Phosphatidylserine-induced modulation of the immune response in mice: effect of intravenous administration

The action of phosphatidylserine on the immune response has been examined in mice after the intravenous administration of phospholipid or exposing cultured splenocytes to the action of phosphatidylserine vesicles. Phosphatidylserine (5-25 mg/kg) reduces the T-dependent and the T-independent antibody production. This effect is observed when the phospholipid is injected before (4 h) but not after (24 h) the immunization. The decreased influence of phosphatidylserine injected 24 h before the immunization indicates the reversibility of the action of phospholipid. The effect on the immune system may in part reflect a direct interaction with lymphocytes, since phosphatidylserine (12-60 microM) decreases the production of T-cell growth factors (mainly interleukin-2) elicited by mitogens in cultured spleen cells and reduces the expression of growth factor receptors in the same cells activated by mitogens. In addition, the activity of T-helper cells is found to be reduced in mice receiving the injection of phosphatidylserine. By contrast, the antigen processing and presentation by macrophages is not affected. The data suggest that the intravenous injection of phosphatidylserine vesicles in mice is followed by a transient decrease of lymphocyte activity.     

Regulation of interleukin-2 production and phosphatidylserine synthesis in Jurkat T lymphocytes by K+ channel antagonists

Modification of phospholipid metabolism during T cell activation has been repeatedly reported. Recently, we have shown that phytohaemagglutinin, CD3 and CD2 mAbs, which are potent in vitro activators of helper T lymphocytes, markedly inhibit phosphatidylserine synthesis concomitantly as they induce the secretion of IL-2. In this paper, we show evidence that in T lymphocytes K+ channels, which have been shown to participate in the cell activation process, are also reciprocally related to phosphatidylserine synthesis. In fact, in resting T cells the drugs affecting the activity of K+ channels, such as quinine and 4-aminopyridine, induce a rise of phosphatidylserine synthesis. In activated cells, quinine and 4-amidopyridine also caused a rise in phosphatidylserine synthesis which paralleled a decreased production of IL-2, strongly suggesting that these two events are correlated in a reciprocal manner. More precisely, phosphatidylserine synthesis was stimulated by drugs which have been reported to inhibit potassium channels in lymphocytes, e.g. quinine, 4-aminopyridine, tetraethylammonium. These data suggest that the decreased PS synthesis observed during T cell activation intervenes in the cascade of events leading to IL-2 secretion. The decrease in the biosynthesis of this phospholipid seems to be dependent on the activity of K+ channels.     

Role of protein kinase C in the phosphatidylserine-induced inhibition of DNA synthesis in blood mononuclear cells.

The mechanism of immunosuppressant activity of phosphatidylserine has been studied in peripheral blood mononuclear cells depleted or not of monocytes. After the addition of phosphatidylserine, mass determinations and uptake of labeled compound demonstrate its transfer into the cells. Phosphatidylserine incorporation causes a 2.5-fold increase of membrane-bound protein kinase C activity. The activation of translocated enzyme is indicated by the inhibition of phosphoinositide hydrolysis, and early feedback effect induced by activated protein kinase C. This action of phosphatidylserine is reproduced by tetradecanoylphorbolacetate and is prevented by the protein kinase C inhibitor, staurosporine. Consistently, phosphatidylserine (8 nmol/10(6) cells) decreases by 46% the production of inositol phosphates in cells responding to phytohemagglutinin. The decrease of phosphoinositide signal pathway as well as the inhibition of mitogen-induced DNA synthesis are produced at the same phosphatidylserine concentration and are equally manifest in total mononuclear cells or in preparations depleted of monocytes. However, only in the presence of monocytes does tetradecanoylphorbolacetate enhance the action of phospholipid, decreasing its IC50 from 13-15 microM to 7 microM. Thus, the data suggest that a reaction driven by protein kinase-C and a factor released by activated monocytes are involved in the phosphatidylserine-induced inhibition of lymphocyte DNA synthesis.     

