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.

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.

Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.

The cellular resistance against oxidative stress (H2O2) is independent of neutral trehalase (Ntc1p) activity in Candida albicans.

The protective role of trehalose against oxidative stress caused by hydrogen peroxide in Candida albicans has been investigated in the homozygous mutant ntc1Delta/ntc1Delta, disrupted in the NTC1 gene, which encodes the neutral (cytosolic) trehalase (Ntc1p). After a severe oxidative exposure (50 mM H(2)O(2)), both parental (CAI-4) and ntc1Delta/ntc1Delta exponential-phase cells stored large amounts of intracellular trehalose. In turn, the degree of cell survival was roughly equivalent in both strains, although slightly higher in ntc1Delta/ntc1Delta cultures. The mechanism of 'adaptive tolerance' was functional in the two strains. Thus, a gently oxidative pretreatment (5 mM H(2)O(2)) increased the recovery of cellular viability when it was followed by a severe challenge (50 mM H(2)O(2)); this phenomenon was accompanied by a significant elevation of the endogenous trehalose content. Oxidative stress also induced specific activation of the antioxidant enzymes catalase and glutathione reductase upon gentle oxidative treatment (5 mM H(2)O(2)), whereas superoxide dismutase activity was only activated upon prolonged exposure. Taken together, these results strongly suggest that in C. albicans neutral trehalase activity does not play an essential role in the protective response against oxidative stress. They also suggest that a diminished Ntc1p activity might favour the growth of C. albicans cells subjected to a strong oxidative exposure.

Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor.

H(2)O(2) and other reactive oxygen species are key regulators of many intracellular pathways. Within mammalian skin, H(2)O(2) is formed as a byproduct of melanin synthesis, and following u.v. irradiation. We therefore analyzed its effects on melanin synthesis. The activity of the rate-limiting melanogenic enzyme, tyrosinase, decreased in H(2)O(2)-treated mouse and human melanoma cells. This inhibition was concentration- and time-dependent in the B16 melanoma model. Maximal inhibition (50-75%) occurred 8-16 hours after a 20 minute exposure to 0.5 mM H(2)O(2). B16 cells withstand this treatment adequately, as shown by a small effect on glutathione levels and a rapid recovery of basal lipid peroxidation levels. Enzyme activities also recovered, beginning to increase 16-20 hours after the treatment. Inhibition of enzyme activities reflected decreased protein levels. mRNAs for tyrosinase, tyrosinase-related protein 1, dopachrome tautomerase, silver protein and melanocortin 1 receptor also decreased after H(2)O(2) treatment, and recovered at different rates. Downregulation of melanocyte differentiation markers mRNAs was preceded by a decrease in microphthalmia transcription factor (Mitf) gene expression, which was quantitatively similar to the decrease achieved using 12-O-tetradecanoylphorbol-13-acetate. Recovery of basal Mitf mRNA levels was also observed clearly before that of tyrosinase. Therefore, oxidative stress may lead to hypopigmentation by mechanisms that include a microphthalmia-dependent downregulation of the melanogenic enzymes.

Transforming growth factor beta1 mediates hypopigmentation of B16 mouse melanoma cells by inhibition of melanin formation and melanosome maturation.

