Treatment with atorvastatin alters the ratio of interleukin-12/interleukin-10 gene expression [corrected

BACKGROUND: Statins have been shown to have pleiotropic effects extending beyond their ability to lower cholesterol. MATERIAL AND METHODS: Seventeen patients with heterozygous familial hypercholesterolaemia participated in a single-blind placebo controlled study. The patients underwent three treatment regimens: placebo (4 weeks), atorvastatin 10 mg day(-1) (4 weeks) and atorvastatin 40 mg day(-1) (12 weeks). Following each treatment period, serum lipids and plasma mevalonic acid were measured, mononuclear leukocytes were isolated and total RNA was prepared. The content of mRNA for IL-12p35 and IL-10 was assayed, blinded, by real-time quantitative polymerase chain reactions. RESULTS: Treatment of the subjects with atorvastatin decreased the abundance of IL-12p35 mRNA in mononuclear cells, but did not alter that of IL-10, so that the ratio of the IL-12p35 to IL-10 mRNA content was significantly reduced (P < 0.0026). The IL-12p35/IL-10 ratio correlated significantly with plasma mevalonic acid concentrations but not with serum LDL concentrations. CONCLUSIONS: This study provides evidence that atorvastatin exerts an immunomodulatory effect in vivo, characterized by a decrease in the ratio of IL-12 mRNA to IL-10 mRNA in leukocytes. The immunomodulatory effect of statins, in addition to their cholesterol-lowering properties, may contribute to the rapid cardiovascular benefit observed during treatment with statins and reduced the rate of rejection in patients with solid organ transplantation.     

The expression of IL-2 receptor subunits on various leukemic cells]

Interleukin-2 receptors are composed of at least two polypeptide chains of alpha (55KD) and beta (75KD). The IL-2R beta chain is an essential component of the functional receptor for signal transduction of IL-2. We previously reported the distribution of IL-2R subunits among peripheral blood mononuclear cells. We here present some data regarding the expression of IL-2R subunits on various hemopoietic malignant cells. Fresh leukemic cells obtained from adult T cell leukemia patients expressed both alpha and beta chains, and leukemic cells derived from some patients with T cell leukemia, B cell leukemia or myeloid leukemia expressed the alpha and/or beta chain of IL-2R. The IL-2R beta chain on these leukemic cells were demonstrated to be functional for cell growth signaling. IL-2R alpha and beta chains should be tumor markers.     

Expression and release of IL-2 receptor and production of IL-2 by activated T lymphocyte subsets.

T lymphocyte subsets differ in expression of cell surface antigen and functional properties. Both CD4+ and CD8+ subsets express interleukin-2 receptor (IL-2R) following their activation in vitro. In the present investigation T lymphocyte subsets were activated by different mitogens and IL-2R expression was enumerated on these stimulated subsets. Peripheral blood mononucleated cells (PBMC) were stimulated with phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) and then stained with anti-CD4 or anti-CD8 antibodies conjugated with fluorescein isothiocyanate and anti-IL-2R monoclonal antibody conjugated with phycoerythrin using a direct immunofluorescence technique. The percentage of IL-2R positive lymphocytes was enumerated by flow cytometry. The results showed that mitogen activated lymphocytes expressed variable degrees of IL-2R which were significantly higher than the control. 53% of CD4+ lymphocytes and 28% of CD8+ expressed IL-2R following PHA stimulation in vitro. Similarly, 47% of CD4+ lymphocytes and 23% of CD8+ lymphocytes expressed IL-2R following PWM stimulation. The present study also revealed that the release of soluble IL-2R (sIL-2R) and IL-2 production in supernatant from cultured PBMC varied with different mitogen stimulation. Using the same concentration of PHA and PWM as used to study IL-2R expression, higher activity of sIL-2R was detected in PHA stimulated lymphocytes as compared to PWM treated lymphocytes. However, IL-2 production was more in culture medium from PMW treated PBMC. Thus, there was a significant correlation between the cellular and soluble IL-2R but the production of IL-2 from activated PBMC cells had no good correlation with either the cellular IL-2R expression or the release of sIL-2R.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interleukin 2 receptor gamma chain expression on resting and activated lymphoid cells

