CD1d-restricted natural killer T (NKT) cells, expressing the invariant T cell antigen receptor (TCR) chain encoded by Valpha14-Jalpha18 gene segments in mice and Valpha24-Jalpha18 in humans [invariant NKT (iNKT) cells], contribute to immunoregulatory processes, such as tolerance, host defense, and tumor surveillance. iNKT cells are positively selected in the thymus by CD1d molecules expressed by CD4(+)/CD8(+) cortical thymocytes. However, the identity of the endogenous lipid(s) responsible for positive selection of iNKT cells remains unclear. One candidate lipid proposed to play a role in positive selection is isoglobotrihexosylceramide (iGb3). However, no direct evidence for its physiological role has been provided. Therefore, to directly investigate the role of iGb3 in iNKT cell selection, we have generated mice deficient in iGb3 synthase [iGb3S, also known as alpha1-3galactosyltransferase 2 (A3galt2)]. These mice developed, grew, and reproduced normally and exhibited no overt behavioral abnormalities. Consistent with the notion that iGb3 is synthesized only by iGb3S, lack of iGb3 in the dorsal root ganglia of iGb3S-deficient mice (iGb3S(-/-)), as compared with iGb3S(+/-) mice, was confirmed. iGb3S(-/-) mice showed normal numbers of iNKT cells in the thymus, spleen, and liver with selected TCR Vbeta chains identical to controls. Upon administration of alpha-galactosylceramide, activation of iNKT and dendritic cells was similar in iGb3S(-/-) and iGb3S(+/-) mice, as measured by up-regulation of CD69 as well as intracellular IL-4 and IFN-gamma in iNKT cells, up-regulation of CD86 on dendritic cells, and rise in serum concentrations of IL-4, IL-6, IL-10, IL-12p70, IFN-gamma, TNF-alpha, and Ccl2/MCP-1. Our results strongly suggest that iGb3 is unlikely to be an endogenous CD1d lipid ligand determining thymic iNKT selection.

Prostate cancer continues to be the most common nonskin cancer diagnosed and the second leading cause of cancer death in men in the United States. Prostate cancer that has metastasized to bone remains incurable. The interactions between prostate cancer cells and the various cells of the host microenvironment result in enhanced growth of tumor cells and activation of host cells that together culminate in osteoblastic bone metastases. These dynamic tumor-host interactions are mediated by cancer and host-produced cytokines and chemokines. Among them, chemokine (C-C motif) ligand 2 (CCL2) has been identified as a prominent modulator of metastatic growth in the bone microenvironment. CCL2 is produced by bone marrow osteoblasts, endothelial cells, stromal cells, and prostate cancer cells. It has been demonstrated to modulate tumor-associated macrophage migration and promote osteoclast maturation. In addition, CCL2 functions through binding to its receptor CCR2 to induce prostate cell proliferation, migration, and invasion in both autocrine and paracrine manners. CCL2 protects prostate cancer cells from autophagic death by activating survivin through a PI3K/AKT (phosphatidylinositol 3-kinase/protein kinase B)-dependent mechanism. Inhibition of CCL2 substantially decreases macrophage infiltration, decreases osteoclast function, and inhibits prostate cancer growth in bone in preclinical animal models. The multiple roles of CCL2 in the tumor microenvironment make it an attractive therapeutic target in metastatic prostate cancer as well as in other cancers.

Aluminum hydroxide (alum) and the oil-in-water emulsion MF59 are widely used, safe and effective adjuvants, yet their mechanism of action is poorly understood. We assessed the effects of alum and MF59 on human immune cells and found that both induce secretion of chemokines, such as CCL2 (MCP-1), CCL3 (MIP-1alpha), CCL4 (MIP-1beta), and CXCL8 (IL-8), all involved in cell recruitment from blood into peripheral tissue. Alum appears to act mainly on macrophages and monocytes, whereas MF59 additionally targets granulocytes. Accordingly, monocytes and granulocytes migrate toward MF59-conditioned culture supernatants. In monocytes, both adjuvants lead to increased endocytosis, enhanced surface expression of MHC class II and CD86, and down-regulation of the monocyte marker CD14, which are all phenotypic changes consistent with a differentiation toward dendritic cells (DCs). When monocyte differentiation into DCs is induced by addition of cytokines, these adjuvants enhanced the acquisition of a mature DC phenotype and lead to an earlier and higher expression of MHC class II and CD86. In addition, MF59 induces further up-regulation of the maturation marker CD83 and the lymph node-homing receptor CCR7 on differentiating monocytes. Alum induces a similar but not identical pattern that clearly differs from the response to LPS. This model suggests a common adjuvant mechanism that is distinct from that mediated by danger signals. We conclude that during vaccination, adjuvants such as MF59 may increase recruitment of immune cells into the injection site, accelerate and enhance monocyte differentiation into DCs, augment Ag uptake, and facilitate migration of DCs into tissue-draining lymph nodes to prime adaptive immune responses.