Induction of apoptosis in human mitogen-activated peripheral blood T-lymphocytes by the ether phospholipid ET-18-OCH3: involvement of the Fas receptor/ligand system.

1. Activated T-cells constitute a target for treatment of autoimmune diseases. We have found that the antitumour ether phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) induced dose- and time-dependent apoptosis in human mitogen-activated peripheral blood T-lymphocytes, but not in resting T-cells. T-lymphocytes were stimulated with phytohemagglutinin and interleukin-2 or with concanavalin A. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses, as well as through visualization of internucleosomal DNA fragmentation in agarose gels. 2. The ET-18-OCH3-mediated apoptotic response in activated T-lymphocytes was less intense than in human leukaemic T cell lines, such as Jurkat cells and Peer cells; namely about 25% apoptosis in activated T-cells versus about 46-61% apoptosis in T leukaemic cells after 24 h treatment with 10 microM ET-18-OCH3. 3. The ET-18-OCH3 thioether analogue BM 41.440 (ilmofosine) showed a similar apoptotic capacity to that found with ET-18-OCH3 in activated T-cells, whereas the phospholipid analogue hexadecylphosphocholine (miltefosine) failed to promote this response. 4. The uptake of [3H]-ET-18-OCH3 was much larger in activated T-cells than in resting lymphocytes. 5. Using a cytofluorimetric approach we have found that ET-18-OCH3 induced disruption of the mitochondrial transmembrane potential and production of reactive oxygen species in activated T-cells, but not in resting lymphocytes. 6. ET-18-OCH3 induced an increase in Fas (APO-1/CD95) ligand mRNA expression in activated T-cells, and incubation with a blocking anti-Fas (APO-1/CD95) antibody partially inhibited the ET-18-OCH3-induced apoptosis of activated T-lymphocytes. 7. These results demonstrate that mitogen-activated T-cells, unlike resting lymphocytes, are able to take up significant amounts of ET-18-OCH3, and are susceptible to undergo apoptosis by the ether lipid via, in part, the Fas (APO-1/CD95) receptor/ligand system. This ET-18-OCH3 apoptotic action can be of importance in the therapeutic action of this ether lipid in certain autoimmune diseases.     

Effect of cordycepin on interleukin-10 production of human peripheral blood mononuclear cells

Therapeutic options for controlling autoimmune diseases are still very limited. Interleukin-10 has been reported to be a promising approach to therapeutic intervention. In the search for a drug which results in the selective upregulation of interleukin-10, we investigated the immunoregulative effects of cordycepin. We have measured interleukin-10 and interleukin-2 secretion of human peripheral blood mononuclear cells that were incubated with cordycepin and assessed the influence of cordycepin on the expression of interleukin-10 mRNA, the proliferative response and the expression of surface markers on T lymphocytes. In addition, the subsets of interleukin-10-secreting cells, the influence of anti-interleukin-10 neutralizing antibody and cytotoxicity of cordycepin were evaluated. Our results suggest that cordycepin has a significantly upregulative effect on interleukin-10 production and interleukin-10 mRNA expression. Interleukin-10-producing cells included in CD4+, CD8+, CD19+, CD56+ and CD14+ cells. At the same time, cordycepin inhibited phytohaemagglutinin-induced interleukin-2 production and proliferation of peripheral blood mononuclear cells. A restricted T lymphocyte activation was also reflected by a reduced expression of the surface markers CD25, CD45RO, CD54, CD71 and HLA DR. Anti-interleukin-10 neutralizing antibody could not completely block the suppressive effect of cordycepin on production of interleukin-2. Cordycepin in the effective concentration presented slight cytotoxicity but did not increase apoptosis. These results indicate that cordycepin exerts immunoregulative effects. Further research on it may provide an approach for the development of novel immunomodulatory drugs which directly alter the secretion of cytokines.     

