The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

Murine eotaxin: an eosinophil chemoattractant inducible in endothelial cells and in interleukin 4-induced tumor suppression.

Guinea pig eotaxin is a recently described member of the Cys-Cys family of chemokines and is involved in a guinea pig model of asthma. To determine whether eotaxin is a distinctive member of this family and to understand its physiologic role, we have cloned the mouse eotaxin gene and determined its structure and aspects of its biologic function. The sequence relationship between the mouse and guinea pig genes indicates that eotaxin is indeed a distinct member of the chemokine family. Moreover, murine eotaxin maps to a region of mouse chromosome 11 that encodes other Cys-Cys chemokines. In addition, recombinant murine eotaxin protein has direct chemoattractant properties for eosinophils. The eotaxin gene is widely (but not ubiquitously) expressed in normal mice and is strongly induced in cultured endothelial cells in response to interferon gamma. Eotaxin is also induced locally in response to the transplantation of interleukin 4-secreting tumor cells, indicating that it likely contributes to the eosinophil recruitment and antitumor effect of interleukin 4. Such responses suggest that eotaxin may be involved in multiple inflammatory states.

Transendothelial migration of neutrophils involves integrin-associated protein (CD47).

Inflammation is a primary pathological process. The development of an inflammatory reaction involves the movement of white blood cells through the endothelial lining of blood vessels into tissues. This process of transendothelial cell migration of neutrophils has been shown to involve neutrophil beta 2 integrins (CD18) and endothelial cell platelet-endothelium cell adhesion molecules (PECAM-1; CD31). We now show that F(ab')2 fragments of the monoclonal antibody B6H12 against integrin-associated protein (IAP) blocks the transendothelial migration of neutrophils stimulated by an exogenous gradient of the chemokine interleukin 8 (IL-8; 60% inhibition), by the chemotactic peptide N-formyl-methionylleucylphenylalanine (FMLP; 76% inhibition), or by the activation of the endothelium by the cytokine tumor necrosis factor alpha (98% inhibition). The antibody has two mechanisms of action: on neutrophils it prevents the chemotactic response to IL-8 and FMLP, and on endothelium it prevents an unknown but IL-8-independent process. Blocking antibodies to IAP do not alter the expression of adhesion proteins or production of IL-8 by endothelial cells, and thus the inhibition of neutrophil transendothelial migration is selective. These data implicate IAP as the third molecule essential for neutrophil migration through endothelium into sites of inflammation.

Participação do nicho endosteal na regulação da hemopoese de camundongos submetidos à desnutrição proteica

O nicho endosteal da medula óssea abriga as células-tronco hemopoéticas (CTH) em quiescência/autorrenovação. As CTH podem ser classificadas em dois grupos: células que reconstituem a hemopoese em longo prazo (LT-CTH) e curto prazo (CT-CTH). Investigamos, neste trabalho, os efeitos da desnutrição proteica (DP) no tecido ósseo e a participação do nicho endosteal na sinalização osteoblasto-CTH. Para tanto, utilizamos camundongos submetidos à DP induzida pelo consumo de ração hipoproteica. Os animais desnutridos apresentaram pancitopenia e diminuição nas concentrações de proteínas séricas e albumina. Quantificamos as CTH por citometria de fluxo e verificamos que os desnutridos apresentaram menor porcentagem de LT-CTH, CT-CTH e de progenitores multipotentes (PMP). Avaliamos a expressão das proteínas CD44, CXCR4, Tie-2 e Notch-1 nas LT-CTH. Observamos diminuição da expressão da proteína CD44 nos desnutridos. Isolamos as células LT-CTH por cell sorting e avaliamos a expressão gênica de CD44, CXCR4 e NOTCH-1. Verificamos que os desnutridos apresentaram menor expressão de CD44. Em relação ao ciclo celular, verificamos maior quantidade de LT-CTH nas fases G0/G1. Caracterizamos as alterações do tecido ósseo femoral, in vivo. Observamos diminuição da densidade mineral óssea e da densidade medular nos desnutridos. A desnutrição acarretou diminuição da área média das seções transversais, do perímetro do periósteo e do endósteo na cortical do fêmur dos animais. E na região trabecular, verificou-se diminuição da razão entre volume ósseo e volume da amostra e do número de trabéculas, aumento da distância entre as trabéculas e prevalência de trabéculas ósseas em formato cilíndrico. Avaliamos a expressão de colágeno, osteonectina (ON) e osteocalcina (OC) por imuno-histoquímica, e de osteopontina (OPN) por imunofluorescência no fêmur e verificamos diminuição da marcação para OPN, colágeno tipo I, OC e ON nos desnutridos. Evidenciamos, pela técnica do Picrosírius, desorganização na distribuição das fibras colágenas e presença de fibras tipo III nos fêmures dos desnutridos, além de maior número de osteoclastos evidenciados pela reação da fosfatase ácida tartarato resistente. Os osteoblastos da região femoral foram isolados por depleção imunomagnética, imunofenotipados por citometria de fluxo e cultivados em meio de indução osteogênica. Observamos menor positividade para fosfatase alcalina e vermelho de alizarina nas culturas dos osteoblastos dos desnutridos. Avaliamos, por Western Blotting, a expressão de colágeno tipo I, OPN, osterix, Runx2, RANKL e osteoprotegerina (OPG), e, por PCR em tempo real, a expressão de COL1A2, SP7, CXCL12, ANGPT1, SPP1, JAG2 e CDH2 nos osteoblastos isolados. Verificamos que a desnutrição acarretou diminuição da expressão proteica de osterix e OPG e menor expressão gênica de ANGPT1. Avaliamos a proliferação das células LSK (Lin-Sca1+c-Kit+) utilizando ensaio de CFSE (carboxifluoresceína succinimidil ester). Foi realizada cocultura de células LSK e osteoblastos (MC3T3-E1) na presença e ausência de anti-CD44. Após uma semana, verificamos menor proliferação das LSK dos desnutridos. O bloqueio de CD44 das LSK do grupo controle diminuiu a proliferação destas em três gerações. Entretanto, nos desnutridos, esse bloqueio não afetou a proliferação. Concluímos que a DP promoveu alterações no tecido ósseo e nas CTH. Entretanto, não podemos afirmar que as alterações observadas no sistema hemopoético foram decorrentes de alterações exclusivas do nicho endosteal.