OBJECTIVE

Hepatocellular carcinoma (HCC) is a heterogeneous disease with poor prognosis and limited methods for predicting patient survival. The nature of the immune cells that infiltrate tumours is known to impact clinical outcome. However, the molecular events that regulate this infiltration require further understanding. Here the ability of immune genes expressed in the tumour microenvironment to predict disease progression was investigated.

METHODS

Using quantitative PCR, the expression of 14 immune genes in resected tumour tissues from 57 Singaporean patients was analysed. The nearest-template prediction method was used to derive and test a prognostic signature from this training cohort. The signature was then validated in an independent cohort of 98 patients from Hong Kong and Zurich. Intratumoural components expressing these critical immune genes were identified by in situ labelling. Regulation of these genes was analysed in vitro using the HCC cell line SNU-182.

RESULTS

The identified 14 immune-gene signature predicts patient survival in both the training cohort (p=0.0004 and HR=5.2) and the validation cohort (p=0.0051 and HR=2.5) irrespective of patient ethnicity and disease aetiology. Importantly, it predicts the survival of patients with early disease (stages I and II), for whom classical clinical parameters provide limited information. The lack of predictive power in late disease stages III and IV emphasises that a protective immune microenvironment has to be established early in order to impact disease progression significantly. This signature includes the chemokine genes CXCL10, CCL5 and CCL2, whose expression correlates with markers of T helper 1 (Th1), CD8(+) T and natural killer (NK) cells. Inflammatory cytokines (tumour necrosis factor α, interferon γ) and Toll-like receptor 3 ligands stimulate intratumoural production of these chemokines which drive tumour infiltration by T and NK cells, leading to enhanced cancer cell death.

CONCLUSIONS

A 14 immune-gene signature, which identifies molecular cues driving tumour infiltration by lymphocytes, accurately predicts survival of patients with HCC especially in early disease.

Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non-cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.

Macrophage recruitment to adipose tissue in obesity contributes to enhanced adipose tissue inflammatory activity and thus may underlie obesity-associated metabolic dysfunction. Obese adipose tissue exhibits increases in CC chemokine ligand 2 (CCL2, or monocyte chemoattractant protein-1), an important macrophage-recruiting factor. We therefore hypothesized that elevated CCL2 may contribute to obesity-associated adipose tissue macrophage recruitment. Male 6-week-old CCL2(-/-) and wild-type mice (n = 11-14 per group) were fed standard and high-fat diets until 34 weeks of age. At 12-16 and 25-29 weeks of age, blood was collected for plasma glucose and hormone measurements, and glucose tolerance and insulin tolerance tests were performed. Adipose tissue was collected at 34 weeks for analysis of macrophage infiltration. Surprisingly, CCL2(-/-) mice on high-fat diet showed no reductions in adipose tissue macrophages. CCL2(-/-) mice on standard and high-fat diet were also glucose intolerant and had mildly increased plasma glucose and decreased serum adiponectin levels compared with wild-type mice. On high-fat diet, CCL2(-/-) mice also gained slightly more weight and were hyperinsulinemic compared with wild-type mice. Because macrophage levels were unchanged in CCL2(-/-) mice, the phenotype appears to be caused by lack of CCL2 itself. The fact that metabolic function was altered in CCL2(-/-) mice, despite no changes in adipose tissue macrophage levels, suggests that CCL2 has effects on metabolism that are independent of its macrophage-recruiting capabilities. Importantly, we conclude that CCL2 is not critical for adipose tissue macrophage recruitment. The dominant factor for recruiting macrophages in adipose tissue during obesity therefore remains to be identified.

