Impact of Leukocyte Function-Associated Antigen-1 Blockade on Endogenous Allospecific T Cells to Multiple Minor Histocompatibility Antigen Mismatched Cardiac Allograft.

BACKGROUND: Blocking leukocyte function-associated antigen (LFA)-1 in organ transplant recipients prolongs allograft survival. However, the precise mechanisms underlying the therapeutic potential of LFA-1 blockade in preventing chronic rejection are not fully elucidated. Cardiac allograft vasculopathy (CAV) is the preeminent cause of late cardiac allograft failure characterized histologically by concentric intimal hyperplasia. METHODS: Anti-LFA-1 monoclonal antibody was used in a multiple minor antigen-mismatched, BALB.B (H-2B) to C57BL/6 (H-2B), cardiac allograft model. Endogenous donor-specific CD8 T cells were tracked down using major histocompatibility complex multimers against the immunodominant H4, H7, H13, H28, and H60 minor Ags. RESULTS: The LFA-1 blockade prevented acute rejection and preserved palpable beating quality with reduced CD8 T-cell graft infiltration. Interestingly, less CD8 T cell infiltration was secondary to reduction of T-cell expansion rather than less trafficking. The LFA-1 blockade significantly suppressed the clonal expansion of minor histocompatibility antigen-specific CD8 T cells during the expansion and contraction phase. The CAV development was evaluated with morphometric analysis at postoperation day 100. The LFA-1 blockade profoundly attenuated neointimal hyperplasia (61.6 vs 23.8%; P < 0.05), CAV-affected vessel number (55.3 vs 15.9%; P < 0.05), and myocardial fibrosis (grade 3.29 vs 1.8; P < 0.05). Finally, short-term LFA-1 blockade promoted long-term donor-specific regulation, which resulted in attenuated transplant arteriosclerosis. CONCLUSIONS: Taken together, LFA-1 blockade inhibits initial endogenous alloreactive T-cell expansion and induces more regulation. Such a mechanism supports a pulse tolerance induction strategy with anti-LFA-1 rather than long-term treatment.

A new class of obesity genes encodes leukocyte adhesion receptors

Obesity is a complex disease, and multiple genes contribute to the trait. The description of five genes (ob, db, tub, Ay, and fat) responsible for distinct syndromes of spontaneous monogenic obesity in mice has advanced our knowledge of the genetics of obesity. However, many other genes involved in the expression of this disease remain to be determined. We report here the identification of an additional class of genes involved in the regulation of adipose tissue mass. These genes encode receptors mediating leukocyte adhesion. Mice deficient in intercellular adhesion molecule-1 became spontaneously obese in old age on normal mouse chow or at a young age when provided with a diet rich in fat. Mice deficient in the counterreceptor for intercellular adhesion molecule-1, the leukocyte integrin αMβ2 (Mac-1), showed a similar obesity phenotype. Since all mice consumed approximately the same amount of food as controls, the leukocyte function appears to be in regulating lipid metabolism and/or energy expenditure. Our results indicate that (i) leukocytes play a role in preventing excess body fat deposition and (ii) defects in leukocyte adhesion receptors can result in obesity.