Low DNA damage in peripheral lymphocytes of healthy elderly individuals with inverted CD4:CD8 ratio

The aim of this study was to evaluate the DNA damage in peripheral lymphocytes and the frequencies of CD8+ T cells expressing CD25, CD28 and CD45ro in aged individuals with inverted CD4:CD8 ratio. Blood samples of elderly individuals (aged >65) with normal CD4:CD8 ratio (n = 8) and inverted CD4:CD8 ratio (n = 8) were collected to identify the expression of CD25+, CD28+ and CD45ro+ in CD8+ T cells. DNA damage index was evaluated by the alkaline comet assay which was performed in lymphocytes treated with different concentrations of methyl methanesulfonate (MMS) (control non-treatment, 2 × 10⁻⁵ M, 4 × 10⁻⁵ M) for 1, 2 or 24 h. Elderly individuals with inverted CD4:CD8 ratio presented low frequency of CD8+ CD28+. Moreover, low DNA damage was observed in lymphocytes of elderly with inverted CD4:CD8 ratio in different doses of MMS. Aged individuals with inverted CD4:CD8 ratio presented lower DNA damage events in peripheral lymphocytes, suggesting a resistance for cell death in T cells of individuals with immune risk profile.     

Effect of topical immunomodulators on acute allergic inflammation and bronchial hyperresponsiveness in sensitised rats

We examined the effects of different immunomodulators administered topically on asthmatic responses in a rat model of asthma. Sensitised Brown-Norway rats were administered rapamycin, SAR943 (32-deoxorapamycin), IMM125 (a hydroxyethyl derivative of D-serine(8)-cyclosporine), and budesonide by intratracheal instillation 1 h prior to allergen challenge. Allergen exposure induced bronchial hyperresponsiveness, accumulation of inflammatory cells in bronchoalveolar lavage fluid, and also an increase in eosinophils and CD2+, CD4+ and CD8+ T cells in the airways. Interleukin-2, interleukin-4, interleukin-5, interleukin-10, and interferon-gamma mRNA expression was upregulated by allergen exposure. Budesonide abolished airway inflammation, suppressed the mRNA expression for interleukin-2, interleukin-4, and interleukin-5 (P<0.03), and bronchial hyperresponsiveness (P<0.05). IMM125 suppressed airway infiltration of eosinophils, and CD8+ T cells (P<0.02), and prevented the upregulated mRNA expression for interleukin-4, interleukin-5, and interferon-gamma (P<0.02). Rapamycin suppressed CD8+ T cell infiltration in airway submucosa (P<0.03), and mRNA expression for interleukin-2 (p<0.002), while SAR943 suppressed interleukin-2, interleukin-4, and interferon-gamma mRNA (P<0.05). IMM125, rapamycin and SAR943 did not alter airway submucosal CD2+ and CD4+ T cell infiltration, and bronchial hyperresponsiveness. CD8+ T cells, in contrast to CD4+ T cells, are more susceptible to the inhibition by IMM125 and rapamycin, which also caused greater suppression of Th1 compared to Th2 cytokine mRNA expression. In this acute model of allergic inflammation, differential modulation of Th1 and Th2 cytokines may determine the effects of various immunomodulators on airway inflammation and bronchial hyperresponsiveness.     

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.     

Effects of single intravenous doses of recombinant human interleukin-10 on subsets of circulating leukocytes in humans

Recombinant human interleukin-10 (rhIL-10) is a potent and specific immunomodulatory agent which inhibits endotoxin-stimulated pro-inflammatory cytokine production by monocytes, blocks T-lymphocyte activation by antigen presenting cells, and modulates T(H)1/T(H)2 balance in immune responses. In previous clinical trials, rhIL-10 administered to healthy volunteers induced rapid and transient elevations of neutrophil and monocyte counts and reductions of lymphocyte counts in addition to suppression of endotoxin-stimulated whole blood cytokine synthesis. We sought to better characterize the effects of rhIL-10 on immunophenotypically defined subsets of circulating leukocytes that could be relevant to its immunomodulatory effects. Healthy volunteers were given single doses of 10 microg/kg rhIL-10 (n = 8) or equivalent placebo (n = 4) by intravenous injection. Significant changes of circulating leukocytes included transiently increased neutrophils and monocytes with parallel increases of CD33+ and CD14+ cells. Total lymphocytes as well as total CD3+, CD3+/CD4+ and CD3+/CD8+ cells transiently decreased. Mean fluorescence intensity of CD11a (integrin alpha-chain subunit of lymphocyte function antigen-1, LFA-1) on lymphocytes transiently but significantly decreased, suggesting a mechanism for transient alteration of lymphocyte trafficking. In addition, mean fluorescence intensity of HLA-DR (major histocompatibility class II) on CD14+ cells (predominantly monocytes) transiently but significantly decreased, implying a possible alteration of antigen presenting function. Further study will be required to elucidate the immunomodulatory roles and potential clinical significance of these hematologic changes in therapeutic trials of rhIL-10 in patients with chronic inflammatory and autoimmune diseases.     