Chemokines such as monocyte chemoattractant protein (MCP)-1 are key agonists that attract macrophages to tumors. In melanoma, it has been previously shown that variable levels of MCP-1/CCL2 appear to correlate with infiltrating macrophages and tumor fate, with low to intermediate levels of the chemokine contributing to melanoma development. To work under such conditions, a poorly tumorigenic human melanoma cell line was transfected with an expression vector encoding MCP-1. We found that M2 macrophages are associated to MCP-1+ tumors, triggering a profuse vascular network. To target the protumoral macrophages recruitment and reverting tumor growth promotion, clodronate-laden liposomes (Clod-Lip) or bindarit were administered to melanoma-bearing mice. Macrophage depletion after Clod-Lip treatment induced development of smaller tumors than in untreated mice. Immunohistochemical analysis with an anti-CD31 antibody revealed scarce vascular structures mainly characterized by narrow vascular lights. Pharmacological inhibition of MCP-1 with bindarit also reduced tumor growth and macrophage recruitment, rendering necrotic tumor masses. We suggest that bindarit or Clod-Lip abrogates protumoral-associated macrophages in human melanoma xenografts and could be considered as complementary approaches to antiangiogenic therapy.

The acute respiratory distress syndrome (ARDS) is a life-threatening lung condition resulting from direct and indirect insults to the lung. It is characterized by disruption of the endothelial-epithelial barrier, alveolar damage, pulmonary edema, and respiratory failure. A key feature of ARDS is the accumulation of neutrophils in the lung microvasculature, interstitium, and alveolar space. Despite a clear association between neutrophil influx into the lung and disease severity, there is some debate as to whether neutrophils directly contribute to disease pathogenesis. The primary function of neutrophils is to provide immediate host defense against pathogenic microorganisms. Neutrophils release numerous antimicrobial factors such as reactive oxygen species, proteinases, and neutrophil extracellular traps. However, these factors are also toxic to host cells and can result in bystander tissue damage. The excessive accumulation of neutrophils in ARDS may therefore contribute to disease progression. Central to neutrophil recruitment is the release of chemokines, including the archetypal neutrophil chemoattractant IL-8, from resident pulmonary cells. However, the chemokine network in the inflamed lung is complex and may involve several other chemokines, including CXCL10, CCL2, and CCL7. This review will therefore focus on the experimental and clinical evidence supporting neutrophils as key players in ARDS and the chemokines involved in recruiting them into the lung.

We describe a model of severe acute respiratory syndrome-coronavirus (SARS-CoV) infection in C57BL/6 mice. A clinical isolate of the virus introduced intranasally replicated transiently to high levels in the lungs of these mice, with a peak on day 3 and clearance by day 9 postinfection. Viral RNA localized to bronchial and bronchiolar epithelium. Expression of mRNA for angiotensin converting enzyme 2, the SARS-CoV receptor, was detected in the lung following infection. The virus induced production in the lung of the proinflammatory chemokines CCL2, CCL3, CCL5, CXCL9, and CXCL10 with differential kinetics. The receptors for these chemokines were also detected. Most impressively, mRNA for CXCR3, the receptor for CXCL9 and CXCL10, was massively up-regulated in the lungs of SARS-CoV-infected mice. Surprisingly Th1 (and Th2) cytokines were not detectable, and there was little local accumulation of leukocytes and no obvious clinical signs of pulmonary dysfunction. Moreover, beige, CD1-/-, and RAG1-/- mice cleared the virus normally. Infection spread to the brain as it was cleared from the lung, again without leukocyte accumulation. Infected mice had a relative failure to thrive, gaining weight significantly more slowly than uninfected mice. These data indicate that C57BL/6 mice support transient nonfatal systemic infection with SARS-CoV in the lung, which is able to disseminate to brain. In this species, proinflammatory chemokines may coordinate a rapid and highly effective innate antiviral response in the lung, but NK cells and adaptive cellular immunity are not required for viral clearance.

