Effect of exogenous galectin-1 on leukocyte migration: modulation of cytokine levels and adhesion molecules

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Induction of Bim limits cytokine-mediated prolonged survival of neutrophils

Under inflammatory conditions, neutrophil apoptosis is delayed due to survival-factor exposure, a mechanism that prevents the resolution of inflammation. One important proinflammatory cytokine involved in the regulation of neutrophil survival/activation is granulocyte-macrophage colony-stimulating factor (GM-CSF). Although GM-CSF mediates antiapoptotic effects in neutrophils, it does not prevent apoptosis, and the survival effect is both time dependent and limited. Here, we identified the proapoptotic Bcl-2 family member Bim as an important lifespan limiting molecule in neutrophils, particularly under conditions of survival factor exposure. Strikingly, GM-CSF induced Bim expression in both human and mouse neutrophils that was blocked by pharmacological inhibition of phosphatidylinositol-3 kinase (PI3K). Increased Bim expression was also seen in human immature bone marrow neutrophils as well as in blood neutrophils from septic shock patients; both cell populations are known to be exposed to GM-CSF under in vivo conditions. The functional role of Bim was investigated using Bim-deficient mouse neutrophils in the presence and absence of the survival cytokines interleukin (IL)-3 and GM-CSF. Lack of Bim expression resulted in a much higher efficacy of the survival cytokines to block neutrophil apoptosis. Taken together, these data demonstrate a functional role for Bim in the regulation of neutrophil apoptosis and suggest that GM-CSF and other neutrophil hematopoietins initiate a proapoptotic counterregulation that involves upregulation of Bim.

Human immunodeficiency virus type 1 infection alters chemokine beta peptide expression in human monocytes: implications for recruitment of leukocytes into brain and lymph nodes.

Two chemokine (chemoattractant cytokines) beta peptides, macrophage inflammatory proteins 1 alpha and 1 beta (MIP-1 alpha and MIP-1 beta), were induced in human monocyte cultures following infection with the human immunodeficiency virus type 1 (HIV-1). Induction depended on productive viral infection: not only did the kinetics of MIP-1 peptide induction closely follow those of viral replication, but monocyte cultures inoculated with heat-inactivated virus or infected in the presence of AZT failed to produce these chemokine beta peptides. In addition, HIV infection markedly altered the pattern of beta chemokine expression elicited by tumor necrosis factor (TNF), itself a potent proinflammatory cytokine upregulated during the development of AIDS. Reverse transcription (RT)-PCR and RT-in situ PCR studies on brain tissue from patients with AIDS dementia demonstrated elevated MIP-1 alpha and MIP-1 beta mRNA expression relative to comparable samples from HIV-1-infected patients without dementia. Cells expressing chemokines in HIV-1-infected brains were identified morphologically as microglia and astrocytes. As MIP-1 alpha and MIP-1 beta are potent chemoattractants for both monocytes and specific subpopulations of lymphocytes, this dysregulation of beta chemokine expression may influence the trafficking of leukocytes during HIV infection. These data, taken together, suggest a mechanism by which HIV-1-infected monocytes might recruit uninfected T cells and monocytes to sites of active viral replication or inflammation, notably the brain and lymph nodes.