The immunomodulatory effects of anti-thyroid drugs are mediated via actions on thyroid cells, affecting thyrocyte-immunocyte signalling: a review

The mechanism of action of the immunosuppressive effects of antithyroid drugs has remained a matter of controversy, despite our earlier contention that such effects in vivo were indirect; ie., it was our view that the drugs were acting on the thyroid cells, reducing their thyroid hormone production and other activities, with a consequent reduction in thyrocyte-immunocyte signalling. The reduction in the activation of CD4+ cells,the increased number and activation of CD8+ (and CD8+CDllb+) cells, and the reduction of soluble interleukin-2 receptors, thought once to be direct effects of the medication, are now shown to be due to amelioration of the hyperthyroidism. Thus the reduction in thyroid hormone production induced by the drugs is central to these actions. In addition, the iodination of thyroglobulin is inhibited by these agents, which may affect antigen presentation by the thyrocyte. Furthermore, there is now evidence that the thionamides interfere with thyrocyte expression of such molecules as Class I antigen, interleukin-1, interleukin-6, prostaglandin E2, and heat shock protein. The expression of thyrocyte Class II antigen is probably not inhibited by these drugs, although one group has shown that lectin-stimulated thyrocyte Class II expression is diminished by this treatment; this group postulated that this effect might be mediated by reduced interferon gamma production by T lymphocytes, but in vitro experiments do not corroborate this proposal. In any event, the actions as described of the effects of antithyroid drugs on the thyroid cells (particularly normalization of thyroid function) would certainly suffice to explain the diminution of thyroid antibodies (including thyroid stimulating antibody), the reduced immunological response, and the increased remission rate in Graves disease as a consequence of antithyroid drug therapy, without the need to invoke a direct immunosuppressive effect.     

Quantitation of immunosuppression by tacrolimus using flow cytometric analysis of interleukin-2 and interferon-gamma inhibition in CD8(-) and CD8(+) peripheral blood T cells

The authors have determined the frequency of intracellular interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) synthesis by T-cell subsets in whole blood (WB) and isolated lymphocytes in 16 transplant recipients treated with tacrolimus and 10 control patients who were not transplant recipients. The authors also determined the impact of varying amounts of red blood cells (RBC) on immunosuppression by tacrolimus. Samples were analyzed by two-color flow cytometry, and the results were expressed as a ratio of whole blood to isolated lymphocytes. In healthy subjects who were not transplant recipients, the frequency of IL-2--producing CD8(-) and CD8(+) cells was higher in WB than in isolated lymphocytes (mean +/- SD of whole blood to lymphocytes ratio: 1.24 +/- 0.5 and 1.67 +/- 0.62, respectively). Adding varying amounts of RBC had no significant impact on IL-2 production by CD8(-) and CD8(+) T cells. Adding tacrolimus (10 ng/mL) to lymphocyte cultures inhibited (90%) IL-2 production in isolated T cells but not in the whole-blood assay. The dose of tacrolimus required for a 50% inhibition of IL-2 release in T cells was 10-fold higher in cultures with RBC than without. Peripheral blood mononuclear cells (PBMC) isolated from tacrolimus-treated whole blood (WB) showed less IL-2 inhibition than did lymphocytes in the WB. The authors also tested cytokine production in WB and PBMCs in 16 transplant recipients and observed various patterns of reactivity. The frequency of IL-2--producing CD8(-) and CD8(+) cells was similar using two different methods in 10 of 16 patients tested. By contrast, in the remaining six patients the authors observed a significant inhibition of IL-2 production in both CD8(-) and CD8(+) T-cell subsets in the whole-blood assay but not in the isolated lymphocytes. The frequency of CD8(-) IFN-gamma--producing cells was significantly lower in 9 of 16 patients, but the same individuals showed no inhibition of their CD8(+) IFN-gamma T cells. The trough levels of tacrolimus did not predict the level of cytokine inhibition in the whole-blood assay in these patients. The authors' results show that the whole-blood assay for cytokine production can be used for monitoring the in vivo effect of tacrolimus in transplant recipients.     