Pneumococcal infection of the respiratory tract is often secondary to recent influenza virus infection and accounts for much of the morbidity and mortality during seasonal and pandemic influenza. Here, we show that coinfection of the upper respiratory tract of mice with influenza virus and pneumococcus leads to synergistic stimulation of type I IFNs and that this impairs the recruitment of macrophages, which are required for pneumococcal clearance, due to decreased production of the chemokine CCL2. Type I IFN expression was induced by pneumococcal colonization alone. Colonization followed by influenza coinfection led to a synergistic type I IFN response, resulting in increased density of colonizing bacteria and susceptibility to invasive infection. This enhanced type I IFN response inhibited production of the chemokine CCL2, which promotes the recruitment of macrophages and bacterial clearance. Stimulation of CCL2 by macrophages upon pneumococcal infection alone required the pattern recognition receptor Nod2 and expression of the pore-forming toxin pneumolysin. Indeed, the increased colonization associated with concurrent influenza virus infection was not observed in mice lacking Nod2 or the type I IFN receptor, or in mice challenged with pneumococci lacking pneumolysin. We therefore propose that the synergistic stimulation of type I IFN production during concurrent influenza virus and pneumococcal infection leads to increased bacterial colonization and suggest that this may contribute to the higher rates of disease associated with coinfection in humans.

The intracellular sensor Nod2 is activated in response to bacteria, and the impairment of this response is linked to Crohn's disease. However, the function of Nod2 in host defense remains poorly understood. We found that Nod2-/- mice exhibited impaired intestinal clearance of Citrobacter rodentium, an enteric bacterium that models human infection by pathogenic Escherichia coli. The increased bacterial burden was preceded by reduced CCL2 chemokine production, inflammatory monocyte recruitment, and Th1 cell responses in the intestine. Colonic stromal cells, but not epithelial cells or resident CD11b+ phagocytic cells, produced CCL2 in response to C. rodentium in a Nod2-dependent manner. Unlike resident phagocytic cells, inflammatory monocytes produced IL-12, a cytokine that induces adaptive immunity required for pathogen clearance. Adoptive transfer of Ly6C(hi) monocytes restored the clearance of the pathogen in infected Ccr2-/- mice. Thus, Nod2 mediates CCL2-CCR2-dependent recruitment of inflammatory monocytes, which is important in promoting bacterial eradication in the intestine.

There is growing evidence that tumor-associated macrophages (TAMs) promote tumor growth and dissemination. Many individual reports have focused on the protumor function of molecules linked to the recruitment of macrophages, but little is known about which factor has the strongest impact on recruitment of macrophages in breast cancer. To elucidate this question, we performed RT-PCR using species-specific primers and evaluated tumoral and stromal mRNA expression of macrophage chemoattractants separately in human breast tumor xenografts. The correlation between the tumoral or stromal chemoattractant mRNA expression including monocyte chemoattractant protein-1 (MCP-1) (CCL2), MIP-1alpha (CCL3), RANTES (CCL5), colony-stimulating factor 1, tumor necrosis factor alpha, platelet-derived growth factor (PDGF)-BB and macrophage infiltration were compared. There was significant positive correlation between stromal MCP-1 expression and macrophage number (r = 0.63), and negative correlation between tumoral RANTES expression and macrophage number (r = -0.75). However, no significant correlation was found for the other tumoral and stromal factors. The interaction between the tumor cells and macrophages was also investigated. Tumor cell-macrophage interactions augmented macrophage-derived MCP-1 mRNA expression and macrophage chemotactic activity in vitro. Treatment of immunodeficient mice bearing human breast cancer cells with a neutralizing antibody to MCP-1 resulted in significant decrease of macrophage infiltration, angiogenetic activity and tumor growth. Furthermore, immunohistochemical analysis of human breast cancer tissue showed stromal MCP-1 had a significant correlation with relapse free survival (p = 0.029), but tumoral MCP-1 did not (p = 0.105). These findings indicate that stromal MCP-1 produced as a result of tumor-stromal interactions may be important for the progression of human breast cancer and macrophages may play an important role in this tumor-stroma interaction.

BACKGROUND

Microbial translocation contributes to immune activation and disease progression during chronic human immunodeficiency virus type 1 (HIV-1) infection. However, its role in the African AIDS epidemic remains controversial. Here, we investigated the relationship between markers of monocyte activation, plasma lipopolysaccharide (LPS), and HIV-1 RNA in South Africans prioritized to receive combination antiretroviral therapy (cART).