The interleukin 2 receptor (IL-2R) is known to be comprised of at least three genetically distinct subunits termed alpha, beta, and gamma. These chains can be expressed individually or in various combinations resulting in distinct receptors with different affinities for IL-2. In contrast to alpha and beta, the cell surface expression of the gamma chain protein previously has not been well-characterized. To examine cell surface expression of IL-2R gamma on hematopoietic cells, we developed two new monoclonal antibodies (mAbs) specific for this protein. Both 1A11 (immunoglobulin [IgG1]) and 3G11 (IgM) specifically reacted with murine cells transfected with IL-2R gamma cDNA, and immunoprecipitation studies indicated that both antibodies precipitated a protein of approximately 62-65 kD. Scatchard analysis of IL-2 binding to murine cells transfected with cDNA-encoding combinations of IL-2R components demonstrated that neither beta nor gamma chain bind IL-2 with measurable affinity, but coexpression of both beta and gamma is sufficient to form an intermediate affinity receptor. In the absence of gamma chain, beta chain interacts with alpha chain to form a "pseudo- high" affinity receptor. In contrast, gamma chain does not appear capable of interacting with alpha in the absence of beta chain. Thus, gamma chain appears to interact only with beta, but beta chain is capable of interacting with both alpha and gamma. Using the newly developed mAbs to examine cell surface expression by immunofluorescence, resting T cells were found to express low levels of gamma chain without detectable alpha or beta. Early after mitogen stimulation, T cells expressed higher levels of alpha, beta, and gamma. However, at later time points, T cells expressed alpha and gamma in marked excess over beta. Thus, formation of high affinity IL-2R on activated T cells was primarily limited by beta chain expression. In contrast, resting natural killer (NK) cells constitutively expressed IL- 2R beta without detectable alpha or gamma. After activation with either IL-2 or IL-12, expression of both alpha and gamma transiently increased and then returned to very low levels. Expression of functional IL-2R on resting and activated NK cells, therefore, appeared to be primarily limited by the expression of gamma chain. IL-2 binding studies with resting NK cells confirmed the results of immunofluorescence studies indicating the presence of very low numbers of intermediate affinity (beta gamma) receptors for IL-2 on these cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

丝裂原素在人单核细胞白细胞介素2受体的表达及杀伤机制中的作用

目的：研究丝裂原素在人单核细胞白细胞介素２（ＩＬ－２）受体的表达及杀伤机制中的作用。方法：采用多种丝裂原素处理人单核细胞，观察其对单核细胞ＩＬ－２受体α链表达和杀伤活性的影响。结果：美洲商陆（ＰＷＭ）和脂多糖（ＬＰＳ）在处理２４小时后诱导人单核细胞表达ＩＬ－２受体α链；植物血凝素（ＰＨＡ）只有在处理４８小时后才观察到有诱导α链表达作用；而刀豆素Ａ（ＣｏｎＡ）无明显诱导作用。在以上丝裂原素预处理后，加入ＩＬ－２，在有诱导ＩＬ－２受体α链表达的处理组，可见其单核细胞杀伤肿瘤细胞（Ｕ９３７）的活性明显提高。其中以ＰＷＭ预处理组的单核细胞活性增加最为显著。用流式细胞仪检测到ＰＷＭ可同时与单核细胞和Ｕ９３７靶细胞结合。经ＰＷＭ预处理后，加入ＩＬ－２，明显见到Ｕ９３７细胞发生凋亡的现象（包括核浓缩、凋亡小体的形成和ＤＮＡ的断裂）。结论：通过丝裂原素诱导人单核细胞ＩＬ－２受体的表达，可以促进ＩＬ－２激活的单核细胞的杀伤活性，包括诱导靶细胞的凋亡。     

Tolerance to endotoxin-induced expression of the interleukin-1 beta gene in blood neutrophils of humans with the sepsis syndrome.

