Regulatory role of PI3K-protein kinase B on the release of interleukin-1ß in peritoneal macrophages from the ascites of cirrhotic patients.

Great effort has been paid to identify novel targets for pharmaceutical intervention to control inflammation associated with different diseases. We have studied the effect of signalling inhibitors in the secretion of the proinflammatory and profibrogenic cytokine interleukin (IL)-1ß in monocyte-derived macrophages (M-DM) obtained from the ascites of cirrhotic patients and compared with those obtained from the blood of healthy donors. Peritoneal M-DM were isolated from non-infected ascites of cirrhotic patients and stimulated in vitro with lipopolysaccharide (LPS) and heat-killed Candida albicans in the presence or absence of inhibitors for c-Jun N-terminal kinase (JNK), mitogen-activated protein kinase kinase 1 (MEK1), p38 mitogen-activated protein kinase (MAPK) and phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K). The IL1B and CASP1 gene expression were evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The expression of IL-1ß and caspase-1 were determined by Western blot. IL-1ß was also assayed by enzyme-linked immunosorbent assay (ELISA) in cell culture supernatants. Results revealed that MEK1 and JNK inhibition significantly reduced the basal and stimulated IL-1ß secretion, while the p38 MAPK inhibitor had no effect on IL-1ß levels. On the contrary, inhibition of PI3K increased the secretion of IL-1ß from stimulated M-DM. The activating effect of PI3K inhibitor on IL-1ß release was mediated mainly by the enhancement of the intracellular IL-1ß and caspase-1 content release to the extracellular medium and not by increasing the corresponding mRNA and protein expression levels. These data point towards the role of MEK1 and JNK inhibitors, in contrast to the PI3K-protein kinase B inhibitors, as potential therapeutic tools for pharmaceutical intervention to diminish hepatic damage by reducing the inflammatory response mediated by IL-1ß associated with liver failure.

Prostaglandins from human T suppressor/cytotoxic cells modulate natural killer antibacterial activity.

We have recently described potent antibacterial activity of purified human NK cells. Here we show that this function is regulated by T cytotoxic/suppressor CD8+ cells. Thus, coculture of NK and CD8+ cells for 3 h or longer times abrogated the expression of the NK antibacterial activity, and of two activation markers IL-2R and transferrin receptor (Tf-R). The suppressive activity was mediated by PGE2 as demonstrated by direct PGE2 determination in CD8+ cell free supernatants, and by inhibition of CD8+ cell suppression with indomethacin or piroxicam in vitro. We also found that resting T cytotoxic/suppressor cells purified by negative selection produce higher amounts of PGE2 than adherent cells like monocytes and macrophages, and that these concentration levels are in the range of concentrations known to suppress a significant number of in vitro immunologic functions.

Antibacterial activity of human natural killer cells.

The in vitro effects of human NK cells on viability of Gram-negative and Gram-positive bacteria was investigated. PBLs depleted of glass-adherent cells showed a significant antibacterial activity that was increased as the concentration of NK cells became higher. Leu-11-enriched cells exhibited the most efficient bactericidal activity. Stimulation of NK cells with staphylococcal enterotoxin B for 16 h produced a significant increase in the antibacterial activity of all NK cells tested. The antibacterial activity of monocyte-depleted cells and Leu-11-enriched cells was also enhanced after culturing in vitro for 16-24 h without exogenous cytokines. Dependence of the antibacterial activity on the presence of serum in the culture medium was not found. Ultrastructural studies revealed close contact between NK cell membranes and bacteria, no evidence of phagocytosis, and extracellular bacterial ghosts, after incubation at 37 degrees C. Supernatants from purified NK cells exhibited potent bactericidal activity with kinetics and target specificity similar to that of effector cells. These results document the potent antibacterial activity of purified NK cells and suggest an extracellular mechanism of killing.

Role of trehalose in resistance to macrophage killing: study with a tps1/tps1 trehalose-deficient mutant of Candida albicans.