Régulation de l'expression de PPARγ dans l'arthrose

L’arthrose (OA) est une maladie dégénérative très répondue touchant les articulations. Elle est caractérisée par la destruction progressive du cartilage articulaire, l’inflammation de la membrane synoviale et le remodelage de l’os sous chondral. L’étiologie de cette maladie n’est pas encore bien définie. Plusieurs études ont été menées pour élucider les mécanismes moléculaires et cellulaires impliqués dans le développement de l’OA. Les effets protecteurs du récepteur activé par les proliférateurs de peroxysomes gamma (PPARγ) dans l'OA sont bien documentés. Il a été démontré que PPARγ possède des propriétés anti-inflammatoires et anti-cataboliques. Aussi, plusieurs stimuli ont été impliqués dans la régulation de l’expression de PPARγ dans différents types cellulaires. Cependant, les mécanismes exacts responsables de cette régulation ainsi que le profil de l’expression de ce récepteur au cours de la progression de l’OA ne sont pas bien connus. Dans la première partie de nos travaux, nous avons essayé d’élucider les mécanismes impliqués dans l’altération de l’expression de PPARγ dans cette maladie. Nos résultats ont confirmé l’implication de l’interleukine-1β (IL-1β), une cytokine pro-inflammatoire, dans la réduction de l’expression de PPARγ au niveau des chondrocytes du cartilage articulaire. Cet effet coïncide avec l'induction de l’expression du facteur de transcription à réponse précoce de type 1 (Egr-1). En plus, la diminution de l'expression de PPARγ a été associée au recrutement d'Egr-1 et la réduction concomitante de la liaison de Sp1 au niveau du promoteur de PPARγ. Dans la deuxième partie de nos travaux, nous avons évalué le profil d’expression de ce récepteur dans le cartilage au cours de la progression de cette maladie. Le cochon d’inde avec OA spontanée et le chien avec OA induite par rupture du ligament croisé antérieur (ACLT) deux modèles animaux d’OA ont été utilisés pour suivre l’expression des trois isoformes de PPARs : PPAR alpha (α), PPAR béta (β) et PPAR gamma (γ) ainsi que la prostaglandine D synthase hématopoïétique (H-PGDS) et la prostaglandine D synthase de type lipocaline (L-PGDS) deux enzymes impliquées dans la production de l’agoniste naturel de PPARγ, la 15-Deoxy-delta(12,14)-prostaglandine J(2) (15d-PGJ2). Nos résultats ont démontré des changements dans l’expression de PPARγ et la L-PGDS. En revanche, l’expression de PPARα, PPARβ et H-PGDS est restée stable au fil du temps. La diminution de l’expression de PPARγ dans le cartilage articulaire semble contribuer au développement de l’OA dans les deux modèles animaux. En effet, le traitement des chondrocytes par de siRNA dirigé contre PPARγ a favorisé la production des médiateurs arthrosiques tels que l'oxyde nitrique (NO) et la métalloprotéase matricielle de type 13 (MMP-13), confirmant ainsi le rôle anti-arthrosique de ce récepteur. Contrairement à ce dernier, le niveau d'expression de la L-PGDS a augmenté au cours de la progression de cette maladie. La surexpression de la L-PGDS au niveau des chondrocytes humains a été associée à la diminution de la production de ces médiateurs arthrosiques, suggérant son implication dans un processus de tentative de réparation. En conclusion, l’ensemble de nos résultats suggèrent que la modulation du niveau d’expression de PPARγ, de la L-PGDS et d’Egr-1 au niveau du cartilage articulaire pourrait constituer une voie thérapeutique potentielle dans le traitement de l’OA et probablement d’autres formes d'arthrite.

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

Quercetin inhibits UV irradiation-induced inflammatory cytokine production in primary human keratinocytes by suppressing NF-kappa B pathway

Background: Topical flavonoids, such as quercetin, have been shown to reduce ultraviolet (UV) irradiation-mediated skin damage. However, the mechanisms and signaling pathways involved in this protective effect are not clear. UV irradiation leads to activation of two major signaling pathways, namely nuclear factor kappa B (NF-kappa B) and activator protein-1 (AP-1) pathways. Activation of NF-kappa B pathway by UV irradiation stimulates inflammatory cytokine expression, whereas activation of AP-1 pathway by UV irradiation promotes matrix metalloproteinase (MMP) production. Both pathways contribute to UV irradiation-induced skin damage, such as photoaging and skin tumor formation. Objective: To elucidate the underlying mechanism, we examined the effect of quercetin on UV irradiation induced activation of NF-kappa B and AP-1 pathways. Methods: Primary human keratinocytes, the major skin cell type subjected to physiological solar UV irradiation, were used to study the effects of quercetin on UV irradiation-induced signal transduction pathways. Results: Quercetin decreased UV irradiation-induced NF-kappa B DNA-binding by 80%. Consequently, quercetin suppressed UV irradiation-induced expression of inflammatory cytokines IL-1 beta (similar to 60%), IL-6 (similar to 80%), IL-8 (similar to 76%) and TNF-alpha (similar to 69%). In contrast, quercetin had no effect on UV irradiation activation of three MAP kinases, ERK, JNK, or p38. Accordingly, induction of AP-1 target genes such as MMP-1 and MMP-3 by UV irradiation was not suppressed by quercetin. Conclusion: Our data indicate that the ability of quercetin to block UV irradiation-induced skin inflammation is mediated, at least in part, by its inhibitory effect on NF-kappa B activation and inflammatory cytokine production. (C) 2011 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

