR in peripheral blood of the patients with alcoholic hepatitis increased significantly, respectively. These results showed that the ratio of CD8+ cells positive for Fas antigen in peripheral blood of the patients with alcoholic hepatitis decreased. It was suspected that this finding might be due to the direct effect of intaken alcohol to T cell subsets rather than hepatitis, and/or the result of immunological homeostasis in alcoholic hepatitis.