OBJECTIVE

To quantify the association between systemic levels of the chemokine regulated on activation normal T-cell expressed and secreted (RANTES/CCL5), interferon-γ-inducible protein-10 (IP-10/CXCL10), monocyte chemoattractant protein-1 (MCP-1/CCL2), and eotaxin-1 (CCL11) with future coronary heart disease (CHD) and ischemic stroke events and to assess their usefulness for CHD and ischemic stroke risk prediction in the PRIME Study.

METHODS

After 10 years of follow-up of 9,771 men, 2 nested case-control studies were built including 621 first CHD events and 1,242 matched controls and 95 first ischemic stroke events and 190 matched controls. Standardized hazard ratios (HRs) for each log-transformed chemokine were estimated by conditional logistic regression.

RESULTS

None of the 4 chemokines were independent predictors of CHD, either with respect to stable angina or to acute coronary syndrome. Conversely, RANTES (HR = 1.70; 95% confidence interval [CI] 1.05-2.74), IP-10 (HR = 1.53; 95% CI 1.06-2.20), and eotaxin-1 (HR = 1.59; 95% CI 1.02-2.46), but not MCP-1 (HR = 0.99; 95% CI 0.68-1.46), were associated with ischemic stroke independently of traditional cardiovascular risk factors, hs-CRP, and fibrinogen. When the first 3 chemokines were included in the same multivariate model, RANTES and IP-10 remained predictive of ischemic stroke. Their addition to a traditional risk factor model predicting ischemic stroke substantially improved the C-statistic from 0.6756 to 0.7425 (p = 0.004).

CONCLUSIONS

In asymptomatic men, higher systemic levels of RANTES and IP-10 are independent predictors of ischemic stroke but not of CHD events. RANTES and IP-10 may improve the accuracy of ischemic stroke risk prediction over traditional risk factors.

Multiple sclerosis (MS) is a polygenic disease characterized by inflammation and demyelination in the central nervous system (CNS), which can be modeled in experimental autoimmune encephalomyelitis (EAE). The Eae18b locus on rat chromosome 10 has previously been linked to regulation of beta-chemokine expression and severity of EAE. Moreover, the homologous chemokine cluster in humans showed evidence of association with susceptibility to MS. We here established a congenic rat strain with Eae18b locus containing a chemokine cluster (Ccl2, Ccl7, Ccl11, Ccl12 and Ccl1) from the EAE- resistant PVG rat strain on the susceptible DA background and utilized myelin oligodendrocyte glycoprotein (MOG)-induced EAE to characterize the mechanisms underlying the genetic regulation. Congenic rats developed a milder disease compared to the susceptible DA strain, and this was reflected in decreased demyelination and in reduced recruitment of inflammatory cells to the brain. The congenic strain also showed significantly increased Ccl11 mRNA expression in draining lymph nodes and spinal cord after EAE induction. In the lymph nodes, macrophages were the main producers of CCL11, whereas macrophages and lymphocytes expressed the main CCL11 receptor, namely CCR3. Accordingly, the congenic strain also showed significantly increased Ccr3 mRNA expression in lymph nodes. In the CNS, the main producers of CCL11 were neurons, whereas CCR3 was detected on neurons and CSF producing ependymal cells. This corresponded to increased levels of CCL11 protein in the cerebrospinal fluid of the congenic rats. Increased intrathecal production of CCL11 in congenic rats was accompanied by a tighter blood brain barrier, reflected by more occludin(+) blood vessels. In addition, the congenic strain showed a reduced antigen specific response and a predominant anti-inflammatory Th2 phenotype. These results indicate novel mechanisms in the genetic regulation of neuroinflammation.

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

BACKGROUND

Acute exacerbations (AEs) in chronic obstructive pulmonary disease (COPD) are a major cause of morbidity and mortality in COPD.

OBJECTIVE

The marked heterogeneity in the host defense mechanisms may be attributed to single nucleotide polymorphisms (SNPs) in the inflammatory chemokines that show enhanced expression in the airway of patients with COPD who experience AEs.

METHODS

We investigated four SNPs of the CCL11, CCL1, and CCL5 genes in relation to the frequency and severity of AEs in retrospective and prospective studies of a cohort of 276 male patients with COPD.