The induction of genes of host cells stimulated by microbial products such as endotoxin and the tolerance of cells to endotoxin excitation play critical roles in the pathogenesis of microbial-induced acute disseminated inflammation with multiorgan failure (the sepsis syndrome). One gene that is induced in phagocytic cells by endotoxin and that appears to play an essential role in the pathogenesis of the sepsis syndrome is IL-1 beta. We report here that blood neutrophils (PMN) of patients with the sepsis syndrome (sepsis PMN) are consistently tolerant to endotoxin-induced expression of the IL-1 beta gene, as determined by decreased synthesis of the IL-1 beta protein and reductions in IL-1 beta mRNA. This down-regulation of the IL-1 beta gene in sepsis PMN occurs concomitant with an upregulation in the constitutive expression of the type 2 IL-1 receptor (IL-1R2). These phenotypic changes do not persist in PMN of patients recovering from the sepsis syndrome. Tolerance has stimulus and response specificity since sepsis PMN tolerant to endotoxin can respond normally to Staphylococcus aureus stimulation of IL-1 beta production and they normally secrete elastase. Uninfected patients with severe trauma or shock from causes are not tolerant to endotoxin and tolerance is not limited to patients infected with Gram-negative bacteria. The mechanism responsible for tolerance involves pretranslational events and is not due to loss of the CD14 surface protein, a receptor required for endotoxin induction of IL-1 beta in PMN. The physiological significance of the tolerance to endotoxin and increased expression of IL-1R2 on sepsis PMN is unknown, but may represent an attempt by the host to protect itself from the deleterious effects of disseminated inflammation.     

Glycoprotein VI agonists have distinct dependences on the lipid raft environment

BACKGROUND: It has been reported that the association of glycoprotein VI (GPVI) with lipid rafts regulates GPVI signaling in platelets. OBJECTIVE: Secreted adenosine 5'-diphosphate (ADP) potentiates GPVI-induced platelet aggregation at particular agonist concentrations. We have investigated whether the decrease in GPVI signaling, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. METHODS: We disrupted platelet lipid rafts with methyl-beta-cyclodextrin and measured signaling events downstream of GPVI activation. RESULTS: Lipid raft disruption decreases aggregation induced by low concentrations of convulxin, but this decrease is almost eliminated in the presence of ADP antagonists. Signaling indicators, such as protein phosphorylation and calcium mobilization, were not affected by raft disruption in collagen or convulxin stimulated platelets. Interestingly, however, raft disruption directly reduced GPVI signaling induced by collagen-related peptide. CONCLUSIONS: Lipid rafts do not directly contribute to signaling by the physiologic agonist collagen. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates GPVI signaling.     

T cell receptor ligation induces interleukin (IL) 2R beta chain expression in rat CD4,8 double positive thymocytes, initiating an IL-2- dependent differentiation pathway of CD8 alpha+/beta- T cells

The role of interleukin (IL)2 in intrathymic T cell development is highly controversial, and nothing is known about IL-2R expression on thymocytes of the T cell receptor (TCR) alpha/beta lineage undergoing TCR-driven differentiation events. We analyze here IL-2R alpha and beta mRNA expression in an in vitro system where newly generated rat CD4,8 double positive (DP) thymocytes respond to TCR ligation plus IL-2 (but not to either stimulus alone) with rapid differentiation to functional CD8 single positive T cells (Hunig, T., and R. Mitnacht. 1991. J. Exp. Med. 173:561). TCR ligation induced expression of IL-2R beta (but not alpha) chain mRNA in DP thymocytes. Addition of IL-2 then lead to functional maturation and expression of the IL-2R alpha chain. To investigate if the CD8 T cells generated via this IL-2R beta-driven pathway in vitro correspond to the bulk of CD8 T cells seeding peripheral lymphoid organs in vivo, we compared their phenotype to that of lymph node CD8 T cells. Surprisingly, analysis of CD8 cell surface expression using a novel anti-CD8 monoclonal antibody specific for the alpha/beta heterodimeric isoform, and of CD8 alpha and beta chain mRNA revealed that T cells generated by TCR ligation plus IL-2 resemble thymus-independent rather than thymus-derived CD8 cells in that they express CD8 alpha without beta chains. These findings demonstrate that TCR crosslinking induces functional IL-2R on immature DP rat thymocytes. In addition, they show that at least in vitro, CD8 alpha/alpha T cells are generated from TCR-stimulated DP thymocytes (which express the CD8 alpha/beta in the heterodimeric isoform) along an IL-2-driven pathway of T cell differentiation.     