Accumulation of trehalose by yeast is an important protective mechanism against different stress conditions. This study examined the effect of trehalose on several growth features, as well as its association with the intracellular survival of yeasts exposed to macrophages. A tps1/tps1 mutant and its parental counterpart, CAI4, exhibited similar growth rates and preserved their dimorphic conversion and agglutination ability. However, electron-microscopy of cell-wall architecture showed a partial loss of material from the outer cell-wall layer in the tps1/tps1 mutant. Flow-cytometry revealed that the mutant had lower auto-fluorescence levels and a higher fluorescein isothiocynate staining efficiency. When co-cultured with macrophages, a slight reduction in binding to macrophages and slower ingestion kinetics were revealed for the tps1/tps1 mutant, but these did not interfere significantly with the amount of yeast ingested by macrophages after co-incubation for 2 h. Under the same conditions, CAI4 cells were more resistant to macrophage killing than was the tps1 null mutant, provided that the macrophages had been stimulated previously with interferon-gamma. Measurement of trehalose content and the anti-oxidant activities of yeast cells recovered after phagocytosis revealed that the trehalose content and the glutathione reductase activity were increased only in CAI4 cells, whereas levels of catalase activity were increased similarly in both strains. These results suggest that the presence of trehalose in Candida albicans is a contributory factor that protects the cell from injury caused by macrophages.

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.

Quantitation of effector-target affinity in the human NK cell and K562 tumor cell system.

A quantitative procedure to characterize the affinity of human natural killer (NK) cells for K562 tumor is described. Using highly purified (greater than or equal to 98%) NK cells, measurements of the conjugate frequencies permit the determination of the apparent Michaelis constants (KappM) for the conjugation process. Because no intermediate steps for the lytic process are involved the interpretation of the values of KappM is the simplest one that can be achieved. Thus, we found that a plot of KappM against the number of effector cells allows us to determine the dissociation constant, KS, that characterizes the effector-target affinity. KS is independent of the donor cell source and this value was (1.0 +/- 0.1) x 10(5) cell/tube. In contrast, the KappM values vary among donors, and this could be used to compare the relative activity of different donors in relation to their binding capacity.

The maximum conjugate frequency (alpha max) characterizes killer cell populations.

A quantitative procedure to characterize NK cell populations based on the dependence of the frequency of conjugation (alpha) on the effector-to-target ratio (R) is shown. To this end, a detailed study of the influence exerted by: (a) the value of R; (b) the number of effector and target cells (N, T); and (c) the source (donor) and enrichment of the effector cell population on the frequency of conjugation between NK effector and K562 target cells has been performed. This has demonstrated that for a given value of R large differences in the values of alpha can be obtained for different donors and/or N values. Hence, the usual practice of reporting the frequency of conjugation at a given value of R cannot be used as a valid criterion for comparison, and this could explain the differences in the alpha values reported in the literature for the same effector-target system. Moreover, the frequency of conjugation depends on the enrichment of the effector cell populations, although it has been shown that in all cases a plot of 1/alpha vs. R for N = constant is always linear with intercept 1/alpha max.alpha max represents the maximum frequency of conjugation for an effector-target system and remains constant for all values of R and N, and is also independent on the donor of the cell source. These characteristics make that the values of alpha max can be used as an easy criterion to determine with accuracy conjugate frequencies in an effector-target system, and could also be applied to characterize the activation or inhibition of effector cell populations by monoclonal antibodies or other agents. This criterion was applied to characterize the enrichment of NK cell populations and so, a value of alpha max = 58 +/- 3% has been obtained when highly purified (greater than or equal to 99%) NK effector cells obtained by panning with the monoclonal antibodies Leu-2, Leu-3 and Leu-4 are used. However, the corresponding value for MDC (14% NK cells) was lowered to 26 +/- 1%.

Effector-target interactions: saturability, affinity and binding isotherms. A study of such interactions in the human NK cell-K562 tumour cell system.

Effector-target interactions at the cell-to-cell level have been studied. This has revealed that saturability, i.e., the existence of a finite number of specific receptor sites, applies to both the effector and target cell populations and plays a key role in the formation of conjugates. As a result, two maximum conjugate frequencies, alpha max and beta max, are recognised for the effector and target cell populations, respectively. The dissociation constant of the conjugates formed, KD, characterizes effector-target affinity. This constant, together with the maximum conjugate frequencies, are the three parameters which make it possible to describe the binding process quantitatively. The existence of binding isotherms for effector-target interactions has been demonstrated. These isotherms contain all the relevant information necessary to interpret quantitatively the formation of conjugates. Quantitative procedures to determine the three binding parameters are described together with the modifications necessary to use Scatchard plots in the analysis of conjugate frequencies in these kinds of cell-to-cell interactions. A quantitative study of these interactions in the NK-K562 tumour cell system has been performed. For this purpose, nine different cell source donors were used to test the model proposed. Relationships with related phenomena--CMC and the adhesion process--are also discussed.

