Marta Sampaio de Freitas

Heme Inhibits Human Neutrophil Apoptosis: Involvement of Phosphoinositide 3-Kinase, MAPK, and NF-κB

High levels of free heme are found in pathological states of increased hemolysis, such as sickle cell disease, malaria, and ischemia reperfusion. The hemolytic events are often associated with an inflammatory response that usually turns into chronic inflammation. We recently reported that heme is a proinflammatory molecule, able to induce neutrophil migration, reactive oxygen species generation, and IL-8 expression. In this study, we show that heme (1–50 μM) delays human neutrophil spontaneous apoptosis in vitro. This effect requires heme oxygenase activity, and depends on reactive oxygen species production and on de novo protein synthesis. Inhibition of ERK and PI3K pathways abolished heme-protective effects upon human neutrophils, suggesting the involvement of the Ras/Raf/MAPK and PI3K pathway on this effect. Confirming the involvement of these pathways in the modulation of the antiapoptotic effect, heme induces Akt phosphorylation and ERK-2 nuclear translocation in neutrophils. Futhermore, inhibition of NF-κB translocation reversed heme antiapoptotic effect. NF-κB (p65 subunit) nuclear translocation and IκB degradation were also observed in heme-treated cells, indicating that free heme may regulate neutrophil life span modulating signaling pathways involved in cell survival. Our data suggest that free heme associated with hemolytic episodes might play an important role in the development of chronic inflammation by interfering with the longevity of neutrophils.

Phosphoinositide-3 kinases critically regulate the recruitment and survival of eosinophils in vivo : importance for the resolution of allergic inflammation

The phosphatidylinositol‐3 kinase (PI3K) family of signaling enzymes plays a crucial role in leukocyte recruitment and activation and hence, likely regulates the induction and propagation phases of inflammation. However, little data have emerged showing a role for these processes in the resolution phase in models of in vivo inflammation. Here, we have evaluated the role of PI3K for the migration and survival of eosinophils in a model of allergic pleurisy in mice. Eosinophil accumulation in PI3Kγ‐deficient mice was inhibited at 48 h, as compared with wild‐type mice but not at earlier time‐points (6 and 24 h). Experiments with adoptive transfer of bone marrow showed that PI3Kγ in eosinophils but not in non‐bone marrow‐derived cells was required for their accumulation. Systemic treatment with PI3K inhibitors before antigen challenge prevented the recruitment of eosinophils. This was associated with decreased Akt phosphorylation, interleukin‐5 production, and eosinophil release from the bone marrow. Treatment with PI3K inhibitors 24 h after antigen challenge markedly cleared the accumulated eosinophils, an effect associated with inhibition of Akt phosphorylation and an increased number of apoptotic events. Altogether, our data demonstrate an important role of PI3Kγ for the maintenance of eosinophilic inflammation in vivo, whereas other isoforms of PI3K may be relevant for the recruitment process.

Induction of NOS in rat blood PMN in vivo and in vitro: modulation by tyrosine kinase and involvement in bactericidal activity.

Intravenous administration of lipopolysaccharide (LPS) to rats increased the production of nitric oxide (NO) metabolites (NOx) by blood polymorphonuclear neutrophils (PMN) in vitro. Both dexamethasone and L‐NMMA, added in vitro to neutrophil cultures, inhibited the production of NO. On the other hand, the production of NO was not affected by the treatment, in vivo or in vitro, with different inhibitors of cyclooxygenase or 5‐lipoxygenase or with a platelet‐activating factor (PAF) antagonist. The incubation of blood PMN from normal rats in vitro with neutrophil activators (PAF, leukotriene B4, and interleukin‐8) and different cytokines [interleukin‐1, tumor necrosis factor α, and interferon‐γ (IFN‐γ)] showed that only IFN‐γ was able to induce the production of high amounts of NO. This induction was directly correlated with the expression of iNOS and an increase in in the enzyme activity in blood PMN. The tyrosine kinase inhibitor genistein inhibited NO production induced by IFN‐γ, suggesting that the signal transduction pathway leading to NOS induction in rat PMN involves phosphorylation by tyrosine kinase. We also showed that NO produced by IFN‐γ activated rat blood PMN involved in the killing of Pseudomonas aeruginosa. J. Leukoc. Biol. 65: 508–514; 1999.