A balance between positive and negative signals in cytotoxic lymphocytes regulates the polarization of lipid rafts during the development of cell-mediated killing

Plasma membrane microdomains containing sphingolipids and cholesterol (lipid rafts) are enriched in signaling molecules. The cross-linking of certain types of cell surface receptors initiates the redistribution of these lipid rafts, resulting in the formation of signaling complexes. However, little is known about the regulation of the initial raft redistribution and whether negative regulatory signaling pathways target this phase of cellular activation. We used natural killer (NK) cells as a model to investigate the regulation of raft redistribution, as both positive and negative signals have been implicated in the development of their cellular function. Here we show that after NK cells form conjugates with sensitive tumor cells, rafts become polarized to the site of target recognition. This redistribution of lipid rafts requires the activation of both Src and Syk family protein tyrosine kinases. In contrast, engagement of major histocompatibility complex (MHC)-recognizing killer cell inhibitory receptors (KIRs) on NK cells by resistant, MHC-bearing tumor targets blocks raft redistribution. This inhibition is dependent on the catalytic activity of KIR-associated SHP-1, a Src homology 2 (SH2) domain containing tyrosine phosphatase. These results suggest that the influence of integrated positive and negative signals on raft redistribution critically influences the development of cell-mediated cytotoxicity.     

Differential regulation of lymphokine production by distinct subunits of the T cell interleukin 2 receptor.

Most biologic responses to IL-2 have been attributed to interaction of IL-2 with a high affinity receptor which consists of a heterodimer composed of two distinct IL-2-binding proteins (IL-2R alpha/IL-2R beta). However, both low affinity IL-2R alpha (55 kD) and intermediate affinity IL-2R beta (70-75 kD) also appear to be expressed independently on the cell surface. We investigated the receptor-specific regulatory effects of IL-2 on cytokine production in unstimulated and activated T cells. T cells were activated by stimulation of the antigen receptor complex with anti-CD3 mAb. IL-2 (10(2) U/ml, 1 nM) stimulation of resting cells resulted in a fivefold increase in GM-CSF release but in only minimal IFN-gamma release. IL-2 markedly augmented mRNA expression of GM-CSF but not IFN-gamma in unstimulated T cells. IL-2R beta mAb but not IL-2R alpha mAb decreased IL-2-induced GM-CSF release and mRNA expression from unstimulated T cells. IL-2 concentrations required for GM-CSF release from resting cells suggested ligand binding to an intermediate affinity receptor. GM-CSF and IFN-gamma release from activated T cells increased four- to fivefold in response to 1 nM IL-2 and IL-2 augmented both GM-CSF and IFN-gamma mRNA. IL-2R beta mAb but not IL-2R alpha mAb reduced GM-CSF release and mRNA expression in activated T cells stimulated with 1 nM IL-2. IL-2R alpha blockade markedly decreased IL-2-induced IFN-gamma release and mRNA expression from activated cells, while IL-2R beta blockade had little effect on IFN-gamma production in activated cells. IL-2R alpha blockade altered the affinity of the receptor mediating activated cell GM-CSF release from a high affinity to an intermediate affinity state. These studies indicate an independent role for IL-2R beta in mediating GM-CSF production from T cells. They also suggest that unstimulated and activated T cells, which express distinct IL-2 receptor moieties, mediate release of separate lymphokines and that different subunits of the IL-2 receptor may play an important role in the regulation of cytokine production.     

Functional interleukin-2 receptors on intestinal epithelial cells.

The presence of receptors for the cytokine IL-2 was assessed in the IEC-6 cell line established from normal rat crypt epithelium and primary intestinal epithelial cells. 125I-IL-2 was found to specifically bind to subconfluent IEC-6 cells. Maximal binding was observed within 30 min after addition of the ligand; binding could be inhibited by excess unlabeled IL-2 or addition of antibody to the IL-2 receptor. Both intermediate and low affinity receptors with approximate Kd of 10 and 100 pM, respectively were present. Kinetic analysis were consistent with the results of Western blot analysis using an antisera to the 75-kD IL-2 receptor beta chain. IL-2 receptors appeared to be functional; addition of IL-2 led to modulation of proliferation with initial stimulation at 24 h followed by inhibition at 48 h. This effect could be blocked by addition of antibody to the IL-2 receptor beta chain. IL-2 treatment could be shown to enhance expression (range = 4- to 50-fold stimulation) of TGF-beta, as well as the lectin protein mac-2, in IEC-6 cells. The relevance of observations in the IEC-6 cell line to intestinal mucosa in vivo was supported by the demonstration of a gradient of expression of the IL-2 receptor in primary rat intestinal epithelial cells by Western blot analysis. In addition, mRNA for the IL-2 receptor-beta chain was demonstrated by Northern blot analysis using mRNA from primary rat intestinal epithelial cells depleted of detectable contaminating intraepithelial lymphocytes by two cycles of fractionation on Percoll gradients. Collectively, these observations suggest that the range of cellular targets of the putative lymphokine IL-2 is broader than appreciated, and IL-2 may serve to integrate epithelial and lymphocyte responses in the intestinal mucosa.     