Effects of short chain fatty acids on effector mechanisms of neutrophils

Short chain fatty acids (SCFAs) are metabolic by products of anerobic bacteria fermentation. These fatty acids, despite being an important fuel for colonocytes, are also modulators of leukocyte function. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate, and butyrate) on function of neutrophils, and the possible mechanisms involved. Neutrophils obtained from rats by intraperitoneal lavage 4 h after injection of oyster glycogen solution (1%) were treated with non toxic concentrations of the fatty acids. After that, the following measurements were performed: phagocytosis and destruction of Candida albicans, production of ROS (O(2)(center dot-), H(2)O(2), and HOCl) and degranulation. Gene expression (p47(phox) and p22(phox)) and protein phosphorylation (p47(phox)) were analyzed by real time reverse transcriptase chain reaction (RT-PCR) and Western blotting, respectively. Butyrate inhibited phagocytosis and killing of C. albicans. This SCFA also had an inhibitory effect on production of O(2)(center dot-), H(2)O(2), and HOCI by neutrophils stimulated with PMA or fMLP. This effect of butyrate was not caused by modulation of expression of NADPH oxidase subunits (p47(phox) and p22(phox)) but it was in part due to reduced levels of p47(phox) phosphorylation and an increase in the concentration of cyclic AMP. Acetate increased the production of O(2)(center dot-) and H(2)O(2), in the absence of stimuli but had no effect on phagocytosis and killing of C. albicans. Propionate had no effect on the parameters studied. These results suggest that butyrate can modulate neutrophil function, and thus could be important in inflammatory neutrophil-associated diseases. Copyright (C) 2008 John Wiley & Sons, Ltd.

Suppressive effect of short-chain fatty acids on production of proinflammatory mediators by neutrophils

Short chain fatty acids (SCFAs) are fermentation products of anaerobic bacteria. More than just being an important energy source for intestinal epithelial cells, these compounds are modulators of leukocyte function and potential targets for the development of new drugs. The aim of this study was to evaluate the effects of SCFAs (acetate, propionate and butyrate) on production of nitric oxide (NO) and proinflammatory cytokines [tumor necrosis factor alpha (TNF-alpha) and cytokine-induced neutrophil chemoattractant-2 (CINC-2 alpha beta)] by rat neutrophils. The involvement of nuclear factor kappa B (NF-kappa B) and histone deacetylase (HDAC) was examined. The effect of butyrate was also investigated in vivo after oral administration of tributyrin (a pro-drug of butyrate). Propionate and butyrate diminished TNF-alpha, CINC-2 alpha beta and NO production by LPS-stimulated neutrophils. We also observed that these fatty acids inhibit HDAC activity and NF-kappa B activation, which might be involved in the attenuation of the LPS response. Products of cyclooxygenase and 5-lipoxygenase are not involved in the effects of SCFAs as indicated by the results obtained with the inhibitors of these enzymes. The recruitment of neutrophils to the peritonium after intraperitoneal administration of a glycogen solution (1%) and the ex vivo production of cytokines and NO by neutrophils were attenuated in rats that previously received tributyrin. These results argue that this triglyceride may be effective in the treatment of inflammatory conditions. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Expression Patterns of Cell Adhesion Molecules in Mice`s Lung After Administration of Meso-2,3-Dimercaptosuccinic Acid-Coated Maghemite Nanoparticles

We studied the expression pattern of cell adhesion molecules associated to transendothelial migration of leukocytes in different lung`s vascular compartments after administration of a magnetic fluid sample containing maghemite nanoparticles surface-coated with meso-2,3-dimercaptosuccinic acid. The analyses were conducted in mice 4 and 12 h after endovenous administration of the magnetic fluid in control mice. Firstly, the migratory activity of leukocytes after magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration was confirmed using broncho-alveolar lavage and light microscopy. Then, the expression of cell adhesion molecules in the lung`s vascular compartments was investigated by immunofluorescence microscopy of frozen sections, using antibodies against L-selectin, P-selectin, E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1. L- and P-selectin showed similar pattern of expression in the pulmonary vasculature in animals treated with magnetic fluid and in the control group. In contrast, macrophage antigen-1 and leukocyte function associated antigen-1 were found in capillary only in animals treated with magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration. In addition, after magnetic fluid administration E-selectin was found in post-capillary sites. Our findings demonstrated that magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration exhibits modulation effects on expression patterns of E-selectin, macrophage antigen-1, and leukocyte function associated antigen-1 in the lung`s vascular compartments. These findings are very important in a strategy to reduce the potential toxicity of magnetic fluid surface-coated with meso-2,3-dimercaptosuccinic acid administration for medical applications.