RESULTS

In the 2-yr retrospective study , one SNP (National Center for Biotechnology Information SNP reference: rs2282691) in the predicted enhancer region of the CCL1 gene, encoding a chemotactic factor for a series of leukocytes, was significantly associated with the frequency of AEs in a dominant model (Fisher's exact test: odds ratio [OR], 2.70; 95% confidence interval [CI], 1.36-5.36; p=0.004; logistic regression: OR, 3.06; 95% CI, 1.46-6.41; p=0.003; and Kruskal-Wallis test: p=0.003). In the 30-mo prospective study, the "A" allele was a significant risk allele for the severity of AEs, with a gene-dosage effect (Kaplan-Meier method with log-rank test: AA vs. TT; log-rank statistic: 7.67, p=0.006; Cox proportional hazards regression method: OR, 5.93; 95% CI, 1.28-27.48; p=0.023). The electromobility shift assay showed that C/EBPbeta, a key transcriptional factor in response to pulmonary infections, binds to the "T" allele, but not to the "A" allele.

CONCLUSIONS

Variants in the CCL1 gene are associated with susceptibility to AEs through their potential implication in the host defense mechanisms against AEs.

The form of (1-3)-beta-D glucan found in the cell walls of the anamorphic Trichocomaceae that grow on damp building materials is considered to have potent toxic and inflammatory effects on cells of the respiratory system. It is also considered to have a potential role in the development of non-allergenic respiratory health effects. While human studies involving experimental exposures all point to the inflammatory potential of pure curdlan, a linear (1-3)-beta-D glucan in a triple helix configuration, animal experiments result in conflicting conclusions concerning the inflammatory potency of this glucan. However, because mice appear to be a better model than guinea pigs for exploring the respiratory effects of curdlan and because molecular mechanisms associated with this glucan remain largely unknown, we conducted further work to clarify the role of curdlan on the inflammatory response using our mouse model of lung disease. This study used in situ hybridization (ISH) to probe dectin-1 mRNA transcription with a digoxigenin-labeled cDNA probe, with reverse transcription (RT)-PCR based arrays used to measure inflammation gene and receptor transcriptional responses. Also, immunohistochemistry (IHC) was used to probe dectin-1 as well as anti-mouse Ccl3, Il1-alpha, and TNF-alpha expression to evaluate dose and time-course (4 and 12 h) postexposure (PE) response patterns in the lungs of intratracheally instilled mice exposed to a single 50 mul dose of curdlan at 10(-7), 10(-8), 10(-9), and 10(-10) M/animal (=4 mug to 4 ng curdlan/kg lung wt). Dectin-1 mRNA transcription and expression was observed in bronchiolar epithelium, alveolar macrophages (AMs), and alveolar type II cells (ATIIs) of lungs exposed to 4 mug to 40 ng curdlan/kg lung wt, at both time points. Compared to controls, array analysis revealed that 54 of 83 genes assayed were significantly modulated by curdlan. mRNA transcription patterns showed both dose and time dependency, with highest transcription levels in 10(-7) and 10(-8) M treatment animals, especially at 4-h PE. Nine gene mRNA transcripts (Ccl3, Ccl11, Ccl17, Ifng, Il1alpha, Il-20, TNF-alpha, Tnfrsf1b, and CD40lg) were significantly expressed at all doses suggesting they may have a central role in immunomodulating curdlan exposures. IHC revealed Ccl3, Il1-alpha, and TNF-alpha expression in bronchiolar epithelium, AMs and ATIIs illustrate the important immunomodulatory role that these cells have in the recognition of, and response to glucan. Collectively, these results confirm the inflammatory nature of curdlan and demonstrate the complex of inflammation-associated gene responses induced by (1-3)-beta-D glucan in triple helical forms. These observations also provide a biological basis for the irritant and inflammatory response to curdlan observed in humans and animals in experimental studies.

Borna disease virus (BDV) frequently causes meningoencephalitis and fatal neurological disease in young but not old mice of strain MRL. Disease does not result from the virus-induced destruction of infected neurons. Rather, it is mediated by H-2(k)-restricted antiviral CD8 T cells that recognize a peptide derived from the BDV nucleoprotein N. Persistent BDV infection in mice is not spontaneously cleared. We report here that N-specific vaccination can protect wild-type MRL mice but not mutant MRL mice lacking gamma interferon (IFN-gamma) from persistent infection with BDV. Furthermore, we observed a significant degree of resistance of old MRL mice to persistent BDV infection that depended on the presence of CD8 T cells. We found that virus initially infected hippocampal neurons around 2 weeks after intracerebral infection but was eventually cleared in most wild-type MRL mice. Unexpectedly, young as well as old IFN-gamma-deficient MRL mice were completely susceptible to infection with BDV. Moreover, neurons in the CA1 region of the hippocampus were severely damaged in most diseased IFN-gamma-deficient mice but not in wild-type mice. Furthermore, large numbers of eosinophils were present in the inflamed brains of IFN-gamma-deficient mice but not in those of wild-type mice, presumably because of increased intracerebral synthesis of interleukin-13 and the chemokines CCL1 and CCL11, which can attract eosinophils. These results demonstrate that IFN-gamma plays a central role in host resistance against infection of the central nervous system with BDV and in clearance of BDV from neurons. They further indicate that IFN-gamma may function as a neuroprotective factor that can limit the loss of neurons in the course of antiviral immune responses in the brain.