METHODS

Ten HIV-1-negative African controls and 80 HIV-1-infected patients with CD4 T cell counts <200 cells/microL were sampled prior to (n=60) or during (n=20) receipt of effective cART. Viral load was measured by Nuclisens; LPS by the Limulus amoebocyte lysate assay; monocyte and T cell subsets by flow cytometry; and soluble CD14, cytokines, and chemokines by enzyme-linked immunosorbent assay and customized Bio-Plex plates.

RESULTS

Three distinct sets of markers were identified. CCL2, CXCL10, and CD14(+)CD16(+) monocyte levels were positively correlated with HIV-1 viremia. This finding, together with cART-induced normalization of these markers, suggests that their upregulation was driven by HIV-1. Plasma interleukin-6 was associated with the presence of opportunistic coinfections. Soluble CD14 and tumor necrosis factor were linked to plasma LPS levels and, as observed for LPS, remained elevated in patients receiving effective cART.

CONCLUSIONS

Microbial translocation is a major force driving chronic inflammation in HIV-infected Africans receiving cART. Prevention of monocyte activation may be especially effective at enhancing therapeutic outcomes.

BACKGROUND

Recent experimental approaches have unraveled essential migratory and functional differences of monocyte subpopulations in mice. In order to possibly translate these findings into human physiology and pathophysiology, human monocyte subsets need to be carefully revisited in health and disease. In analogy to murine studies, we hypothesized that human monocyte subsets dynamically change during ageing, potentially influencing their functionality and contributing to immunosenescence.

RESULTS

Circulating monocyte subsets, surface marker and chemokine receptor expression were analyzed in 181 healthy volunteers (median age 42, range 18-88). Unlike the unaffected total leukocyte or total monocyte counts, non-classical CD14+CD16+ monocytes significantly increased with age, but displayed reduced HLA-DR and CX(3)CR1 surface expression in the elderly. Classical CD14++CD16- monocyte counts did not vary dependent on age. Serum MCP-1 (CCL2), but not MIP1alpha (CCL3), MIP1beta (CCL4) or fractalkine (CX(3)CL1) concentrations increased with age. Monocyte-derived macrophages from old or young individuals did not differ with respect to cytokine release in vitro at steady state or upon LPS stimulation.

CONCLUSIONS

Our study demonstrates dynamic changes of circulating monocytes during ageing in humans. The expansion of the non-classical CD14+CD16+ subtype, alterations of surface protein and chemokine receptor expression as well as circulating monocyte-related chemokines possibly contribute to the preserved functionality of the monocyte pool throughout adulthood.

The objective of this study was to examine the expression of TLR by human primary uterine epithelial cells (UEC) and to determine whether exposure to the TLR agonist poly(I:C) would induce an antiviral response. The secretion of several cytokines and chemokines was examined as well as the mRNA expression of human beta-defensin-1 and -2 (HBD1 and HBD2), IFN-beta, and the IFN-beta-stimulated genes myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase. The expression of TLR1-9 by UEC was demonstrated by RT-PCR, with only TLR10 not expressed. Stimulation of UEC with the TLR3 agonist poly(I:C) induced the expression of the proinflammatory cytokines TNF-alpha, IL-6, GM-CSF, and G-CSF, as well as the chemokines CXCL8/IL-8, CCL2/MCP-1, and CCL4/MIP-1beta. In addition, poly(I:C) exposure induced the mRNA expression of HBD1 and HBD2 by 6- and 4-fold, respectively. Furthermore, upon exposure to poly(I:C) UEC initiated a potent antiviral response resulting in the induction of IFN-beta mRNA expression 70-fold and myxovirus resistance gene 1 and 2',5' oligoadenylate synthetase mRNA expression (107- and 96-fold), respectively. These results suggest that epithelial cells that line the uterine cavity are sensitive to viral infection and/or exposure to viral dsRNA released from killed epithelial cells. Not only do UEC release proinflammatory cytokines and chemokines that mediate the initiation of an inflammatory response and recruitment of immune cells to the site of infection, but they also express beta-defensins, IFN-beta, and IFN-beta-stimulated genes that can have a direct inhibiting effect on viral replication.

OBJECTIVE

Obesity increases the risk of developing major diseases such as diabetes and cardiovascular disease. Adipose tissue, particularly adipocytes, may play a major role in the development of obesity and its comorbidities. The aim of this study was to characterise, in adipocytes from obese people, the most differentially expressed genes that might be relevant to the development of obesity.