Transforming growth factor-beta1 (TGFbeta1) downregulates tyrosinase in B16 melanoma cells by decreasing gene expression and the intracellular half-life of the enzyme, but does not block tyrosinase stimulation by alpha-melanocyte stimulating hormone (alphaMSH). In the presence of both agents, the enzymatic activity is intermediate between the one of cells treated with either agent alone. Here we show that TGFbeta1 equally inhibits the melanogenic activities of melan-a melanocytes and B16 melanoma cells, thus validating the B16 model. In both cell types, TGFbeta1 (10(-10) M, 48 h) inhibited to comparable levels tyrosine hydroxylation and melanin formation from L-tyrosine. Thus, the inhibitory effect is exerted mainly at the rate limiting step of the pathway. By means of quantitative image analysis techniques, we also studied the effects of TGFbeta1 and alphaMSH on melanosome number, volume density and maturation degree. alphaMSH (10(-7) M, 48 h) increased 7-fold melanosome volume density, whereas TGFbeta1 by itself had no significant effect. However, melanosomal volume density was intermediate in cells treated with both agents, as compared to control or alphaMSH-treated cells. Moreover, TGFbeta1 blocked the alphaMSH-elicited increase in the number of melanosomes. Control and alphaMSH-treated melanocytes contained more stage I+II premelanosomes and stage IV, fully melanized organelles than partially melanized stage III melanosomes. TGFbeta1 increased the percentage of stage III melanosomes. This trend was even more marked in cells treated with alphaMSH and TGFbeta1. The accumulation of incompletely melanized melanosomes is consistent with the inhibition of melanin formation activity by TGFbeta1 and with its hypopigmenting effect.

New insights on the structure of the mouse silver locus and on the function of the silver protein.

The melanosomal proteins encoded by the silver locus play important roles in melanogenesis. The human locus yields two proteins, PMEL17 and GP100, by alternative mRNA splicing. The mouse si locus was reported to encode a Pmel17 protein, and later gp87, a GP100 homologue. When we re-examined the products of wild-type and silver-mutant mouse si loci, RT-PCR of wild-type RNA and genomic DNA sequence accounted for gp87 but excluded the occurrence of Pmel17. Analysis of cDNA from the silver (si/si) melanocyte line, melan-si, showed that the pathogenic mutation is a G to A substitution at nt 1808, which yields a premature stop codon and a predicted protein truncated in the C-terminus. This was confirmed by reaction of a specific anti-gp87 antiserum with si/si melanocyte extracts. To further explore gp87 function, we compared the DHICA oxidase activity of extracts from B16, melan-si (heterozygous for the brown mutation and homozygous for the silver mutation) and Cloudman S91 cells (homozygous for the brown mutation), since both TRP1 and gp87 are thought to be involved in DHICA oxidation/polymerization. Cloudman extracts do not oxidize significantly DHICA and its methyl ester, supporting the involvement of native mouse TRP1 in DHICA oxidation. Extracts from B16 and melan-si do not show significant differences for the oxidation of free acid and methylated dihydroxyindoles, indicating that the mechanism is not decarboxylative. Melan-si extracts are very efficient in catalyzing dihydroxyindole oxidation, in spite of being heterozygous for the TRP1 mutation, consistent with a stablin effect for the wild-type gp87 protein. On the other hand, aggregated and degraded forms of that mutant gp87 protein are found in the cytosolic fraction of melan-si, suggesting that misrouting and aberrant processing of the gp87 and tyrosinase may also be related to the high DHICA oxidase activity of these melanocytes.

Regulation of the murine silver locus product (gp87) by the hypopigmenting cytokines TGF-beta1 and TNF-alpha.

The melanosomal proteins encoded by the silver (si, SILV, or PMEL17) locus play important roles in melanogenesis and are actively investigated as targets for melanoma immunotherapy. The human silver locus yields two proteins, gp100 and PMEL17, by alternative splicing of a common mRNA precursor. Mouse melanocytes exclusively express the gp100 orthologue, here termed gp87, thus providing a simpler model with which to study the silver locus products. We have analyzed the effects of [Nle4, D-Phe7]-alpha melanocyte-stimulating hormone (alphaMSH) and two hypopigmenting cytokines, tumor necrosis factor (TNF)-alpha and transforming growth factor (TGF)-beta1, on the expression of gp87 in B16 mouse melanoma cells. TNF-alpha and TGF-beta1 (at saturating doses for 48 hr) decreased gp87 mRNA by 50%. The gp87 protein was almost undetectable by Western immunoblotting after TNF-alpha treatment, but was not affected by TGF-beta1. alphaMSH increased the mRNA and the gp87 protein approximately 2-fold. Moreover, the amount of gp87 was not reduced by TNF-alpha in the presence of the hormone, in spite of a 50%, decrease in its mRNA. Therefore, the levels of mRNA and gp87 protein did not correlate after treatment with the cytokines. Overall, our data suggest that the silver locus product is not regulated exclusively at the transcriptional level, and highlight the importance of still-uncharacterized regulatory translational and/or post-translational events.