Chemokines have been reported to play crucial roles in tumor progression. Eotaxin-1 (CCL11), a member of the CC chemokine family, is elevated in many types of human cancer. Here, to reveal the molecular mechanisms of eotaxin-1 in prostate cancer cell invasion, the expression of eotaxin-1 receptors [CC chemokine receptor (CCR)2, CCR3 and CCR5] were silenced by small interfering RNA (siRNA). The ERK pathway was inhibited by the specific MEK inhibitor U0126. The role of eotaxin-1 and the CCR3-ERK pathway in prostate cancer cell invasion was assessed by invasion and migration assays. MMP-3 expression was detected by real-time PCR and ELISA assay. The results demonstrated that eotaxin-1 promoted the invasion and migration of DU-145 cells, and increased ERK1/2 activation and MMP-3 expression. Knockdown of CCR3 inhibited the invasion and migration of prostate cancer cells, and attenuated the eotaxin-1-induced ERK1/2 activation and MMP-3 expression. Furthermore, inactivation of the ERK pathway suppressed the eotaxin‑1-promoted invasion and migration, and decreased MMP-3 expression in the prostate cancer cells. Together, the present study suggests that eotaxin-1 increases MMP-3 expression via the CCR3-ERK pathway, thereby promoting prostate cancer cell invasion and migration. Thus, therapies that block eotaxin-1 and CCR3 may be effective interventions for prostate cancer.

Chemokines are thought to play an important role in several aspects of bone metabolism including the recruitment of leukocytes and the formation of osteoclasts. We investigated the impact of diabetes on chemokine expression in normal and diabetic fracture healing. Fracture of the femur was performed in streptozotocin-induced diabetic and matched normoglycemic control mice. Microarray analysis was carried out and chemokine mRNA levels in vivo were assessed. CCL4 were examined in fracture calluses by immunohistochemistry and the role of TNF in diabetes-enhanced expression was investigated by treatment of animals with the TNF-specific inhibitor, pegsunercept. In vitro studies were conducted with ATDC5 chondrocytes. Diabetes significantly upregulated mRNA levels of several chemokines in vivo including CCL4, CCL8, CCL6, CCL11, CCL20, CCL24, CXCL2, CXCL5 and chemokine receptors CCR5 and CXCR4. Chondrocytes were identified as a significant source of CCL4 and its expression in diabetic fractures was dependent on TNF (P<0.05). TNF-α significantly increased mRNA levels of several chemokines in vitro which were knocked down with FOXO1 siRNA (P<0.05). CCL4 expression at the mRNA and proteins levels was induced by FOXO1 over-expression and reduced by FOXO1 knockdown. The current studies point to the importance of TNF-α as a mechanism for diabetes enhanced chemokine expression by chondrocytes, which may contribute to the accelerated loss of cartilage observed in diabetic fracture healing. Moreover, in vitro results point to FOXO1 as a potentially important transcription factor in mediating this effect.

OBJECTIVE

To compare serum levels of MCP-1/CCL2, RANTES/CCL5, and Eotaxin/CCL11 between female patients with recurrent major depressive disorder (MDD) and healthy controls, verifying if there is a difference in the levels of these mediators between those with or without current suicidal ideation.

METHODS

Thirty female outpatients with recurrent MDD were divided in two groups accordingly the presence or absence of suicidal ideation. These groups were compared with 16 healthy controls. Serum levels of MCP-1/CCL2, RANTES/CCL5, and Eotaxin/CCL11 were determined. Depression severity was evaluated by Beck Depression Inventory (BDI). Suicidal ideation was assessed by SCID-I and BDI.

RESULTS

Patients with recurrent MDD and healthy controls did not differ in age, socioeconomic status, and education. All patients reported high scores of BDI (mean, SD, n; 29.75, 10.55, 28). Multivariable analysis of covariance adjusted for age and BMI showed that MDD patients with suicidal ideation presented lower levels of MCP-1/ CCL2 and RANTES/CCL5 (p < 0.001) and higher levels of Eotaxin/CCL11 (p = 0.04) compared to healthy controls. These differences remained significant after adjusting for depression severity.

CONCLUSIONS

The findings of this study indicated that the presence of recurrent MDD with suicidal ideation is associated with differences in inflammatory chemokines when compared to those without suicidal ideation.