Inhibition of NF-kB activation and cytokines production in THP-1 monocytes by 2-styrylchromones

Nuclear factor kappa B (NF-kB) is one of the most important transcription factors whose modulation triggers a cascade of signaling events, namely the expression of many cytokines, enzymes, chemokines, and adhesion molecules, some of which being potential key targets for intervention in the treatment of inflammatory conditions. The 2-styrylchromones (2-SC) designation represents a well-recognized group of natural and synthetic chromones, vinylogues of flavones (2-phenylchromones). Several 2-SC were recently tested for their anti-inflammatory potential, regarding the arachidonic acid metabolic cascade, showing some motivating results. In addition, several flavones with structural similarities to 2-SC have shown NF-kB inhibitory properties. Hence, the aim of the present work was to continue the investigation on the interference of 2-SC in inflammatory pathways. Herein we report their effects on lipopolysaccharide (LPS)-induced NF-kB activation and consequent production of proinflammatory cytokines/chemokine, using a human monocytic cell line (THP-1). From the twelve 2-SC tested, three of them were able to significantly inhibit the NF-kB activation and to reduce the production of the proinflammatory cytokines/chemokine. The compound 3',4',5-trihydroxy-2- styrylchromone stood up as the most active in both assays, being a promising candidate for an anti-inflammatory drug.

Multifaceted Role of Heme during Severe Plasmodium falciparum Infections in India

Several immunomodulatory factors are involved in malaria pathogenesis. Among them, heme has been shown to play a role in the pathophysiology of severe malaria in rodents, but its role in human severe malaria remains unclear. Circulating levels of total heme and its main scavenger, hemopexin, along with cytokine/chemokine levels and biological parameters, including hemoglobin and creatinine levels, as well as transaminase activities, were measured in the plasma of 237 Plasmodium falciparum-infected patients living in the state of Odisha, India, where malaria is endemic. All patients were categorized into well-defined groups of mild malaria, cerebral malaria (CM), or severe noncerebral malaria, which included acute renal failure (ARF) and hepatopathy. Our results show a significant increase in total plasma heme levels with malaria severity, especially for CM and malarial ARF. Spearman rank correlation and canonical correlation analyses have shown a correlation between total heme, hemopexin, interleukin-10, tumor necrosis factor alpha, gamma interferon-induced protein 10 (IP-10), and monocyte chemotactic protein 1 (MCP-1) levels. In addition, canonical correlations revealed that heme, along with IP-10, was associated with the CM pathophysiology, whereas both IP-10 and MCP-1 together with heme discriminated ARF. Altogether, our data indicate that heme, in association with cytokines and chemokines, is involved in the pathophysiology of both CM and ARF but through different mechanisms.