Independent regulation of tyrosinase by the hypopigmenting cytokines TGF beta1 and TNF alpha and the melanogenic hormone alpha-MSH in B16 mouse melanocytes.

In B16 melanocytes, tyrosinase activity and melanin formation are upregulated by alpha-MSH and downregulated by TGF beta1 and TNF alpha. Since TGF beta1 or TNF alpha block the differentiation programs induced by throphic hormones in other cell types, we studied tyrosinase regulation by alpha-MSH in the presence of the hypopigmenting cytokines, as well as the effects of the cytokines on several aspects of alpha-MSH signaling. TGF beta1 and TNF alpha only slightly diminished MC1 receptor gene expression, and had no effect on the intracellular levels of cAMP, or on the alpha-MSH-dependent cAMP rise. The intracellular levels of tyrosinase mRNA, protein and enzymatic activities were also upregulated by alpha-MSH in cells pretreated with TGF beta1 or TNF alpha. Therefore the cytokines do not block the response to alpha-MSH. However, the cytokine-induced inhibition of tyrosinase gene expression, protein levels and the reduction of tyrosinase intracellular half-life also occurred in the presence of alpha-MSH, indicating that the hormone does not override TGF beta1 or TNF alpha inhibition. Thus, tyrosinase activity and the rate of melanin formation in B16 melanocytes might reflect simply the balance between alpha-MSH stimulation and TGF beta1 or TNF alpha inhibition, acting by independent mechanisms.

Mechanisms of melanogenesis inhibition by tumor necrosis factor-alpha in B16/F10 mouse melanoma cells.

The inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) is present in the dermal and epidermal layers of normal skin [Kilgus, O., Payer, E., Schreiber, S., Elbe, A., Strohal, R. & Stingl, G. (1993) J. Invest. Dermatol. 100, 674-680]. Its local concentrations are modified by several stimuli, including wound healing and ultraviolet irradiation. Moreover, TNF-alpha inhibits melanogenesis in normal melanocytes [Swope, V., Abdel-Malek, Z., Kassem, L. & Norlund, J. (1991) J. Invest. Dermatol. 96, 180-185], and is, therefore, a potential autocrine/paracrine regulator of pigmentation. We have analyzed the mechanisms of this effect using B16/F10 melanoma cells as a model. Nanomolar concentrations of TNF-alpha inhibit the tyrosine hydroxylase and dopa oxidase activities of B16/F10 melanocytes, to less than 30% control levels, without effects on tyrosinase-related protein 2/dopachrome tautomerase (TRP2/DCT). The 50% inhibition was obtained at 1 nM TNF-alpha and 48 h treatment. The effect of TNF-alpha was noticeable after 6 h treatment, and maximal after 24 h. This inhibition is explained by decreased intracellular levels of tyrosinase and tyrosinase-related protein 1 (TRP1), but not of TRP2/DCT as detected by Western blotting. Northern-blot experiments showed that the inhibitory effect is partially explained by a reduced accumulation of the corresponding mRNAs, that dropped to about 50% of control values (48 h treatment, 5 nM TNF-alpha). Moreover, the tyrosine hydroxylase and dopa oxidase activities decreased more rapidly in TNF-alpha-treated cells than in control cells, under conditions of inhibition of protein synthesis. This suggests a TNF-mediated reduction of tyrosinase half-life. However, the possibility of an inhibitory post-translational modification of the enzyme induced by TNF cannot be ruled out. Therefore, the inhibitory effect of TNF-alpha on tyrosinase and TRP-1 results from combined effect on mRNA levels and enzymatic activity or protein stability.

Molecular interactions within the melanogenic complex: formation of heterodimers of tyrosinase and TRP1 from B16 mouse melanoma.