Binding units (BU) and the area under binding isotherms (AUI). New indices of effector-target conjugation.

New methods for simplified quantitation of effector-target conjugation have been developed. The binding unit (BU) is defined as the number of target cells required to bind a specified percentage of effector cells. The number of binding units is determined from binding isotherms in which effector conjugate frequencies are measured by holding constant the number of effector cells and by varying the number of target cells. Alternately, a binding unit can be defined as the number of effector cells required to bind a specified percentage of target cells. In this case, BU is computed from binding isotherms in which target conjugate frequencies are measured at different values of effector cells by holding constant the number of target cells. Also, the area under the curve (AUI) of these isotherms is another index that can be used as an overall measure of the binding capacity in an effector-target system. The experimental values of BU and AUI determined from effector and target isotherms agree well with theoretical predictions based on our previously developed binding model (J. Immunol. Methods (1992) 155, 133-147). The relationship between BU and AUI, and procedures to determine these parameters are shown. The value of these indices to express effector-target conjugation quantitatively has been confirmed by determining the values of BU and AUI for the NK-K562 effector-target system.

The derivation of binding parameters from effector and target conjugate frequency data using linear and non-linear data-fitting transformations. Application of such transformations to the NK-MOLT4 and NK-K562 effector-target systems.

Effector-target conjugation is described quantitatively by binding isotherms which are characterized by three parameters, the maximum effector and target conjugate frequencies, alpha max and beta max, and the dissociation constant of the conjugates formed, KD. In this paper the application of non-linear data-fitting techniques, as well as linear transformations of the binding isotherms that permit us to use standard regression analysis, has been tested to calculate estimates of these parameters in the NK-MOLT4 and NK-K562 effector-target systems. Both unweighted and weighted data were used to calculate alpha max, beta max and KD for six different donors which were used as a source of NK cells. The results obtained have shown that these regression methods are useful for revealing potential disparities between binding efficiencies in effector-target systems.

Determination of parameters that characterize effector-target conjugation of human NK and LAK cells by flow cytometry.

Effector-target conjugation between different cell populations of human NK cells and K562 tumor cells has been studied from binding isotherms obtained from data of effector (alpha) and target (beta) conjugate frequencies measured by flow cytometry analysis at different effector-to-target ratios. Non-linear and linear regression methods were applied to these isotherms to calculate the binding parameters that characterize the process of conjugation, namely, the maximum effector and target conjugate frequencies, the dissociation constant of the conjugates formed, the binding units and the area under the binding isotherms. The results obtained show that: (1) flow cytometry analysis of effector-target conjugation is faster, unbiased and more suitable than microscopic counting of conjugates, thereby permitting the analysis of larger number of conjugates in shorter times, (2) the binding parameters derived from conjugate frequencies obtained by flow cytometry analysis differ from those obtained by microscopy, (3) the discrepancies between the two methods are due to the presence of several cells engaged in multicellular conjugates that are detected as single particles by flow cytometry and (4) the analysis of population distributions of the conjugates formed at different values of the effector-to-target ratio permit the above discrepancies to be corrected.

Kinetic analysis of effector cell recycling and effector-target binding capacity in a model of cell-mediated cytotoxicity.

A kinetic analysis of cell-mediated cytotoxicity (CMC) at the population level based on theoretical models has been performed. This analysis considers that the binding process and the kinetics of the lytic process occur through different types of conjugates: LTn, i.e., conjugates containing one effector cell and n target cells, and LmT conjugates which contain m effector cells bound to one target cell. This allowed us to provide a quantitative description of the conjugation process, and of the binding capacities of the effector and target cells. Thus, it has been shown that these processes are governed by an equilibrium in which are involved the unbound effector and target cells, and the conjugates formed. This implies that, when the equilibrium concentrations are reached, the total number of conjugates cannot be increased from the unbound effector and target cells. However, it does not mean that the free effector cells are nonbinding, and so, when the conditions of equilibrium are perturbated (as occurs for example in CMC), all effector cells, virgin and those who have already participated in the lytic process, are able to form new conjugates. The existence of this equilibrium has also important consequences when different subpopulations are separated from an effector-target system. Thus, it explains the observation reported in the literature that, although cytometric techniques can be used to detect and count different types of conjugates, the conjugates formed cannot be separated by cell sorting (unless special precautions are taken). Finally, we have found that the number of target cells killed by one effector cell, which has been previously considered as the recycling capacity of an effector population, is in reality the result of two different mechanisms. One of these mechanisms is due to the recycling process, whereas the other (which has the same effect) is due to the multiple killing capacity of the LTn conjugates which kill more than one target cell. The average number of target cells killed per conjugate has been determined, and this allowed us to obtain the relative contributions of these two mechanisms to the total killing capacity of one effector cell. It appears that the recycling capacities of effector cell populations previously determined had been 1- to 3-fold overestimated.