Human eosinophil adhesion and degranulation stimulated with eotaxin and RANTES in vitro: Lack of interaction with nitric oxide

Background: Airway eosinophilia is considered a central event in the pathogenesis of asthma. The toxic components of eosinophils are thought to be important in inducing bronchial mucosal injury and dysfunction. Previous studies have suggested an interaction between nitric oxide (NO) and chemokines in modulating eosinophil functions, but this is still conflicting. In the present study, we have carried out functional assays (adhesion and degranulation) and flow cytometry analysis of adhesion molecules (VLA-4 and Mac-1 expression) to evaluate the interactions between NO and CC-chemokines (eotaxin and RANTES) in human eosinophils. Methods: Eosinophils were purified using a percoll gradient followed byimmunomagnetic cell separator. Cell adhesion and degranulation were evaluated by measuring eosinophil peroxidase (EPO) activity, whereas expression of Mac-1 and VLA-4 was detected using flow cytometry. Results: At 4 h incubation, both eotaxin (100 ng/ml) and RANTES (1000 ng/ml) increased by 133% and 131% eosinophil adhesion, respectively. L-NAME alone (but not D-NAME) also increased the eosinophil adhesion, but the co-incubation of L-NAME with eotaxin or RANTES did not further affect the increased adhesion seen with chemokines alone. In addition, L-NAME alone (but not D-NAME) caused a significant cell degranulation, but it did not affect the CC-chemokine-induced cell degranulation. Incubation of eosinophils with eotaxin or RANTES, in absence or presence of L-NAME, did not affect the expression of VLA-4 and Mac-1 on eosinophil surface. Eotaxin and RANTES (100 ng/ml each) also failed to elevate the cyclic GMP levels above baseline in human eosinophils. Conclusion: Eotaxin and RANTES increase the eosinophil adhesion to fibronectin-coated plates and promote cell degranulation by NO-independent mechanisms. The failure of CC-chemokines to affect VLA-4 and Mac-1 expression suggests that changes in integrin function (avidity or affinity) are rather involved in the enhanced adhesion. © 2008 Lintomen et al; licensee BioMed Central Ltd.

Neutrophil dysfunction varies with the stage of canine visceral leishmaniosis

Canine visceral leishmaniosis (CVL) causes a dependent-stage alteration in neutrophil oxidative metabolism. When production of reactive oxygen species (ROS) exceeds the antioxidant capacity of neutrophils, apoptosis is triggered, impairing the viability and function of these cells, which can predispose dogs to infection. However, the uremic condition observed in late-stage CVL can also alter the viability and function of human neutrophils. To more clearly understand this relationship, the apoptosis rate and oxidative metabolism of neutrophils from control dogs (n= 20) were compared to dogs in moderate (n= 15) and very severe (n= 15) stage CVL, classified according to LeishVet Consensus. To assess neutrophil oxidative metabolism, superoxide production was measured using the nitroblue tetrazolium reduction test (NBT) in isolated neutrophils. The apoptosis rate of neutrophils was estimated using the morphological method. Moderate-stage dogs presented increased superoxide production, while dogs with very severe stage CVL presented decreased superoxide production and an increase neutrophil apoptosis rate. Leishmaniosis causes differential neutrophil dysfunction according to disease stage. In moderate stage CVL, increased superoxide production is observed with no change in neutrophil viability. However, in very severe stage CVL, decreased superoxide production and increased apoptosis occur associated with uremia. © 2013 Elsevier B.V.