METHODS

We carried out microarray gene profiling of isolated abdominal subcutaneous adipocytes from 20 non-obese (BMI 25+/-3 kg/m2) and 19 obese (BMI 55+/-8 kg/m2) non-diabetic Pima Indians using Affymetrix HG-U95 GeneChip arrays. After data analyses, we measured the transcript levels of selected genes based on their biological functions and chromosomal positions using quantitative real-time PCR.

RESULTS

The most differentially expressed genes in adipocytes of obese individuals consisted of 433 upregulated and 244 downregulated genes. Of these, 410 genes could be classified into 20 functional Gene Ontology categories. The analyses indicated that the inflammation/immune response category was over-represented, and that most inflammation-related genes were upregulated in adipocytes of obese subjects. Quantitative real-time PCR confirmed the transcriptional upregulation of representative inflammation-related genes (CCL2 and CCL3) encoding the chemokines monocyte chemoattractant protein-1 and macrophage inflammatory protein 1alpha. The differential expression levels of eight positional candidate genes, including inflammation-related THY1 and C1QTNF5, were also confirmed. These genes are located on chromosome 11q22-q24, a region with linkage to obesity in the Pima Indians.

CONCLUSIONS

This study provides evidence supporting the active role of mature adipocytes in obesity-related inflammation. It also provides potential candidate genes for susceptibility to obesity.

OBJECTIVE

To analyze genome-wide changes in chondrocyte gene expression in a surgically induced model of early osteoarthritis (OA) in rats, to assess the similarity of this model to human OA, and to identify genes and mechanisms leading to OA pathogenesis.

METHODS

OA was surgically induced in 5 rats by anterior cruciate ligament transection and partial medial meniscectomy. Sham surgery was performed in 5 additional animals, which were used as controls. Both groups underwent 4 weeks of forced mobilization, 3 times per week. RNA was extracted directly from articular chondrocytes in the OA (operated), contralateral, and sham-operated knees. Affymetrix GeneChip expression arrays were used to assess genome-wide changes in gene expression. Expression patterns of selected dysregulated genes, including Col2a1, Mmp13, Adamts5, Ctsc, Ptges, and Cxcr4, were validated by real-time polymerase chain reaction, immunofluorescence, or immunohistochemistry 2, 4, and 8 weeks after surgery.

RESULTS

After normalization, comparison of OA and sham-operated samples showed 1,619 differentially expressed probe sets with changes in their levels of expression>> or = 1.5-fold, 722 with changes>> or = 2-fold, 135 with changes>> or = 4-fold, and 20 with changes of 8-fold. Dysregulated genes known to be involved in human OA included Mmp13, Adamts5, and Ptgs2, among others. Several dysregulated genes (e.g., Reln, Phex, and Ltbp2) had been identified in our earlier microarray study of hypertrophic chondrocyte differentiation. Other genes involved in cytokine and chemokine signaling, including Cxcr4 and Ccl2, were identified. Changes in gene expression were also observed in the contralateral knee, validating the sham operation as the appropriate control.

CONCLUSIONS

Our results demonstrate that the animal model mimics gene expression changes seen in human OA, supporting the relevance of newly identified genes and pathways to early human OA. We propose new avenues for OA pathogenesis research and potential targets for novel OA treatments, including cathepsins and cytokine, chemokine, and growth factor signaling pathways, in addition to factors controlling the progression of chondrocyte differentiation.

Rearrangements of the RET receptor tyrosine kinase gene generating RET/PTC oncogenes are specific to papillary thyroid carcinoma (PTC), the most frequent thyroid tumor. Here, we show that the RET/PTC1 oncogene, when exogenously expressed in primary normal human thyrocytes, induces the expression of a large set of genes involved in inflammation and tumor invasion, including those encoding chemokines (CCL2, CCL2CCL2CCL2, CXCL8, CXCR4, L-selectin, GM-CSF, IL1B, MMP9, UPA, and SPP1/OPN) were found up-regulated also in clinical samples of PTC, particularly those characterized by RET/PTC activation, local extrathyroid spread, and lymph node metastases, when compared with normal thyroid tissue or follicular thyroid carcinoma. These results, demonstrating that the RET/PTC1 oncogene activates a proinflammatory program, provide a direct link between a transforming human oncogene, inflammation, and malignant behavior.

