Inhibición de la actividad citotóxica no restringida por MHC de células NK y de clones T CD8+alfa-beta por la agregación de moléculas MHC de clase I / Inhibition of non MHC-restricted cytotoxicity of human NK cells and a CD8+alfa-beta T cell clone by MHC class I cross-linking

La lista de ligandos del sistema inmune que se convierten en receptores es cada día más amplia. Nuestros datos experimentales muestran que la agregación de moléculas MHC de clase I humanas por anticuerpos monoclonales inhibe la lisis no específica de células Natural Killer (NK) y de linfocitos T citotóxicos. Estos resultados sugieren que las moléculas MHC de clase I humanas expresadas por células efectoras T citotóxicas (CTL) o células NK pueden transmitir señales negativas tras su interacción con ligandos expresados por las células que necesitan protegerse de la citotoxicidad no específica. En consecuencia, las moléculas MHC I no son únicamente ligandos del receptor de antígeno T o de receptores NK no reordenados, sino que también son moléculas transmisoras de señales por sí mismas probablemente tras co-localización en grupos de activación supramolecular o asociación con receptores con capacidad de transducción de señales. Además, nuestros resultados experimentales pueden tener consecuencias en el estudio de las señales inhibitorias que modulan la respuesta citotóxica de linfocitos T y células NK en donde está generalizado el uso de anticuerpos anti-MHC I. En resumen, nuestros datos sugieren una nueva función de las moléculas MHC de clase I humanas relevante en el estudio de las señales reguladoras que controlan la actividad de las células efectoras NK y citotóxicas T (AU)

Interleukin-10, interleukin-6 and interferon-gamma gene polymorphisms in melanoma patients.

The immune response against melanoma can be influenced by cytokines with potentially opposite effects on tumour cell growth, such as interleukin-10 (IL10), interleukin-6 (IL6) and interferon-gamma (IFNgamma). Our objective in this study was to investigate whether polymorphisms in the regulatory regions of IL10, IL6 and IFNgamma genes are associated with the development of primary cutaneous melanoma and/or the prognosis of this tumour. We studied genotypic variations at positions -1082, -819 and -592 in the IL10 promoter, -174 in the IL6 promoter and +874 in the IFNgamma intron 1 in 42 melanoma patients and 48 healthy controls. These two populations showed very similar genotypic frequencies for IL10, IL6 and IFNgamma gene polymorphisms. There was a significant increase in the prevalence of IL10 low expression genotypes, specially the ACC/ATA genotype, among patients with a poorer prognosis. In contrast, IL6 promoter and IFNgamma intron 1 gene polymorphisms did not correlate with melanoma prognosis. These data indicate that investigation of polymorphisms in the regulatory regions of IL10, IL6 and INFgamma genes does not seem to be useful for predicting the risk of development of primary cutaneous melanoma. However, IL10 low expression genotypes may be associated with a poorer outcome in melanoma patients.

Implication of reactive oxygen species in the antibacterial activity against Salmonella typhimurium of hepatocyte cell lines.

We recently described the antibacterial activity of a murine hepatocyte cell line stimulated with interferon-gamma (IFN-gamma), interleukin-1 (IL-1), and lipopolysaccharide (LPS) against intracellular Salmonella organisms. Here we show for the first time the existence of basal antibacterial activity in cultured hepatocyte cell lines. Thus treatment of resting and stimulated hepatocytes with catalase or superoxide dismutase increased bacterial number recovered per monolayer, which suggests that the mechanism involved with antibacterial activity of hepatocytes is mediated by reactive oxygen species (ROS). Also, the capacity of these cell lines to generate intracellular peroxides under resting and stimulated conditions was investigated. This revealed that IL-1 and LPS did not induce any increase in the amount of intracellular peroxides by themselves, but they primed IFN-gamma for maximal induction of peroxides. The intracellular amount of peroxides was highly increased on stimulation with IFN-gamma, IL-1, and LPS, and it was strongly inhibited by catalase. This explains that the mechanism whereby this enzyme inhibits antibacterial activity takes place by decreasing the intracellular pool of peroxides. In turn, experiments performed in the presence of several inhibitors of metabolic pathways involved in ROS generation suggested that cyclo-oxygenase are a source of these species in hepatocyte cell lines. These results attribute a prominent role to the generation of peroxides as effector molecules of antibacterial activity in hepatocyte cell lines. Thus these cells displayed a moderate basal level, which increased on stimulation with proinflammatory cytokines such as IFN-gamma, IL-1, and bacterial products such as LPS. Finally, it has been also shown for the first time that IFN-gamma stimulation induces production of peroxides in human and murine hepatocyte cell lines.

Specific regulation of VLA-4 and alpha 4 beta 7 integrin expression on human activated T lymphocytes.