Estresse e imuno modulação por beta-glucano em Pacu (Piaractus mesopotamicus)

Pós-graduação em Aquicultura - FCAV

Valor do teste de redução do Tetrazólio Nitroazul (NBT) no diagnóstico de processos inflamatórios caninos

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

Kindlin-3 regulates integrin activation and adhesion reinforcement of effector T cells

Activated T cells use very late antigen-4/α4β1 integrin for capture, rolling on, and firm adhesion to endothelial cells, and use leukocyte function-associated antigen-1/αLβ2 integrin for subsequent crawling and extravasation. Inhibition of α4β1 is sufficient to prevent extravasation of activated T cells and is successfully used to combat autoimmune diseases, such as multiple sclerosis. Here we show that effector T cells lacking the integrin activator Kindlin-3 extravasate and induce experimental autoimmune encephalomyelitis in mice immunized with autoantigen. In sharp contrast, adoptively transferred autoreactive T cells from Kindlin-3-deficient mice fail to extravasate into the naïve CNS. Mechanistically, autoreactive Kindlin-3-null T cells extravasate when the CNS is inflamed and the brain microvasculature expresses high levels of integrin ligands. Flow chamber assays under physiological shear conditions confirmed that Kindlin-3-null effector T cells adhere to high concentrations of vascular cell adhesion molecule-1 and intercellular adhesion molecule-1, albeit less efficiently than WT T cells. Although these arrested T cells polarize and start crawling, only few remain firmly adherent over time. Our data demonstrate that the requirement of Kindlin-3 for effector T cells to induce α4β1 and αLβ2 integrin ligand binding and stabilization of integrin-ligand bonds is critical when integrin ligand levels are low, but of less importance when integrin ligand levels are high.

The structure of the two amino-terminal domains of human ICAM-1 suggests how it functions as a rhinovirus receptor and as an LFA-1 integrin ligand

The normal function of human intercellular adhesion molecule-1 (ICAM-1) is to provide adhesion between endothelial cells and leukocytes after injury or stress. ICAM-1 binds to leukocyte function-associated antigen (LFA-1) or macrophage-1 antigen (Mac-1). However, ICAM-1 is also used as a receptor by the major group of human rhinoviruses and is a catalyst for the subsequent viral uncoating during cell entry. The three-dimensional atomic structure of the two amino-terminal domains (D1 and D2) of ICAM-1 has been determined to 2.2-Å resolution and fitted into a cryoelectron microscopy reconstruction of a rhinovirus–ICAM-1 complex. Rhinovirus attachment is confined to the BC, CD, DE, and FG loops of the amino-terminal Ig-like domain (D1) at the end distal to the cellular membrane. The loops are considerably different in structure to those of human ICAM-2 or murine ICAM-1, which do not bind rhinoviruses. There are extensive charge interactions between ICAM-1 and human rhinoviruses, which are mostly conserved in both major and minor receptor groups of rhinoviruses. The interaction of ICAMs with LFA-1 is known to be mediated by a divalent cation bound to the insertion (I)-domain on the α chain of LFA-1 and the carboxyl group of a conserved glutamic acid residue on ICAMs. Domain D1 has been docked with the known structure of the I-domain. The resultant model is consistent with mutational data and provides a structural framework for the adhesion between these molecules.

A mutant cholera toxin B subunit that binds GM1- ganglioside but lacks immunomodulatory or toxic activity

GM1-ganglioside receptor binding by the B subunit of cholera toxin (CtxB) is widely accepted to initiate toxin action by triggering uptake and delivery of the toxin A subunit into cells. More recently, GM1 binding by isolated CtxB, or the related B subunit of Escherichia coli heat-labile enterotoxin (EtxB), has been found to modulate leukocyte function, resulting in the down-regulation of proinflammatory immune responses that cause autoimmune disorders such as rheumatoid arthritis and diabetes. Here, we demonstrate that GM1 binding, contrary to expectation, is not sufficient to initiate toxin action. We report the engineering and crystallographic structure of a mutant cholera toxin, with a His to Ala substitution in the B subunit at position 57. Whereas the mutant retained pentameric stability and high affinity binding to GM1-ganglioside, it had lost its immunomodulatory activity and, when part of the holotoxin complex, exhibited ablated toxicity. The implications of these findings on the mode of action of cholera toxin are discussed.