TLR2-dependent mast cell activation contributes to the control of Mycobacterium tuberculosis infection

Mast Cells (MCs) express toll-like receptor 2 (TLR2), a receptor known to be triggered by several major mycobacterial ligands and involved in resistance against Mycobacterium tuberculosis (MTB) infection. This study investigated whether adoptive transfer of TLR2 positive MCs (TLR2(+/+)) corrects the increased susceptibility of TLR2(-/-) mice to MTB infection. TLR2(-/-) mice displayed increased mycobacterial burden, diminished myeloid cell recruitment and proinflammatory cytokine production accompanied by defective granuloma formation. The reconstitution of these mice with TLR2(+/+) MCs, but not TLR2(-/-), confers better control of the infection, promotes the normalization of myeloid cell recruitment associated with reestablishment of the granuloma formation. In addition, adoptive transfer of TLR2(+/+) MC to TLR2(-/-) mice resulted in regulation of the pulmonary levels of IL-beta, IL-6, TNF-alpha, enhanced Th1 response and activated CD8(+) T cell homing to the lungs. Our results suggest that activation of MCs via TLR2 is required to compensate the defect in protective immunity and inability of TLR2(-/-) mice to control MTB infection. (C) 2009 Elsevier Masson SAS. All rights reserved.

A comparison of lipopolysaccharide-induced hepatitis in ethanol-fed Wistar and Lewis rats

Elevated concentrations of plasma proinflammatory cytokines have been detected in patients with alcoholic hepatitis (AH) and in a model of lipopolysaccharide-induced hepatitis in ethanol-fed Wistar rats. These cytokines have been implicated in the pathogenesis of the liver damage. Considering the likely involvement of the immune system in AH, and the frequent use of Lewis rats in autoimmune disease models, Lewis rats were examined in the model to determine whether they would more closely mimic the immune status of a chronic alcoholic and be a preferable strain for use in future experiments. Lipopolysaccharide-induced hepatic tumor necrosis factor-cu, interleukin-1 alpha, interleukin-1 beta, and interleukin-6 mRNA expression was examined in both rat strains. The overall pattern of histological (panlobular piecemeal necrosis) and biochemical liver damage (plasma ALT levels), and cytokine expression was similar in both strains. Thus, it would appear that, despite the known susceptibility of Lewis rats to autoimmune phenomena, they do not respond to the experimental regime significantly better than Wistar rats. This study confirms that unknown mediators are contributing to the liver damage seen in this model and possibly in AH.

IL-1 beta breaks tolerance through expansion of CD25(+) effector T cells

IL-1 is a key proinflammatory driver of several autoimmune diseases including juvenile inflammatory arthritis, diseases with mutations in the NALP/cryopyrin complex and Crohn's disease, and is genetically or clinically associated with many others. IL-1 is a pleiotropic proinflammatory cytokine; however the mechanisms by which increased IL-1 signaling promotes autoreactive T cell activity are not clear. Here we show that autoimmune-prone NOD and IL-1 receptor antagonist-deficient C57BL/6 mice both produce high levels of IL-1, which drives autoreactive effector cell expansion. IL-1 beta drives proliferation and cytokine production by CD4(+)CD25(+)FoxP3(-) effector/memory T cells, attenuates CD4(+)CD25(+)FoxP3(+) regulatory T cell function, and allows escape of CD4(+)CD25(-) autoreactive effectors from suppression. Thus, inflammation or constitutive overexpression of IL-1 beta in a genetically predisposed host can promote autoreactive effector T cell expansion and function, which attenuates the ability of regulatory T cells to maintain tolerance to self.

Interleukin-33 attenuates sepsis by enhancing neutrophil influx to the site of infection

Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options(1,2). Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33-treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (T(H)1) to T(H)2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection(3). Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration(4). We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors(5,6). Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis.