CCR5-Delta 32 mutation is strongly associated with primary sclerosing cholangitis

CCR5 plays a key role in the distribution of CD45RO+ T cells and contributes to generation of a T helper 1 immune response. CCR5-Delta32 is a 32-bp deletion associated with significant reduction in cell surface expression of the receptor. We investigated the role of CCR5-Delta32 on susceptibility to ulcerative colitis (UC), Crohn's disease ( CD) and primary sclerosing cholangitis (PSC). Genotype and allelic association analyses were performed in 162 patients with UC, 131 with CD, 71 with PSC and 419 matched controls. There was a significant difference in CCR5 genotype (OR 2.27, P = 0.003) between patients with sclerosing cholangitis and controls. Similarly, CCR5-Delta32 allele frequency was significantly higher in sclerosing cholangitis (17.6%) compared to controls (9.9%, OR 2.47, P = 0.007) and inflammatory bowel disease patients without sclerosing cholangitis ( 11.3%, OR 1.9, P = 0.027). There were no significant differences in CCR5 genotype or allele frequency between those with either UC or CD and controls. Genotypes with the CCR5-Delta32 variant were increased in patients with severe liver disease defined by portal hypertension and/or transplantation (45%) compared to those with mild liver disease (21%, OR 3.17, P = 0.03). The CCR5-Delta32 mutation may influence disease susceptibility and severity in patients with PSC.

Virus-specific CD8+ T lymphocytes within the normal human liver

The frequency and phenotype of human antiviral memory CD8(+) T cells in blood are well studied, yet little is known about their distribution within tissues. Analysis of antiviral CD8(+) T cell populations derived from a unique set of normal liver and blood samples identified a consistent population of virus-specific cells within the liver. In comparison to the circulating T cells, the liver-derived T cells were present at frequencies which were variably enriched compared to that in the blood, and showed significant differences with regard to the expression of CD45RA, CD45RO, CD95, CCR7, CD27 and CD28. The differences in these cell surface markers are consistent with a mature 'effector memory' phenotype of antigen-specific CD8(+) T cells within the liver. An enrichment of an activated subset of NKT cells (Valpha24/Vbeta11) was also observed, a finding which may be relevant to the regulation of the antiviral population:.

Characterization of heat shock protein-specific T cells in atherosclerosis

A role for infection and inflammation in atherogenesis is widely accepted. Arterial endothelium has been shown to express heat shock protein 60 (HSP60) and, since human (hHSP60) and bacterial (GroEL) HSP60s are highly conserved, the immune response to bacteria may result in cross-reactivity, leading to endothelial damage and thus contribute to the pathogenesis of atherosclerosis. In this study, GroEL-specific T-cell lines from peripheral blood and GroEL-, hHSP60-, and Porphyromonas gingivalis-specific T-cell lines from atherosclerotic plaques were established and characterized in terms of their cross-reactive proliferative responses, cytokine and chemokine profiles, and T-cell receptor (TCR) V beta expression by flow cytometry. The cross-reactivity of several lines was demonstrated. The cytokine profiles of the artery T-cell lines specific for GroEL, hHSP60, and P. gingivalis demonstrated Th2 phenotype predominance in the CD4 subset and Tc0 phenotype predominance in the CD8 subset. A higher proportion of CD4 cells were positive for interferon-inducible protein 10 and RANTES, with low percentages of cells positive for monocyte chemoattractant protein 1 and macrophage inflammatory protein la, whereas a high percentage of CD8 cells expressed all four chemokines. Finally, there was overexpression of the TCR V beta 5.2 family in all lines. These cytokine, chemokine, and V beta profiles are similar to those demonstrated previously for P. gingivalis-specific lines established from periodontal disease patients. These results support the hypothesis that in some patients cross-reactivity of the immune response to bacterial HSPs, including those of periodontal pathogens, with arterial endothelial cells expressing hHSP60 may explain the apparent association between atherosclerosis and periodontal infection.