PTX3 is an essential component of the humoral arm of innate immunity, playing a nonredundant role in resistance against selected microbes and in the regulation of inflammation. PTX3 activates and regulates the Complement cascade by interacting with C1q and with Factor H. PTX3 deficiency was associated with increased susceptibility to mesenchymal and epithelial carcinogenesis. Increased susceptibility of Ptx3(-/-) mice was associated with enhanced macrophage infiltration, cytokine production, angiogenesis, and Trp53 mutations. Correlative evidence, gene-targeted mice, and pharmacological blocking experiments indicated that PTX3 deficiency resulted in amplification of Complement activation, CCL2 production, and tumor-promoting macrophage recruitment. PTX3 expression was epigenetically regulated in selected human tumors (e.g., leiomyosarcomas and colorectal cancer) by methylation of the promoter region and of a putative enhancer. Thus, PTX3, an effector molecule belonging to the humoral arm of innate immunity, acts as an extrinsic oncosuppressor gene in mouse and man by regulating Complement-dependent, macrophage-sustained, tumor-promoting inflammation.

Genetic analyses of cancer in humans indicate that chemokines and their receptors are unlikely to play direct roles in pathogenesis. However, these molecules have pleiotropic effects that impact on cancer pathobiology in animal models, and there is evidence that they may do the same in humans. Given their protean properties, chemokines could have tumor-promoting, tumor-suppressing activities, or either depending on context. An example is found in CCL2, a chemokine that attracts and activates mononuclear cells. In some settings, it stimulates host anti-tumor activities. However, tumor cells themselves secrete CCL2 suggesting that it has growth promoting effects. These have been documented in animal models and clinical epidemiological studies. If CCL2's protumorigenic activities can be validated, then CCL2 and its receptor CCR2 may be therapeutic targets in cancer.

OBJECTIVE

Drusen, which are defined clinically as yellowish white spots in the outer retina, are cardinal features of age-related macular degeneration (AMD). Ccl2-knockout (Ccl2(-/-)) mice have been reported to develop drusen and phenotypic features similar to AMD, including an increased susceptibility to choroidal neovascularization (CNV). This study was conducted to investigate the nature of the drusenlike lesions in vivo and further evaluate the Ccl2(-/-) mouse as a model of AMD.

METHODS

The eyes of 2- to 25-month-old Ccl2(-/-) and C57Bl/6 mice were examined in vivo by autofluorescence scanning laser ophthalmoscopy (AF-SLO) and electroretinography, and the extent of laser-induced CNV was measured by fluorescein fundus angiography. The retinal morphology was also assessed by immunohistochemistry and quantitative histologic and ultrastructural morphometry.

RESULTS

The drusenlike lesions of Ccl2(-/-) mice comprised accelerated accumulation of swollen CD68(+), F4/80(+) macrophages in the subretinal space that were apparent as autofluorescent foci on AF-SLO. These macrophages contained pigment granules and phagosomes with outer segment and lipofuscin inclusions that may account for their autofluorescence. Only age-related retinal pigment epithelium (RPE) damage, photoreceptor loss, and sub-RPE deposits were observed but, despite the accelerated accumulation of macrophages, we identified no spontaneous development of CNV in the senescent mice and found a reduced susceptibility to laser-induced CNV in the Ccl2(-/-) mice.

CONCLUSIONS

These findings suggest that the lack of Ccl2 leads to a monocyte/macrophage-trafficking defect during aging and to an impaired recruitment of these cells to sites of laser injury. Other, previously described features of Ccl2(-/-) mice that are similar to AMD may be the result of aging alone.

BACKGROUND

Mood disturbances are associated with an activated inflammatory response system.

OBJECTIVE

To identify a discriminating and coherent expression pattern of proinflammatory genes in monocytes of patients with bipolar disorder.

METHODS

A quantitative polymerase chain reaction (Q-PCR) case-control gene expression study on purified monocytes of bipolar patients, the offspring of bipolar patients, and healthy control participants after having selected 22 discriminating inflammatory genes using whole genome analyses.

METHODS

Academic research setting in The Netherlands.