Cytotoxic T lymphocyte antigen 4 and CD28 modulate cell surface raft expression in their regulation of T cell function.

Coreceptors CD28 and cytotoxic T lymphocyte antigen (CTLA)-4 have opposing effects on TcR/CD3 activation of T cells. While CD28 enhances and CTLA-4 inhibits activation, the underlying molecular basis of these effects has yet to be established. In this context, ganglioside and cholesterol enriched membrane microdomains (rafts, GEMs) serve as centers of signaling in T cells. Although CD28 can promote TcR/raft colocalization, evidence is lacking on whether the surface expression of membrane rafts can be targeted by CTLA-4 in its modulation of T cell responses. In this study, we demonstrate that both CD28 and CTLA-4 profoundly alter the surface expression of membrane rafts during T cell activation. While CD28 increased expression and the number of peripheral T cells induced to express surface rafts in response to TcR ligation, CTLA-4 potently inhibited both TcR and TcR x CD28 induced raft expression on the surface of T cells. Consistent with this, CD28 increased the presence of the linker of activated T cells (LAT) in purified membrane rafts, while CTLA-4 coligation effectively blocked this increase. Further, the reversal of the CTLA-4 block with CD3/CD28 ligation was accompanied by an increase in surface raft expression and associated LAT. Our observations demonstrate for the first time that CTLA-4 targets the release of rafts to the surface of T cells, and provides a mechanism for the opposing effects of CD28 and CTLA-4 on costimulation.     

Cholesterol deficiency in a mouse model of Smith-Lemli-Opitz syndrome reveals increased mast cell responsiveness

Mutation of the 3beta-hydroxysterol delta7-reductase gene (Dhcr7-/-) results in Smith-Lemli-Opitz syndrome (SLOS). Patients, and genetically altered mice, are unable to produce cholesterol and accumulate 7-dehydrocholesterol (DHC) in serum and tissue. This causes multiple growth and developmental abnormalities as well as immune system anomalies including allergy. Because cholesterol is a key component of liquid-ordered membranes (lipid rafts) and these domains have been implicated in regulating mast cell activation, we examined whether mast cell responsiveness is altered in this model. Mast cells derived from Dhcr7-/- mice (DHCR KO) showed constitutive cytokine production and hyper-degranulation after stimulation of the high affinity IgE receptor (Fc epsilonRI). DHCR KO mast cells, but not wild-type mast cells, accumulated DHC in lipid rafts. DHC partially disrupted lipid raft stability and displaced Lyn kinase protein and activity from lipid rafts. This led to down-regulation of some Lyn-dependent signaling events but increased Fyn kinase activity and Akt phosphorylation. The Lyn-dependent phosphorylation of Csk-binding protein, which negatively regulates Fyn activity, was decreased. This phenotype reproduces some of the characteristics of Lyn-null mast cells, which also demonstrate hyper-degranulation. These findings provide the first evidence of lipid raft dysfunction in SLOS and may explain the observed association of allergy with SLOS.     

Pseudo-high affinity interleukin 2 (IL-2) receptor lacks the third component that is essential for functional IL-2 binding and signaling