Contribution of heparan sulfate to the non-permissive role of the midline glia to the growth of midbrain neurites

Radial glial cells and astrocytes are heterogeneous with respect to morphology, cytoskeletal‐ and membrane‐associated molecules and intercellular interactions. Astrocytes derived from lateral (L) and medial (M) midbrain sectors differ in their abilities to support neuritic growth of midbrain neurons in coculture (Garcia‐Abreu et al. J Neurosci Res 40:471, 1995). There is a correlation between these abilities and the differential patterns of laminin (LN) organization that is fibrillar in growth‐permissive L astrocytes and punctate in the non‐permissive M astroglia (Garcia‐Abreu et al. NeuroReport 6:761, 1995). There are also differences in the production of glycosaminoglycans (GAGs) by L and M midbrain astrocytes (Garcia‐Abreu et al. Glia 17:339, 1996). We show that the relative amounts of the glycoproteins laminin LN, fibronectin (FN) and tenascin (TN) are virtually identical in L and M glia, thus, confirming that an abundant content of LN is not sufficient to promote neurite growth. To further analyze the role of GAGs in the properties of M and L glia, we employed enzymatic degradation of the GAGs chondroitin sulfate (CS) and heparan sulfate (HS). Treatment with chondroitinase has little effect on the non‐permissive properties of M glia but reduces the growth‐supporting ability of L glia. By contrast, heparitinase I produces no significant changes on L glia but leads to neurite growth promotion by M glia. Taken together, these results suggest that glial CS helps to promote neurite growth and, more importantly, they indicate that a HS proteoglycan is, at least, partially responsible for the non‐permissive role of the midline glia to the growth of midbrain neurites. GLIA 29:260–272, 2000.

Interaction of disintegrins with human neutrophils induces cytoskeleton reorganization, focal adhesion kinase activation, and extracellular-regulated kinase-2 nuclear translocation, interfering with the chemotactic function.

We previously demonstrated that jarastatin (JT), a new disintegrin from Bothrops jararaca venom, altered actin dynamics in human polymorphonuclear neutrophils (PMNs) and inhibited cell migration in vivo and in vitro (14). In this study, we evaluated the effects of JT and two other monomeric disintegrins, kistrin (KR) and flavoridin (FL), on PMN chemotaxis and chemokinesis in vitro and on the activation of integrin‐mediated pathways. Although JT, but not KR or FL, was chemotactic, only KR was chemokinetic to PMN. However, preincubation of PMN with any disintegrins inhibited chemotaxis for fMLP. Treatment of PMN with JT and KR increased actin polymerization, whereas FL reduced the content of F‐actin. The effects of JT and KR on actin dynamics were inhibited (50%) by genistein, a tyrosine kinase inhibitor. Accordingly, JT and KR induced an increase in tyrosine phosphorylation, whereas FL had no effect. The three disintegrins were able to induce focal adhesion kinase activation. However, JT and KR promoted Erk‐2 nuclear translocation, and FL inhibited Erk‐2 activation. The data suggest that although the disintegrins JT and KR directly activate integrin‐coupled signaling, FL may interact differently with integrins, triggering an inhibitory pathway modulated by focal adhesion kinase activation.

Thyroid hormone action on astroglial cells fromdistinct brain regions during development

Astrocytes are target to triiodothyronine (T3) hormone action during rat braindevelopment. In this work, we show that astrocytes from distinct developing brain regions aredifferently responsive to thyroid hormone. Distinctly from embryonic or newborn cerebralhemisphere and mesencephalic astrocytes, newborn cerebellar and embryonic hippocampalastrocytes do not change their morphology in response to hormone treatment. We also analysedprotein synthesis and secretion from these T3‐treated astrocytes. The results showed a significantincrease in protein synthesis in astrocytes from older brain regions. Maximum effect, however,was observed in cerebral hemisphere astrocytes from newborn rats. The protein secretion effectwas also more evident in the cerebral hemisphere as well as in cerebellar astrocytes fromnewborn rats. In addition, we examined T3 effects on GFAP/vimentin expression by culturing6‐day old cerebellar astrocytes. In this case T3 seems to induce GFAP expression which might beoccurring as a first step to astrocyte differentiation.

Selective effect of hypothyroidism on expression of myelin markers during development