METHODS

Forty-two bipolar patients with 25 healthy controls, 54 offspring of a bipolar parent (13 had a mood disorder and 3 developed one during follow-up), and 70 healthy children underwent Q-PCR.

METHODS

Inflammatory gene expression levels in monocytes.

RESULTS

We detected in the monocytes of bipolar patients a coherent mutually correlating set (signature) of 19 aberrantly expressed (P < .01) messenger RNAs of inflammatory (PDE4B, IL1B, IL6, TNF, TNFAIP3, PTGS2, and PTX3), trafficking (<em>CCL2</em>, CCL7, <em>CCL2</em>0, CXCL2, CCR2, and CDC42), survival (BCL2A1 and EMP1), and mitogen-activated protein kinase pathway (MAPK6, DUSP2, NAB2, and ATF3) genes. Twenty-three of 42 bipolar patients (55%) had a positive signature test result vs 7 of 38 healthy controls (18%) (positive test result: positivity for PDE4B, ie, a messenger RNA 1 SD higher than the mean level found in healthy controls, plus 25% of the other genes with similar positive findings). Positive signature test results were also present in 11 of 13 offspring with a mood disorder (85%), 3 of 3 offspring developing a mood disorder (100%), and 17 of 38 euthymic offspring (45%) vs 13 of 70 healthy children (19%). Lithium carbonate and antipsychotic treatment downregulated the gene expression of most inflammatory genes.

CONCLUSIONS

The monocytes of a large proportion of bipolar patients and offspring of bipolar parents showed an inflammatory gene expression signature. This coherent set of genes opens new avenues for biomarker development with possibilities for disease prediction in individuals genetically at risk and for the subclassification of bipolar patients who could possibly benefit from anti-inflammatory treatment.

Influenza A virus pneumonia is characterized by severe lung injury and high mortality. Early infection elicits a strong recruitment of monocytes from the peripheral blood across the endo-/epithelial barrier into the alveolar air space. However, it is currently unclear which of the infected resident lung cell populations, alveolar epithelial cells or alveolar macrophages, elicit monocyte recruitment during influenza A virus infection. In the current study, we investigated whether influenza A virus infection of primary alveolar epithelial cells and resident alveolar macrophages would elicit a basal-to-apical monocyte transepithelial migration in vitro. We found that infection of alveolar epithelial cells with the mouse-adapted influenza A virus strain PR/8 strongly induced the release of monocyte chemoattractants CCL2 and CCL5 followed by a strong monocyte transepithelial migration, and this monocytic response was strictly dependent on monocyte CCR2 but not CCR5 chemokine receptor expression. Analysis of the adhesion molecule pathways demonstrated a role of ICAM-1, VCAM-1, integrin-associated protein (CD47), and junctional adhesion molecule-c on the epithelial cell surface interacting with monocyte beta(1) and beta(2) integrins and integrin-associated protein in the monocyte transmigration process. Importantly, addition of influenza A virus-infected alveolar macrophages further enhanced monocyte transmigration across virus-infected epithelium in a TNF-alpha-dependent manner. Collectively, the data show an active role for virus-infected alveolar epithelium in the regulation of CCL2/CCR2-dependent monocyte transepithelial migration during influenza infection that is essentially dependent on both classical beta(1) and beta(2) integrins but also junctional adhesion molecule pathways.

Increased expression of the chemokine CCL2 in tumor cells correlates with enhanced metastasis, poor prognosis, and recruitment of CCR2(+)Ly6C(hi) monocytes. However, the mechanisms driving tumor cell extravasation through the endothelium remain elusive. Here, we describe CCL2 upregulation in metastatic UICC stage IV colon carcinomas and demonstrate that tumor cell-derived CCL2 activates the CCR2(+) endothelium to increase vascular permeability in vivo. CCR2 deficiency prevents colon carcinoma extravasation and metastasis. Of note, CCR2 expression on radio-resistant cells or endothelial CCR2 expression restores extravasation and metastasis in Ccr2(-/-) mice. Reduction of CCR2 expression on myeloid cells decreases but does not prevent metastasis. CCL2-induced vascular permeability and metastasis is dependent on JAK2-Stat5 and p38MAPK signaling. Our study identifies potential targets for treating CCL2-dependent metastasis.