Functional studies of the interleukin 2 receptor (IL-2R) of two (ED515-D and Kit225) IL-2-dependent and three (ED515-I, 3T3-alpha beta 11, and Hut102) IL-2-independent cell lines were done. All of these cell lines appeared to express high as well as low affinity IL-2R. However, ED515-I and 3T3-alpha beta 11, which expressed the IL-2R beta chain, did not bind IL-2 at all when IL-2 binding to their IL-2R alpha chain was blocked with anti-Tac monoclonal antibody, whereas the intermediate affinity binding in ED515-D, Kit225, and Hut102 cells remained. We tentatively called the high affinity IL-2R of the former cells pseudo-high affinity IL-2R. The dissociation constant of pseudo-high affinity IL-2R was higher than that of ordinary high affinity IL-2R. Internalization of cell-bound 125I-IL-2 into ED515-I and 3T3-alpha beta 11 cells was less efficient than that into ED515-D cells. The addition of IL-2 neither promoted cell growth nor upregulated IL-2R alpha chain expression in ED515-I and 3T3-alpha beta 11 cells. Furthermore, tyrosine phosphorylation of the cellular proteins (p120, p98, p96, p54, and p38) was induced or enhanced in response to the addition of IL-2 in ED515-D and Kit225 cells, but not in the cell lines expressing pseudo-high affinity IL-2R. Finally, 125I-IL-2 crosslinking followed by SDS-PAGE analysis showed an 80-kD band corresponding to p65 + IL-2, in addition to bands corresponding to IL-2R alpha and beta chain + IL-2 in cells bearing ordinary high affinity IL-2R but not in cells with pseudo-high affinity IL-2R. Taken together, we consider that another protein whose molecular mass is approximately 65 kD is functionally important in IL-2 binding and subsequent signal transduction and may be the third component of IL-2R.     

Cardioprotection of interleukin-2 is mediated via kappa-opioid receptors

We examined whether interleukin-2 (IL-2) protects the myocardium against injury induced by ischemia and reperfusion via the kappa-opioid receptor (OR). The cardioprotective effect of IL-2 was evaluated by measuring infarct size and lactate dehydrogenase (LDH) release in response to ischemia and reperfusion in the isolated rat heart. IL-2 at an optimal dose of 50 U/ml mimicked the effect of ischemic preconditioning by reducing infarct size and LDH release. The infarct and LDH-reducing effects of IL-2 were blocked by nor-binaltorphimine (5 microM), a kappa-OR antagonist, but not naltrindole (5 microM), a delta-OR antagonist known to block the action of its stimulation. Moreover, blockade of the mitochondrial ATP-sensitive potassium (mito-K(ATP)) channel with a selective antagonist, 5-hydroxydecanoate (100 microM), or a nonselective antagonist of K(ATP) channels, glybenclamide (100 microM), or blockade of protein kinase C (PKC) with its inhibitors chelerythrine (5 microM) or GF 109203X (10 microM) [3-[1-[3-(dimethylaminopropyl]-1H-indol-3-yl]-4-(1H-indol-3-yl)-1H-pyrrole-2,5-dione monohydrochloride] abolished the protective effect of IL-2. Administration of free radical scavengers N-acetylcysteine (4 mM) or N-(2-mercaptopropionyl)-glycine (1 mM) also abolished the protective effects of IL-2 and U50,488H [(trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)-cyclohexyl]benzeneacetamide], a selective kappa-OR agonist. This study provides the first evidence that IL-2 confers cardioprotection against injury induced by ischemia/reperfusion. The effect of IL-2 is mediated via kappa-OR as evidenced by kappa-OR antagonism and similar signaling mechanisms, mito-K(ATP), PKC, and reactive oxygen species involved in the cardioprotective effects of both IL-2 and kappa-OR stimulation.     

Elevated interleukin-2 production by cord blood mononuclear cells from premature newborns

This study was undertaken to delineate the elements of the interleukin-2 (IL-2) system in premature newborns by quantitating IL-2 production and IL-2 receptor (IL-2R) expression and density in cord blood mononuclear cells (CBMC) from 19 premature (estimated gestational age = 27-36 weeks, mean = 33.7), but otherwise healthy, infants and 25 term newborns. Phytohemagglutinin (PHA)-induced IL-2 production by premature CBMC was significantly greater (p less than 0.05) than that produced by term newborn CBMC with a mean value of 52.0 mu/ml for prematures versus 18.2 mu/ml for terms. The mean percentage of CBMC expressing PHA-induced cell-surface IL-2R was 23.4 and 23.0% while the IL-2R density per cell was 4.72 x 10(4) and 6.93 x 10(4) for premature and term newborns, respectively. PHA-induced proliferation was similar in both groups. These results show a significantly increased PHA-induced IL-2 production, but similar IL-2R expression, in CBMC from premature as compared to term newborns, demonstrating that the preterm newborn possesses a competent IL-2 system at birth.     