Penetration of host cell lines by bacteria. Characteristics of the process of intracellular bacterial infection.

A model which describes the characteristics of the penetration of the cells by bacteria is presented. Since the process of invasion is preceded necessarily by the step in which the bacteria adhere to the cells, the proposed model is based on the expressions previously derived for the process of adhesion, which allow us to determine the number of attached bacteria under different conditions. Thus, the model considers that invasion occurs irreversibly from attached bacteria to specific receptors located on the cell surface with a rate coefficient = ki so that the invasive capacity in a given bacterium-host cell system is mainly determined by the value of this coefficient. Once internalized, the bacteria can follow three different time courses, namely: 1) intracellular growth is hindered so that the bacteria remain in stationary phase, 2) there is a lag phase during which the bacteria stay in stationary phase before they are able to grow exponentially with a rate coefficient = kc, and 3) the bacteria exhibit a growth exponential phase as they enter the cells. In turn, the time course followed by extracellular bacteria also has a decisive influence on the process of invasion and, in this regard, unbound bacteria are considered either in stationary or in exponential phase. Expressions for these different situations have been derived, and from them, procedures to determine the levels of bacterial infection and for quantitative invasive data analysis are presented.

Experimental and theoretical kinetics study of antibacterial killing mediated by human natural killer cells.

We have recently shown that purified human NK cells, both resting and activated, efficiently kill gram-negative and gram-positive bacteria in vitro. To investigate the mechanism of NK cell-mediated cytotoxicity against Escherichia coli we have developed a mathematical model of the kinetics using the experimental data. The kinetics of killing are characterized by initial target bacterial multiplication, followed by rapid bacterial death. Experiments demonstrates that for each donor there is a threshold number of effector cells necessary to observe a net killing effect. Below the threshold, even use of high effector-to-target ratios lack killing activity and the bacterial growth cannot be stopped. In contrast, if the number of NK cells is larger than the threshold, complete killing is achieved, even at ratios as low as 1/1000. The threshold number varies among donors, ranging between 1200 and 12000 purified NK cells/tube, and provides a quantitative measure of antibacterial activity. Performing the assay at 4 degrees C raised the threshold number required for killing. Experiments performed in Boyden chambers confirm that NK cell-bacteria contact is not necessary for efficient killing, although the kinetics of bacterial lysis is slower. The fit between model and data supports the hypothesis that the bactericidal mechanism is extracellular and is mediated by an anti-microbial factor released from NK cells. Accumulated evidence also indicates that this factor is distinguishable from the mechanisms mediating tumor cell cytotoxicity.

Population distributions of natural killer and K562 target cell conjugates.

We studied the types of conjugates formed at different effector-to-target ratios (R) in the NK-K562 target system. Using purified natural killer (NK) effector cells prepared by a panning technique (greater than 98% CD16), distributions of conjugates are formed such that LTn (one NK cell bound to one or more target cells) are more common at R values less than 1, and LmT (one or more NK cells bound to one target cell) are observed at R greater than 1. Population distributions of these types of conjugates are in agreement with the predictions of the model previously reported for CTL target cell conjugates. In contrast, monocyte-depleted peripheral blood lymphocytes form conjugates in which the LT type is the predominant subpopulation for all values of R. The impact of cell purity on the kinetics and dynamics of cell-mediated cytotoxicity at the population level is discussed.

Computer simulation and data analysis of effector-target interactions: the extraction of binding parameters from effector and target conjugate frequencies data by using linear and nonlinear data-fitting transformations.