肌肉注射白介素-10质粒DNA预防小鼠自身免疫性糖尿病

目的:研究编码白介素-10(IL-10)质粒DNA对由多次、低剂量链脲佐菌素(STZ)诱发的自身免疫性糖尿病小鼠的作用。方法:连续5天将STZ(每次40 mg/kg)腹腔注射入小鼠体内,于第1、14天将100 mg表达人IL-10-pcDNA3质粒(IL-10处理组)或pcDNA3质粒(对照组)注射入骨骼肌内。测定小鼠血糖水平。第28天处死小鼠,检测小鼠血清干扰素-γ(IFN-γ)水平、胰腺IL 1β和TNF-α mRNA表达,测定脾CD4~+和CD8~+淋巴细胞的数量,同时进行胰腺组织学检查。结果:IL-10质粒DNA注射后,骨骼肌有IL-10 mRNA的持久表达,血浆IL-10水平明显升高,而且对迟发性超敏反应具抑制作用。IL-10处理组,在14、21和28天小鼠血糖显著降低,在21、28天时糖尿病发病率分别为33.3％和40.0％,显著低于对照组(P<0.05);胰腺IL-1β和TNF-αmRNA表达、血清IFN-γ水平,以及脾CD4~+和CD8~+淋巴细胞数量均低于对照组,胰岛炎症程度也明显轻于对照组(P<0.01)。结论:IL-10基因治疗能减轻实验性自身免疫性糖尿病小鼠 的胰岛炎症,降低糖尿病发生的机率。     

木瓜苷对环磷酰胺增强的小鼠接触性超敏反应的影响(英文)

目的　探讨胸腺在环磷酰胺 (Cy)增强的小鼠接触性超敏反应 (CHS)中的作用及木瓜苷 (GCS)对胸腺T淋巴细胞亚型的影响。方法　采用了 2 ,4 二硝基氟苯 (DNFB)诱导小鼠CHS模型及Cy诱导小鼠增强CHS模型 ,检测ConA诱导的小鼠脾脏T淋巴细胞增殖、胸腺T淋巴细胞亚型和ConA诱导的胸腺T淋巴细胞培养上清中TGF β1,IL 4和IL 2水平。结果　小鼠CHS模型中 ,ConA诱导的脾淋巴细胞增殖增强 ,CD4 +CD8+双阳性胸腺T淋巴细胞比例增加 ,胸腺细胞产生的Th1和Th3型细胞因子IL 2和TGF β1水平增高而Th2型细胞因子IL 4水平降低。DNFB初次致敏前 3d腹腔注射Cy (2 5 0mg·kg- 1)可以增强CHS反应。GCS(12 0和 2 4 0mg·kg- 1)连续灌胃 12d可以提升Cy增强的小鼠CHS胸腺T淋巴细胞中CD4 - CD8- 和CD4 +CD8- 细胞比例 ,降低CD4 +CD8+细胞比例 ;并提高胸腺淋巴细胞培养上清中IL 4水平 ,降低IL 2和TGF β1水平。结论　GCS对Cy增强的小鼠CHS有明显抑制作用 ;可有效调节小鼠胸腺CD4 /CD8和Th 淋巴细胞亚群及细胞因子产生平衡。     

Suppressor function of lymphocytes activated in in vitro co-culture against autologous tumor cells and expanded in interleukin-2.