Formation and aggregation of lipid rafts in γδ T cells following stimulation with Mycobacterium tuberculosis antigens

Lipid rafts are plasma membrane microdomains that are implicated in diverse signaling pathways in immune cells. Based on the distinct types of T-cell receptors, two T-cell subpopulations have been identified: αβ and γδ T cells. In humans, γδ T cells represent a relatively rare T lymphocyte population but play a critical role in the immune response to infection by Mycobacterium tuberculosis. It has been demonstrated that Mycobacterium tuberculosis antigens (Mtb-Ag) preferentially activate γδ T cells. Thus, we investigated whether lipid rafts are involved in the Mtb-Ag-mediated activation of γδ T cells. Human peripheral blood mononuclear cells (PBMCs) were stimulated with Mtb-Ag, and expression of a lipid raft marker ganglioside GM1 (GM1) was determined by flow cytometry. The aggregation of lipid rafts was evaluated by laser confocal microscopy. Non-stimulated fresh PBMCs minimally expressed GM1 (6.55 ± 2.01%) and had no aggregated rafts in γδ T cells. Mtb-Ag stimulation gradually increased the expression of GM1 in a time-dependent manner. At 72 h, the majority of γδ T cells expressed GM1 (88.69 ± 7.55%). Furthermore, accompanied with the increased expression of GM1, aggregation of lipid rafts became gradually visible in γδ T cells. The aggregated rafts, however, were not evenly distributed and only occurred over a small portion of GM1-positive cells. Pretreatment with methyl-β-cyclodextrin, a cholesterol-depleting reagent, completely inhibited the Mtb-Ag-mediated aggregation of lipid rafts. These results demonstrate that lipid raft aggregation occurs in Mtb-Ag-activated γδ T cells, suggesting that lipid rafts are involved in activation of γδ T cells.     

Differential effects of interleukin-15 and interleukin-2 on differentiation of bipotential T/natural killer progenitor cells

Bipotential T/natural killer (NK) progenitor cells are destined to differentiate mainly into T cell receptor (TCR) alpha beta and TCR gamma delta cells in a thymic microenvironment, whereas extrathymically they selectively develop into NK cells. The exact environmental conditions that are required for differentiation into these three leukocyte populations are largely unknown. In this report, we have investigated and compared the effect of interleukin (IL)-15 and IL-2 in this process. The IL-15 receptor is composed of the gamma and beta chains of the IL-2 receptor (IL-2R gamma and IL-2R beta) and of a specific alpha chain (IL-15R alpha). Here, it is shown that IL-15 mRNA is mainly expressed in thymic epithelial stromal cells, whereas IL-2 mRNA is exclusively expressed in thymocytes. IL-2R beta-expressing cells were present in the fetal thymus with a CD25-CD44+Fc gamma R+HSA- /low TCR- phenotype, which is characteristic of progenitor cells. These cells also expressed IL-15R alpha messenger RNA. Sorted IL-2R beta + TCR- cells differentiated into TCR alpha beta and TCR gamma delta cells after transfer to alymphoid thymic lobes, whereas culture of the same sorted cells in cell suspension in the presence of IL-15 resulted in the generation of functional NK cells. This shows that IL-2R beta +TCR- cells of the fetal thymus contain bipotential T/NK progenitors. Addition of low concentrations of IL-15 to fetal thymic organ culture (FTOC) resulted in an increase of all T cell subpopulations. The largest expansion occurred in the TCR gamma delta compartment. In contrast, low concentrations of IL-2 did not result in a higher total cell number and did not induce outgrowth of TCR gamma delta cells. High concentrations of IL-15 blocked TCR alpha beta development and shifted differentiation towards NK cells. Differentiation towards TCR gamma delta cells still proceeded. High concentrations of IL-2 similarly induced development into NK cells, but the cell number was fourfold lower than in IL-15 cultures. Importantly, blocking of IL-2R alpha in IL-2-treated FTOC resulted in a drastic increase in cell number, indicating that IL-2R alpha negatively regulates cell expansion. Collectively, these experiments provide direct evidence that IL-15 and IL-2 differentially affect the differentiation of bipotential T/NK progenitors.