Transcription factor Tfec contributes to the IL-4-inducible expression of a small group of genes in mouse macrophages including the granulocyte colony-stimulating factor receptor

Expression of the mouse transcription factor EC (Tfec) is restricted to the myeloid compartment, suggesting a function for Tfec in the development or function of these cells. However, mice lacking Tfec develop normally, indicating a redundant role for Tfec in myeloid cell development. We now report that Tfec is specifically induced in bone marrow-derived macrophages upon stimulation with the Th2 cytokines, IL-4 and IL-13, or LPS. LPS induced a rapid and transient up-regulation of Tfec mRNA expression and promoter activity, which was dependent on a functional NF-kappa B site. IL-4, however, induced a rapid, but long-lasting, increase in Tfec mRNA, which, in contrast to LPS stimulation, also resulted in detectable levels of Tfec protein. IL-4-induced transcription of Tfec was absent in macrophages lacking Stat6, and its promoter depended on two functional Stat6-binding sites. A global comparison of IL-4-induced genes in both wild-type and Tfec mutant macrophages revealed a surprisingly mild phenotype with only a few genes affected by Tfec deficiency. These included the G-CSFR (Csf3r) gene that was strongly up-regulated by IL-4 in wild-type macrophages and, to a lesser extent, in Tfec mutant macrophages. Our study also provides a general definition of the transcriptome in alternatively activated mouse macrophages and identifies a large number of novel genes characterizing this cell type.

CXCR4/SDF1 interaction inhibits the primordial to primary follicle transition in the neonatal mouse ovary

The molecular mechanisms behind the entry of the primordial follicle into the growing follicle pool remain poorly understood. To investigate this process further, a microarray-based comparison was undertaken between 2-day postpartum mouse ovaries consisting of primordial follicles/naked oocytes only and those with both primordial follicles and newly activated follicles (7-day postpartum). Gene candidates identified included the chemoattractive cytokine stromal derived factor-1 (SDF1) and its receptor CXCR4. SDF1 and CXCR4 have been implicated in a variety of physiological processes including the migration of embryonic germ cells to the gonads. SDF1-alpha expression increased with the developmental stage of the follicle. Embryonic expression was found to be dichotomous post-genii cell migration, with low expression in the female. Immunohistochemical studies nonetheless indicate that the autocrine pattern of expression ligand and receptor begins during embryonic life. Addition of recombinant SDF1-alpha to neonatal mouse ovaries in vitro resulted in significantly higher follicle densities than for control ovaries. TUNEL analysis indicated no detectable difference in populations of apoptotic cells of treated or control ovaries. Treated ovaries also contained a significantly lower percentage of activated follicles as determined by measurement of oocyte diameter and morphological analysis. Treatment of cultured ovaries with an inhibitor of SDF1-alpha, AMD3100, ablated the effect of SDF1-alpha. By retaining follicles in an unactivated state, SDF1/CXCR4 signaling may play an important role in maintaining the size and longevity of the primordial follicle pool. (c) 2006 Elsevier Inc. All rights reserved.

Toward the discovery of vaccine adjuvants:coupling in silico screening and in vitro analysis of antagonist binding to human and mouse CCR4 receptors

Background Adjuvants enhance or modify an immune response that is made to an antigen. An antagonist of the chemokine CCR4 receptor can display adjuvant-like properties by diminishing the ability of CD4+CD25+ regulatory T cells (Tregs) to down-regulate immune responses. Methodology Here, we have used protein modelling to create a plausible chemokine receptor model with the aim of using virtual screening to identify potential small molecule chemokine antagonists. A combination of homology modelling and molecular docking was used to create a model of the CCR4 receptor in order to investigate potential lead compounds that display antagonistic properties. Three-dimensional structure-based virtual screening of the CCR4 receptor identified 116 small molecules that were calculated to have a high affinity for the receptor; these were tested experimentally for CCR4 antagonism. Fifteen of these small molecules were shown to inhibit specifically CCR4-mediated cell migration, including that of CCR4+ Tregs. Significance Our CCR4 antagonists act as adjuvants augmenting human T cell proliferation in an in vitro immune response model and compound SP50 increases T cell and antibody responses in vivo when combined with vaccine antigens of Mycobacterium tuberculosis and Plasmodium yoelii in mice.

Gingipains from Porphyromonas gingivalis increase the chemotactic and respiratory burst-priming properties of the 77-amino-acid interleukin-8 variant

Porphyromonas gingivalis, a gram-negative anaerobe which is implicated in the etiology of active periodontitis, secretes degradative enzymes (gingipains) and sheds proinflammatory mediators (e.g., lipopolysaccharides [LPS]). LPS triggers the secretion of interleukin-8 (IL-8) from immune (72-amino-acid [aa] variant [IL-8(72aa)]) and nonimmune (IL-8(77aa)) cells. IL-8(77aa) has low chemotactic and respiratory burst-inducing activity but is susceptible to cleavage by gingipains. This study shows that both R- and K-gingipain treatments of IL-8(77aa) significantly enhance burst activation by fMLP and chemotactic activity (P < 0.05) but decrease burst activation and chemotactic activity of IL-8(72aa) toward neutrophil-like HL60 cells and primary neutrophils (P < 0.05). Using tandem mass spectrometry, we have demonstrated that R-gingipain cleaves 5- and 11-aa peptides from the N-terminal portion of IL-8(77aa) and the resultant peptides are biologically active, while K-gingipain removes an 8-aa N-terminal peptide yielding a 69-aa isoform of IL-8 that shows enhanced biological activity. During periodontitis, secreted gingipains may differentially affect neutrophil chemotaxis and activation in response to IL-8 according to the cellular source of the chemokine.