Interieukin-17 and Kidney Allograft Outcome

Acute rejection episodes (ARE) are important complications that involve the interplay between mechanisms that maintain graft tolerance and promote rejection. The proinflammatory cytokine interieukin-17 (IL-17) has been implicated in many conditions in humans and mice. In kidney transplant patients, the evaluation IL-17 levels has been performed in only a few patients. We performed a cross-sectional study correlating quantitative IL-17 levels and clinical outcomes. Patients and methods. We studied 19 specimens from biopsies performed in patients (n = 19) who received isolated kidney grafts. ARE signs were present in 9 (47%) patients who provide specimens; whereas, 10 (53%) others showed no signs of rejection. Eighteen healthy control sample IL-17 underwent measurement, all of which were performed by an enzyme-linked immunosorbent assay method. We assessed other factors, such as the recipients demographic data, cold ischemia time, HLA mismatches, time elapsed from transplantation to the biopsy, posttransplantation status, antibody panel, donor type, and immunosuppressive treatment. Results. IL-17 levels were clearly increased among samples derived from patients with ongoing rejection (125.7 +/- 27.06 pg/mL) in contrast, to the nonrejection group, (30 +/- 13.32 pg/mL) (P < .05). Healthy controls showed no detectable IL-17 levels. Conclusions. These findings suggested that IL-17 was important in the pathophysiology of acute kidney rejection.

Expression of TLR2 and TLR4 in lesions of patients with tegumentary american leishmaniasis

OBJECTIVES: The aim of this study was to describe the pattern of expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) in skin biopsies of patients with American tegumentary leishmaniasis (ATL) caused by Leishmania braziliensis. METHODS: This prospective study evaluated 12 patients with ATL caused by Leishmania braziliensis confirmed by polymerase chain reaction. Immunohistochemistry was performed to determine the expression of TLR2 and TLR4. The number of NK cells, dendritic cells and macrophages in the tissue were calculated. The cytokine expression was determined using the anti-TNF-α, anti-IFN-Γ, anti-IL-1 and anti-IL-6. Double immunostaining reactions were used to determine the cell expressing TLR2 and TLR4. RESULTS: The numbers of cells expressing TLR2 and TLR4 were 145.48 ± 82.46 cell/mm² and 3.26 ± 4.11 cell/mm² respectively (p < 0.05). There was no correlation of TLR2 and TLR4 with the amount of cytokines and the number of NK cells, dendritic cells or macrophages. The double immunostaining revealed that TLR2 was expressed by macrophages. CONCLUSION: In human cutaneous leishmaniasis caused by Leishmania braziliensis, TLR2 is the most common TLR expressed during active disease, mainly by macrophages although without correlation with the amount of cytokines and number of cells.

The Effect of Statins on Mortality in Septic Patients: a Meta-Analysis of Randomized Controlled Trials

OBJECTIVE: Statins are among the most prescribed drugs worldwide and their recently discovered anti-inflammatory effect seems to have an important role in inhibiting proinflammatory cytokine production, chemokines expression and counteracting the harmful effects of sepsis on the coagulation system. We decided to perform a meta-analysis of all randomized controlled trials ever published on statin therapy in septic patients to evaluate their effect on survival and length of hospital stay. DATA SOURCES AND STUDY SELECTION: Articles were assessed by four trained investigators, with divergences resolved by consensus. BioMedCentral, PubMed, Embase and the Cochrane Central Register of clinical trials were searched for pertinent studies. Inclusion criteria were random allocation to treatment and comparison of statins versus any comparator in septic patients. DATA EXTRACTION AND SYNTHESIS: Data from 650 patients in 5 randomized controlled studies were analyzed. No difference in mortality between patients receiving statins versus control (44/322 [14%] in the statins group vs 50/328 [15%] in the control arm, RR = 0.90 [95% CI 0.65 to 1.26], p = 0.6) was observed. No differences in hospital stay (p = 0.7) were found. CONCLUSIONS: Published data show that statin therapy has no effect on mortality in the overall population of adult septic patients. Scientific evidence on statins role in septic patients is still limited and larger randomized trials should be performed on this topic.

The role of IFNγ in higher brain function: in health and under chronic stress

Tese de Doutoramento em Ciências da Saúde