Early-onset multifocal inflammation in the transforming growth factor beta 1-null mouse is lymphocyte mediated.

Transforming growth factor beta 1 (TGF beta 1)-null mice die fro complications due to an early-onset multifocal inflammatory disorder. We show here that cardiac cells are hyperproliferative and that intercellular adhesion molecule 1 (ICAM-1) is elevated. To determine which phenotypes are primarily caused by a deficiency in TGF beta 1 from those that are secondary to inflammation, we applied immunosuppressive therapy and genetic combination with the severe combined immunodeficiency (SCID) mutation to inhibit the inflammatory response. Treatment with antibodies to the leukocyte function-associated antigen 1 doubled longevity, reduced inflammation, and delayed heart cell proliferation. TGF beta 1-null SCID mice displayed no inflammation or cardiac cell proliferation, survived to adulthood, and exhibited normal major histocompatibility complex II (MHC II) and ICAM-1 levels. TGF beta 1-null pups born to a TGF beta 1-null SCID mother presented no gross congenital heart defects, indicating that TGF beta 1 alone does not play an essential role in heart development. These results indicate that lymphocytes are essential for the inflammatory response, cardiac cell proliferation, and elevated MHC II and ICAM-1 expression, revealing a vital role for TGF beta 1 in regulating lymphocyte proliferation and activation, which contribute to the maintenance of self tolerance.

THE IMPORTANCE OF MHC-INDEPENDENT NATURAL KILLER CELL RECEPTORS IN THE IMMUNOREGULATION OF MURINE PREGNANCY

Numerous leukocyte populations are essential for pregnancy success. Uterine natural killer (uNK) cells are chief amongst these leukocytes and represent a unique lineage with limited cytotoxicity but abundant angiokine production. They possess a distinct phenotype of activating and inhibitory receptors that recognize major histocompatibility complex (MHC) molecules, such as the killer immunoglobulin like receptors (KIRs; mouse Ly49), and MHC-independent activating receptors, including the aryl hydrocarbon receptor (AHR) and natural cytotoxicity receptor 1 (NCR1). While the roles of MHC-dependent receptors are widely addressed in pregnancy, MHC-independent receptors are relatively unstudied. This thesis investigated the roles of MHC-independent receptors in promotion of mouse pregnancy and characterized early leukocyte interactions in the presence and absence of NCR1. It was hypothesized that loss of MHC-independent receptors impairs uNK cell development resulting in aberrations in leukocyte function and decidual vasculature. Implantation sites from Ahr-/- and Ncr1Gfp/Gfp mice were assessed using whole mount in situ immunohistochemistry (WM-IHC) and histochemical techniques. Leukocyte interactions identified during preliminary WM-IHC studies were confirmed as immune synapses. The novel identification of immune synapses in early mouse pregnancy compelled further examination of leukocyte conjugates in wildtype C57BL/6 and Ncr1Gfp/Gfp mice. In Ahr-/- and Ncr1Gfp/Gfp mice, receptor loss resulted in reduced uNK cell diameters, impaired decidual vasculature, and failures in spiral artery remodeling. Ahr-/- mice had severe fertility deficits whereas Ncr1Gfp/Gfp mice had increased fetal resorption indicating differing receptor requirements in pregnancy success. NCR1 loss primarily affected uNK cell maturation and function as identified by alterations in granule ultrastructure, lytic protein expression, and angiokine production. Leukocyte conjugates were frequent in early C57BL/6 decidua basalis and included uNK cells conjugating first with antigen presenting cells and then with T cells. Overall conjugate formation was reduced in the absence of NCR1, but specific uNK cell conjugations were unaffected by receptor loss. While KIR-MHC interactions are associated with numerous pregnancy complications in humans, the role of other uNK cell receptors are not well characterized. These results illustrate the importance of MHC-independent receptors in uNK cell activation during early pregnancy in mice and encourage further studies of pregnancy complications that may occur independently of maternal KIR-MHC contributions.