Mechanisms contributing to the enhanced respiratory burst of neutrophils observed in periodontitis

The aim of this thesis is to investigate possible mechanisms that may contribute to neutrophil hyperactivity and hyper-reactivity. One possibility is the presence of a neutrophil priming factors within the peripheral circulation of periodontitis patients. To examine this possibility differentiated HL-60 cells and primary neutrophils were studied in the presence and absence of plasma from periodontitis patients. In independent experiments, plasma was depleted of IL-8, GM-CSF, interferon-a, immunoglobulins and albumin. This work demonstrated that plasma factors such as IL-8, GM-CSF, and interferon-a present during periodontitis may contribute towards the reported hyperactive neutrophil phenotype. Furthermore, this work demonstrated that products from Pg may regulate neutrophil accumulation at infected periodontal sites by promoting gingipain-dependent modification of IL-8-77 into a more biologically active chemokine. To elucidate whether the oxidatively stressed environment that neutrophils are exposed to in periodontitis could influence hyperactivity and hyper-reactivity, neutrophils were depleted of glutathione. This work showed that during oxidative stress, where cellular redox-levels have been altered, neutrophils exhibit an increased respiratory burst. In conclusion, this work highlights the multiple mechanisms that may contribute to neutrophil hyperactivity and hyperreactivity including gingipain-modulated activity of IL-8 variants, the effect of host factors such as IL-8, GM-CSF, interferon-a on neutrophils priming and activation, and the shift of neutrophil GSH:GSSG ratio in favour of a more oxidised environment as observed in periodontitis.

The role of apoptotic cell clearance in a high-lipid environment:links to ageing

Individuals within the aged population show an increased susceptibility to infection, implying a decline in immune function, a phenomenon known as immunosenescence. Paradoxically, an increase in autoimmune disease, such as rheumatoid arthritis, is also associated with ageing, therefore some aspects of the immune system appear to be inappropriately active in the elderly. The above evidence suggests inappropriate control of the immune system as we age. Macrophages, and their precursors monocytes, play a key role in control of the immune system. They play an important role in host defence in the form of phagocytosis, and also link the innate and adaptive immune system via antigen presentation. Macrophages also have a reparative role, as professional phagocytes of dead and dying cells. Clearance of apoptotic cells by macrophages has also been shown to directly influence immune responses in an anti-inflammatory manner. Inappropriate control of macrophage function with regards to dead cell clearance may contribute to pathology as we age. The aims of this study were to assess the impact of lipid treatment, as a model of the aged environment, on the ability of macrophages to interact with, and respond to, apoptotic cells. Using a series of in vitro cell models, responses of macrophages (normal and lipid-loaded) to apoptotic macrophages (normal and lipid-loaded) were investigated. Monocyte recruitment to apoptotic cells, a key process in resolving inflammation, was assessed in addition to cytokine responses. Data here shows, for the first time, that apoptotic macrophages (normal and lipid-loaded) induce inflammation in human monocyte-derived macrophages, a response that could drive inflammation in age-associated pathology e.g. atherosclerosis. Monoclonal antibody inhibition studies suggest the classical chemokine CX3CL1 may be involved in monocyte recruitment to apoptotic macrophages, but not apoptotic foam cells, therefore differential clearance strategies may be employed following lipid-loading. CD14, an important apoptotic cell tethering receptor, was not found to have a prominent role in this process, whilst the role for ICAM-3 remains unclear. Additionally, a small pilot study using macrophages from young (<25) and mid-life (>40) donors was undertaken. Preliminary data was gathered to assess the ability of primary human monocyte-derived macrophages, from young and mid-life donors, to interact with, and respond to, apoptotic cells. MØ from mid-life individuals showed no significant differences in their ability to respond to immune modulation by apoptotic cells compared to MØ from young donors. Larger cohorts would be required to investigate whether immune modulation of MØ by apoptotic cells contribute to inflammatory pathology throughout ageing.