Melanin synthesis in mammals is catalyzed by three structurally related, membrane-bound proteins, tyrosinase, and the tyrosinase-related proteins 1 and 2 (TRP1 and TRP2). Current evidence suggests that in vivo these proteins may form a multienzyme complex. However, neither the precise composition of the complex, nor the specific interactions between its components have been characterized. This study used purified preparations of tyrosinase and TRP1 to analyze their interactions in non ionic detergent solution. Purified tyrosinase and TRP1 behaved as homodimers as judged by gel filtration chromatography and electrophoresis. Upon mixing of the purified proteins, the preferential formation of heterodimers was detected by: i) coelution in gel filtration chromatography with a shift to a common partition coefficient for both proteins, and ii) the occurrence of fluorescent energy transfer between fluorescein-labeled tyrosinase and rhodamine-labeled TRP1. However, the formation of heterodimers did not cause changes in the tyrosine hydroxylase activity of the enzymes, at least under standard assay conditions. Thus, tyrosinase and TRP1 interact strongly and specifically in detergent solution to form an heterodimer that might contribute to the formation of the melanogenic complex.

Comparison of TRPs from murine and human malignant melanocytes.

Most of our knowledge of the mammalian tyrosinase related protein (TRP) activities is derived from studies using murine melanoma models, such as B16 or Cloudman S-91 melanocytes. Owing to the high degree of homology between the murine and human enzymes, it has been assumed that their kinetic behaviour could be similar. However, the protein sequences at the metal binding sites of the murine and human enzymes show some differences of possible functional relevance. These differences are more significant in the metal-A site than in the metal-B site. By using three human melanoma cell lines (HBL, SCL, and BEU), we have studied the catalytic abilities of the human melanogenic enzymes in comparison to those obtained for the counterpart murine enzymes isolated from B16 melanoma. We have found that TRP2 extracted from all cell lines show dopachrome tautomerase activity, although the activity levels in human malignant melanocytes are much lower than in mouse cells. Reconstitution experiments of the human enzyme indicate that TRP2 has Zn at its metal binding-sites. Although mouse tyrosinase does not show DHICA oxidase activity, and this step of the melanogenesis pathway is specifically catalyzed by mouse TRP1, the human enzyme seems to recognize carboxylated indoles. Thus, human tyrosinase could display some residual DHICA oxidase activity, and the function of human TRP1 could differ from that of the murine protein. Attempts to clarify the nature of the metal cofactor in TRP1 were unsuccessful. The enzyme contains mostly Fe and Cu, but the reconstitution of the enzymatic activity from the apoprotein with these ions was not possible.

Transforming growth factor-beta1 inhibits basal melanogenesis in B16/F10 mouse melanoma cells by increasing the rate of degradation of tyrosinase and tyrosinase-related protein-1.

Current evidence suggests that melanogenesis is controlled by epidermal paracrine modulators. We have analyzed the effects of transforming growth factor-beta1 (TGF-beta1) on the basal melanogenic activities of B16/F10 mouse melanoma cells. TGF-beta1 treatment (48 h) elicited a concentration-dependent decrease in basal tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (Dopa) oxidase activities, to less than 30% of the control values but had no effect on dopachrome tautomerase activity (TRP-2). The inhibition affected to similar extents the Dopa oxidase activity associated to tyrosinase-related protein-1 (TRP-1) and tyrosinase. This inhibition was noticeable between 1 and 3 h after the addition of the cytokine, and maximal after 6 h of treatment. The decrease in the enzymatic activity was paralleled by a decrease in the abundance of the TRP-1 and tyrosinase proteins. TGF-beta1 mediated this effect by increasing the rate of degradation of tyrosinase and TRP-1. Conversely, after 48 h of treatment, the expression of the tyrosinase gene decreased only slightly, while TRP-1 and TRP-2 gene expression was not affected. An increased rate of proteolytic degradation of TRP-1 and tyrosinase seems the main mechanism accounting for the inhibitory effect of TGF-beta1 on the melanogenic activity of B16/F10 cells.

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.