Thyroid hormones are critical for maturation of the central nervous system. In a previous study, we showed a change in the pattern of mature myelinated nerve fibers by 2′3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) in developing hypothyroid animals, which suggests a possible role for thyroid hormones in myelin compaction. The classical myelin markers myelin basic protein (MBP) and proteolipidic protein (PLP) are expressed later in oligodendroglial development, when myelin sheath formation is in progress. A myelin constituent designated myelin‐associated/oligodendrocytic basic protein (MOBP) has been identified and related to myelin compaction. We assessed the developmental sequence of appearance of CNPase, MBP, MOPB, and PLP proteins in cerebellum (Cb) and corpus callosum (cc) in an experimental hypothyroidism model. The appearance of both MOBP isoforms occurred at postnatal day (P)25 and P30 in cc and Cb, respectively, followed by an increase with age in the control group. However, all the MOBP isoforms were weakly detectable in both regions at P30 from the hypothyroid (H) group, and the higher molecular weight isoform remains decreased in cc, even at P90. The developmental pattern of expression of CNPase, MBP, and PLP proteins was also delayed in the H group. CNPase and MBP expression was recovered in cc and Cb, whereas PLP remained below control levels at P90 in cc. Our data show that the experimental hypothyroidism affects the developmental pattern of the oligodendrocytic/myelin markers. Furthermore, thyroid hormone may modulate specific genes, as demonstrated by permanent down‐regulation of MOBP and PLP expression in adulthood. J. Neurosci. Res. 66:254–261, 2001.

Low expression of insulin signaling molecules impairs glucose uptake in adipocytes after early overnutrition

Experimental and clinical studies have demonstrated that early postnatal overnutrition represents a risk factor for later obesity and associated metabolic and cardiovascular disturbance. In the present study, we assessed the levels of glucose transporter 4 (GLUT-4), GLUT-1, insulin receptor (IR), IR substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K) and Akt expression, as well as insulin-stimulated glucose transport and Akt activity in adipocytes from adult rats previously raised in small litters (SL). The normal litter (NL) served as control group. We also investigated glycemia, insulinemia, plasma lipid levels, and glucose tolerance. Our data demonstrated that early postnatal overfeeding induced a persistent hyperphagia accompanied by a significant increase in body weight until 90 days of age. The SL group also presented a significant increase ( approximately 42%) in epidydimal fat weight. Blood glucose, plasma insulin, and lipid levels were similar among the animals from the SL and NL groups. While insulin-stimulated glucose uptake was approximately twofold higher in adipocytes from the NL group, no stimulatory effect was observed in the SL group. The impaired insulin-stimulated glucose transport in adipose cells from the SL rats was associated with a significant decrease in GLUT-4, IRS-1 and PI3K expression, and Akt activity. In contrast, IR and Akt expression in adipocytes was not different between the SL and NL groups. Despite these alterations, our results showed no differences in glucose tolerance test in rats raised under different feeding conditions. Our findings reinforce a potent and long-term effect of neonatal overfeeding, which can program major changes in the metabolic regulatory mechanisms.

Expression of inducible nitric oxide synthase in human umbilical vein endothelial cells during primary culture

The adaptive response of endothelial cells to stress may lead to the upregulation of nitric oxide (NO) production. Herein, we report inducible nitric oxide synthase (iNOS) induction in primary cultures of human umbilical vein endothelial cells (HUVEC). The enzyme expression was earlier observed in 12-h cultures, reaching maximal levels after 3 days and decreasing when cells become confluent. The time course of NO production by HUVEC paralleled iNOS expression during the whole culture period, indicating that enzyme was functionally active. Conversely, iNOS induction could not be further detected in HUVEC subcultures passed once from cells presenting maximal levels of iNOS expression in the primary culture. Induction of iNOS in HUVEC was not related to lipopolysaccharide contamination, since the enzyme expression was not affected in the presence of polymyxin B added to primary cultures. Further analysis showed that aminoguanidine, a specific iNOS inhibitor, did not affect cell proliferation, suggesting that the NO produced by HUVEC may not be directly related to cell growth. Platelet endothelial cell adhesion molecule-1 expression was upregulated during cell confluence, in contrast to the decrease of iNOS expression and activity. The data suggest that iNOS expression may be a molecular mechanism mediating the adaptive response of endothelial cells to culture environment.

Cellular distribution of phosphofructokinase activity and implications to metabolic regulation in human breast cancer

Neoplastic cells generally present profound changes in glucose metabolism. The mechanisms underlying such process are numerous and all may involve altered cellular hormonal responses. Here we report the first evidence that cellular location of phosphofructokinase activity in human breast cancer tissues is different from the one observed in control tissues and that this phenomenon may be involved in the increased glycolytic flux observed in those cells. Through co-sedimentation techniques, we observed that 60% of phosphofructokinase activity in neoplastic tissues is located in an actin-enriched fraction, against 36% in control tissues. Additionally, metastatic tumor tissues presented a two fold increase in this particulate activity when compared to non-metastatic tumor samples. We propose that the alteration in cellular distribution of phosphofructokinase activity in human breast cancer tissues is a mechanism associated to the process of cell transformation and may be a consequence of the altered hormonal milieu observed in several types of cancer.