Modulation of VLA integrins was studied in several human T cell clones upon specific and nonspecific cellular activation. Human activated T lymphocytes down-regulated both alpha 4 beta 1 and alpha 4 beta 7 integrins upon specific recognition of alloantigens (cytotoxic T cells) or in the presence of Staphylococcus enterotoxin B (superantigen recognizing noncytotoxic T cells). In contrast, the expression of other membrane integrins, such as VLA-1 and VLA-5 integrins, was not modified. Down-regulation of alpha 4 beta 1 and alpha 4 beta 7 integrins was observed as early as 3 h after stimulation, lasted later than 72 h and was partially inhibited by cytochalasin D. Interestingly, neither target cells nor NK cells modulated CD49d expression after interaction with T cells of K562, respectively, suggesting that CD49d expression was linked to specific T cell activation. The down-regulation of the CD49d chain in T cell clones stimulated with immobilized anti-CD3 mAbs confirmed the role of TCR-mediated activation in CD49d regulation. However, the CD3-independent cellular aggregation induced by soluble anti-CD43 mAb was also able to strongly down-regulate alpha 4 beta 1 and alpha 4 beta 7. The present work shows the first evidence that CD49d subunit-bearing integrin expression is distinctly regulated from other integrins after Ag or superantigen recognition by human activated T cells. CD49d modulation may be relevant for the traffic and tissue localization of locally activated T cells during immune responses.

Interleukin-7 rescues human activated T lymphocytes from apoptosis induced by glucocorticoesteroids and regulates bcl-2 and CD25 expression.

We studied the ability of several interleukins to inhibit the cellular death of IL-2-dependent human T cells deprived of IL-2 testing viability, DNA integrity, and expression of bcl-2 gene product. Our in vitro results showed that the addition of IL-7, and in a far less efficient manner IL-4, augmented the viability of IL-2-dependent T-cell clones of different origin, specificity, and phenotype. Furthermore, IL-7 reduced the percentage of apoptotic T cells inhibiting DNA fragmentation. In addition, IL-7 but not IL-4 was consistently able to suppress the cell death of IL-2-dependent T cells triggered by DEX, a synthetic GC. The suppression of T-cell death triggered by IL-7 was not affected by the addition of anti-IL-2 antibody. Interestingly, IL-7 inhibited the downregulation of bcl-2 gene product expression that appeared on TCCs after IL-2 withdrawal and also shared with IL-2 the ability to induce the upregulation of CD25 antigen on activated T lymphocytes in the presence of DEX. These experiments establish a novel role for IL-7 in regulating viability and GC-induced apoptosis on activated human T cells and suggest that the maintenance of bcl-2 levels is a general mechanism by which interleukins preserve activated T cells from undergoing apoptosis.

ICAM-3 interacts with LFA-1 and regulates the LFA-1/ICAM-1 cell adhesion pathway.

The interaction of lymphocyte function-associated antigen-1 (LFA-1) with its ligands mediates multiple cell adhesion processes of capital importance during immune responses. We have obtained three anti-ICAM-3 mAbs which recognize two different epitopes (A and B) on the intercellular adhesion molecule-3 (ICAM-3) as demonstrated by sequential immunoprecipitation and cross-competitive mAb-binding experiments. Immunoaffinity purified ICAM-3-coated surfaces were able to support T lymphoblast attachment upon cell stimulation with both phorbol esters and cross-linked CD3, as well as by mAb engagement of the LFA-1 molecule with the activating anti-LFA-1 NKI-L16 mAb. T cell adhesion to purified ICAM-3 was completely inhibited by cell pretreatment with mAbs to the LFA-1 alpha (CD11a) or the LFA-beta (CD18) integrin chains. Anti-ICAM-3 mAbs specific for epitope A, but not those specific for epitope B, were able to trigger T lymphoblast homotypic aggregation. ICAM-3-mediated cell aggregation was dependent on the LFA-1/ICAM-1 pathway as demonstrated by blocking experiments with mAbs specific for the LFA-1 and ICAM-1 molecules. Furthermore, immunofluorescence studies on ICAM-3-induced cell aggregates revealed that both LFA-1 and ICAM-1 were mainly located at intercellular boundaries. ICAM-3 was located at cellular uropods, which in small aggregates appeared to be implicated in cell-cell contacts, whereas in large aggregates it appeared to be excluded from cell-cell contact areas. Experiments of T cell adhesion to a chimeric ICAM-1-Fc molecule revealed that the proaggregatory anti-ICAM-3 HP2/19 mAb was able to increase T lymphoblast attachment to ICAM-1, suggesting that T cell aggregation induced by this mAb could be mediated by increasing the avidity of LFA-1 for ICAM-1. Moreover, the HP2/19 mAb was costimulatory with anti-CD3 mAb for T lymphocyte proliferation, indicating that enhancement of T cell activation could be involved in ICAM-3-mediated adhesive phenomena. Altogether, our results indicate that ICAM-3 has a regulatory role on the LFA-1/ICAM-1 pathway of intercellular adhesion.

ICAM-3, the third LFA-1 counterreceptor, is a co-stimulatory molecule for both resting and activated T lymphocytes.