Binding isotherms for effector-target conjugation when effector conjugate frequencies are measured by holding constant the number of effector cells and by varying the number of target cells are characterized by two parameters, the maximum effector conjugate frequency, alpha max, and gamma, which is related to the dissociation constant of the conjugates formed, K d. The suitability of four linear transformations of these binding isotherms, as well as nonlinear data-fitting techniques, to provide estimates of alpha max and gamma is discussed. The strength and weakness of these procedures were investigated by calculating alpha max and gamma from different sets of 100 or 500 replicate "experiments," which were generated by using an algorithm that provides noise contributions to the conjugate frequencies with gaussian distributed errors. Both unweighted and weighted data points were used in these calculations. A similar analysis can also be performed for binding isotherms in which target conjugate frequencies are measured at different values of effector cells by holding constant the number of target cells. In this case, the binding isotherms are characterized by two parameters, the maximum target conjugate frequency, beta max, and delta, which is also related to K d. The results obtained demonstrate that if the experimental conditions are chosen properly, linear transformations and nonlinear fitting techniques provide reliable estimates for the binding parameters. Not all procedures, however, provide estimates with the same accuracy, and special emphasis to this fact must be given if the binding assays are performed at low values of the number of effector cells.

Adhesion, invasion and intracellular replication of Salmonella typhimurium in a murine hepatocyte cell line. Effect of cytokines and LPS on antibacterial activity of hepatocytes.

Elimination of pathogenic microorganisms in the liver may be an important effector mechanism in host defenses. In this paper we describe the adhesion, invasion and multiplication of Salmonella typhimurium in a murine embryonic hepatocyte cell line (ATCC TIB-73). Monolayers of hepatocytes treated with recombinant IFN gamma, IL1 beta, and LPS exhibit antibacterial activity against intracellular Salmonella. The dynamic of the infection process in stimulated vs unstimulated hepatocytes was determined by counting the number of survival bacteria in the cell monolayers at 4 and 28h after gentamicin was added to the infected cells. Salmonella typhimurium is able to adhere, invade and replicate inside the hepatocytes. The maximum number of cell-associated bacteria is approximately 15 bacteria per cell, whereas the invasive capacity of Salmonella is 0.003 bacteria per hepatocyte. Stimulated cultures display antibacterial activity compared to unstimulated controls. The antibacterial activity does not seem to be mediated by nitric oxide (NO) since inhibition of NO production by using NG-Monomethyl-L-Arginine did not revert the antibacterial activity. Also, high amounts of NO induced by adding L-Arginine to the cell cultures did not enhance hepatocyte antibacterial activity.

Model for population distributions of lymphocyte-target cell conjugates.

A quantitative model for the population distributions of the different types of conjugates formed between cytotoxic T lymphocytes and target cells has been developed. The comparison of the theoretical predictions with data of the literature reveals that the transit populations among the different types of conjugates depends on the lymphocyte-to-target ratio, R, and two constants, k and k1. These constants (where k greater than k1) govern, respectively, the transit populations among conjugates of the type LTi (LTn----LTn-1----...LT), and among LjT conjugates (LT----L2T----...----LmT). We have found that high ratios are necessary to obtain conjugates where multiple T lymphocytes are bound to one target cell, and that under these conditions the predominant conjugate, LjT, varies according to j = 1 + k1R. Conversely, for low values of R the predominant population is of the type LTi, where i also shows a linear dependence on R. Our model explains also why the conjugate LT is normally the predominant population under the experimental conditions reported in the literature. A discussion of the influence exerted by the population distributions of lymphocyte-target cell conjugates on the kinetic of the lytic process for these kinds of effector-target systems has also been made.

Effect of conjugate size on the kinetics of cell-mediated cytotoxicity at the population level.

We developed a model for the kinetics of target cell lysis by cytotoxic T-lymphocytes which accounts for most facts observed at the population level. In contrast to previous models, the following facts: conjugate frequency of cytotoxic T-lymphocytes bound to target cell, dependence of this frequency on the lymphocyte-to-target ratio (R), variation of R with time as target cells are destroyed, and population distributions of the different types of conjugates formed between lymphocytes and target cells, which are involved in the kinetics of these kinds of effector-target systems have been contemplated in the model. The relationship with effector-kinetic analogy models for the lytic process has been discussed. Predictions of the model have been explored and compared with experimental observations about target cell lysis reported in the literature.

Effect of different treatments of the endotoxin-induced modifications in serum iron levels.

The effects of hyperthermia, injection of endotoxin and different antipyretics on serum iron levels in rabbits have been determined. Three antipyretics, Ketoprofen (K), Indomethacin (I), and Polymyxin B (P) induced a rise in serum iron concentration. The rise in serum levels induced by Ketoprofen seems to be related to the half life of the compound. Pretreatment with these antipyretics inhibits the rise in body temperature and the fall in serum concentration observed after the administration of bacterial endotoxin. The hyperthermia failed to modify serum iron levels.