The phenotypic and functional nature of the lymphocytes that have been activated against autologous tumor cells and expanded in Interleukin-2 (IL-2) was studied in the context of their suitability for use in adoptive immunotherapy for cancer. While long-term co-cultures between autologous lymphocytes and tumor cells in the presence of exogenous IL-2 occasionally induced CD8+ cytolytic T cells, a substantial majority of such co-cultures generated predominantly CD4+ non-cytolytic or poorly cytolytic effector cells. In addition, these CD4+ non-cytolytic effector populations, and a number of CD4+ T cell clones derived from them, behaved like functional T suppressor (Ts) cells by elaborating a factor(s) that had profound negative effect(s) on activation of fresh T cells (inhibition of IL-2 synthesis, inhibition of IL-2 receptor [IL-2]-alpha expression, and inhibition of proliferation). Accordingly, infusion of these non-cytolytic populations capable of exhibiting such regulatory properties may have a substantial negative effect on host response toward cancer.     

ACUTE EXPOSURE TO MERCURY FROM AMALGAM: NO SHORT-TIME EFFECT ON THE PERIPHERAL BLOOD LYMPHOCYTES IN HEALTHY INDIVIDUALS

Mercury, released from dental amalgam, has been considered to adversely affect the human immune system. This study has been performed in order to evaluate if an acute low-dose mercury exposure, achieved by total amalgam removal in 10 healthy individuals, would affect the immunocompetent cells in human blood when the mercury level in blood and plasma was increasing. Induction of lymphocyte proliferation, measured as spontaneous de novo DNA synthesis, and total T cells, CD4+ T cells, CD8+ T cells, and B cells, was studied prior to and 7, 31, and 48 h after amalgam removal. In addition, the levels of interleukin-6 (IL-6) and C-reactive protein (CRP) in serum/ plasma were measured. Despite a significant increase of the plasma mercury levels within 24 h after intervention, no significant influence on the peripheral blood lymphocytes could be detected during the first 48 h. The serum IL-6 levels increased significantly within 48 h after intervention, but were still low and within normal range. No influence on the CRP levels up to 7 d after amalgam removal was detected.     

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.     

Pharmacodynamics of FTY720, the first member of a new class of immune-modulating therapeutics in transplantation medicine

FTY is a novel immunomodulator currently undergoing clinical investigation and has the potential of improving immunosuppressive therapy after organ transplantation. Previous experimental studies in animals have shown that FTY has a unique mechanism of action. We have studied the pharmacodynamic effects of FTY in stable renal allograft recipients taking part in a phase I clinical trial. As in various animal models including non-human primates, a single oral dose of FTY (0.25 - 3.5 mg) significantly reduced peripheral lymphocyte count by 30 - 70%. The peripheral lymphocyte count returned to baseline within 24 hours. Only in those patients treated with the highest dose of FTY (3.5 mg), did peripheral lymphopenia persist for more than 96 hours. FTY reduced all lymphocyte subsets, T cells more than B cells and CD4+ cells more than CD8+ cells. The reduction in CD3+CD62L+ cell counts was more pronounced, whereas CD3+CCR5+ cell counts were less affected in comparison to the total number of CD3+ lymphocytes. We found only slightly increased apoptosis rates (< 5%) in peripheral lymphocytes, and this change does not explain the marked reduction in lymphocyte count. In cultured human lymphocytes only suprapharmacological doses of 10 microM FTY induced apoptosis (20.6 +/- 2.8%) after a 4-h incubation. More important, clinically relevant doses of 0.1 microM FTY increased lymphocyte mobility 2-fold. No effect of FTY on anti-CD3mAb-stimulated lymphocyte proliferation was detected and there was no change in phagocytosis rates in whole-blood cultures incubated with FTY. Further studies are necessary to investigate the mechanism of action of FTY in detail.