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a beta 2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some gamma delta T cell clones bearing the V delta 1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dendritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.

Immune functions of tumor necrosis factor. I. Tumor necrosis factor induces apoptosis of mouse thymocytes and can also stimulate or inhibit IL-6-induced proliferation depending on the concentration of mitogenic costimulation.

Murine rTNF produces at least three effects on mouse thymocytes in vitro: 1) Is a modest co-stimulator of proliferation with low PHA-P doses. 2) Has a bi-directional interaction with rIL-6-depending on PHA concentration: at low PHA (5 to 10 micrograms/ml) TNF augments and at high PHA (20 to 30 micrograms/ml) inhibits IL-6-induced proliferation. A comparable bidirectional PHA dose-dependent TNF interaction was seen with IL-1 beta, whereas only inhibition at high PHA with IL-2 and only augmentation at low PHA with IL-4 were seen. 3) TNF induces direct thymocyte apoptosis (a property not shared by IL-1 beta, IL-2, IL-4, IL-6 and IL-7). Of the cytokines studied, only IL-7 reduced TNF apoptosis. Thymocyte apoptosis by TNF showed the same species specificity as costimulation (i.e., human TNF had no effect) and was not inhibited by CY. The thymocyte CD4-CD8 phenotype after 72-h cultures showed that TNF decreased mainly double negative (DN) and single positive (SP) subsets, whereas IL-6 with low or high PHA increased DN and SP, especially the SP CD8+ subset. The regulatory and apoptotic effects of TNF were seen only with thymocytes and not with peripheral splenic or lymph node T cells. Four mAb to mouse TNF (2E2, XT22, 1C6, and 1OD9) could abrogate TNF costimulation and the TNF effects on IL-6-induced thymocyte proliferation, at both augmenting and inhibitory PHA conditions. However, only the two antibodies that also neutralize TNF lytic activity (2E2, XT22) could inhibit TNF-mediated apoptosis, implying two different but neighboring functional domains in the TNF molecule mediating apoptosis/lysis and costimulation. Our studies show that TNF might have unique and complex regulatory effects on growth and death of thymocyte populations in adult mice quite different from its effects on T cells in periphery.

Influence of liposome charge and composition on their interaction with human blood serum proteins.

Lipid composition and specially their electrostatic properties, were found to greatly influence the stability of liposomes in human blood serum. The amount and type of serum proteins bound to the liposomes were also clearly influenced by lipid composition and charge of liposomes. A good correlation was found between the amount of serum proteins adsorbed to a given type of liposome and its instability as measured by the release of an encapsulated fluorescent probe. Liposomes that bind the highest amount of protein were the least stable, except for the case of liposomes containing gangliosides, which were fairly stable even at a high amount of bound protein. Liposomes with neutral charge containing phosphatidylcholine were the most stable and bound the lowest amount of protein. Liposomes with positive charge behaved similarly to those with neutral charge. However, the stability of negatively charged liposomes was very dependent on their composition. Those liposomes containing only one class of negatively charged phospholipids bound a great amount of protein and were very unstable. However, those liposomes containing also phosphatidylcholine bound less protein and were more stable. The examination of the electrophoresis patterns of serum proteins bound to the different types of liposomes indicated the presence of specific proteins which correlated with liposome instability.

Stability of liposomes on long term storage.

The effect of the lipid composition of liposomes on their storage for up to one year under different environmental conditions has been examined using 5,6-carboxyfluorescein as a model drug. When cholesterol and/or alpha-tocopherol were included in the liposomes, a significantly greater amount of dye was retained. The presence of alpha-tocopherol decreased the breakdown of phosphatidylcholine to lysophosphatidylcholine and also reduced the level of peroxidation. Carboxyfluorescein retention was further enhanced when liposomes were stored at 4 degrees C or at room temperature (20 degrees C) in an O2-free atmosphere. Lysophosphatidylcholine formation also slowed when the liposomes were kept at 4 degrees C, or in an O2-free atmosphere. It is concluded that egg yolk lecithin liposomes may be stored for long periods at low temperature in an O2-free atmosphere or with added stabilizers such as cholesterol and alpha-tocopherol.

Treatment of Brucella melitensis infection in mice by use of liposome-encapsulated gentamicin.

Liposomes with entrapped gentamicin were used to treat mice with infection attributable to Brucella melitensis. Liposomes bearing positive charge and formed by egg yolk lecithin, cholesterol, and stearylamine were effective in the elimination of B melitensis residing in liver and spleen. Negatively charged liposomes, formed by egg yolk lecithin, cholesterol, and dicetyl phosphate were also effective in suppression of the infection in liver, but were less so in suppression of the infection in the spleen. Free gentamicin was less effective than the encapsulated antibiotic. At 20 hours after administration of gentamicin encapsulated in liposomes, the gentamicin concentrations in liver and spleen were similar, regardless of the charge of the liposomes--neutral, positive, or negative. However, positively charged liposomes were more efficient than were other liposome types for the treatment of brucellosis caused by B melitensis.