Atopic dermatitis is a chronic or chronically relapsing inflammatory skin disease with a prevalence ranging from 10% to 20% in children and 1% to 3% in adults of developed countries. Skin-infiltrating leukocytes play a pivotal role in the initiation and amplification of atopic skin inflammation. Recent studies demonstrated that infiltration of inflammatory cells into tissues is regulated by chemokines. A subset of chemokines including CCL27, CCL17, CCL22, CCL18, CCL11, and CCL13 are highly expressed in atopic dermatitis. The corresponding chemokine receptors are found on the main leukocyte subsets involved in allergic skin inflammation, such as T cells, eosinophils, and dendritic cells. In this article, we provide an overview of the role of chemokines in the complex immunopathogenesis of atopic dermatitis, highlighting potential areas for therapeutic intervention.

CD4(+) T cells play a central role in inflammatory heart disease, implicating a cytokine product associated with Th cell effector function as a necessary mediator of this pathophysiology. IFN-γ-deficient mice developed severe experimental autoimmune myocarditis (EAM), in which mice are immunized with cardiac myosin peptide, whereas IL-17A-deficient mice were protected from progression to dilated cardiomyopathy. We generated IFN-γ(-/-)IL-17A(-/-) mice to assess whether IL-17 signaling was responsible for the severe EAM of IFN-γ(-/-) mice. Surprisingly, IFN-γ(-/-)IL-17A(-/-) mice developed a rapidly fatal EAM. Eosinophils constituted a third of infiltrating leukocytes, qualifying this disease as eosinophilic myocarditis. We found increased cardiac production of CCL11/eotaxin, as well as Th2 deviation, among heart-infiltrating CD4(+) cells. Ablation of eosinophil development improved survival of IFN-γ(-/-)IL-17A(-/-) mice, demonstrating the necessity of eosinophils in fatal heart failure. The severe and rapidly fatal autoimmune inflammation that developed in the combined absence of IFN-γ and IL-17A constitutes a novel model of eosinophilic heart disease in humans. This is also, to our knowledge, the first demonstration that eosinophils have the capacity to act as necessary mediators of morbidity in an autoimmune process.

Cytokines and chemokines contribute to the pathogenesis of acute disseminated encephalomyelitis (ADEM). Using a multiplex immunochemiluminescence ELISA, we measured 8 Th1/Th2 cytokines and 18 chemokines in the cerebrospinal fluid (CSF) and serum of 17 ADEM patients, 14 multiple sclerosis (MS) patients, and 7 healthy controls (HCs). Relative to HCs, ADEM patients had significantly high mean CSF concentrations of chemokines with attractant/activating properties towards neutrophils (CXCL1 and CXCL7), monocytes/T cells (CCL3 and CCL5), Th1 cells (CXCL10), and Th2 cells (CCL1, CCL22, and CCL17). Mean CSF concentrations of CXCL7, CCL1, CCL22, and CCL17 were higher in ADEM than in MS, whereas those of CCL11 were lower in MS than in ADEM and HCs. CSF pleocytosis correlated with CSF concentrations of CXCL1, CXCL10, CCL1, CCL17, and CCL22. Most of the functionally homologous chemokines correlated with each other. CSF Th1/Th2 cytokines were not detectable in most samples. Their mean concentrations did not differ in the three groups, and the same held for serum cytokines and chemokines. Our data suggest that the upregulation of chemokines active on neutrophils and Th2 cells differentiates ADEM from MS inflammation, and that both Th1 and Th2 chemokines might be produced in ADEM. Chemokines upregulated in ADEM could become CSF biomarkers after a posteriori evaluations in unselected case series.

Recent reports suggest the potential role of toll-like receptor 4 (TLR4) in initiation of inflammatory responses and fatty acid-induced insulin resistance. We describe here the synthesis of pro-inflammatory products in 3T3-L1 preadipocyte cell line after stimulation with lipopolysaccharide (LPS), a TLR4 agonist. Expression profiles of mRNA coding for IL6, CCL2, CCL5, CCL11, NOS2, and PTGS2 demonstrated a higher responsiveness to LPS of these transcripts in preadipocytes than in fully differentiated adipocytes, confirming inflammatory features of preadipocytes. IL6, CCL2, CCL5 and CCL11 were secreted in 3T3-L1 supernatants within 4 h after LPS stimulation. In addition, continuous exposure to LPS during adipocyte differentiation impaired this process as was demonstrated by analysis of mRNA profiles of lipogenesis enzymes (FABP4, GPD1, LPL), adipokines (adiponectin, resistin, visfatin, leptin), and of the transcription factor PPARgamma. This suggests that toll-like receptor mediated activation could regulate maintenance of preadipocyte status, and inflammatory environment encountered in inflamed white adipose tissue.

BACKGROUND

Atopic dermatitis (AD) is a chronic skin disease in which environmental factors play a great role. A widely used murine model for AD has provided a useful tool to study the disease.

OBJECTIVE

The purpose of this study is to investigate kinetically the induction of this AD model and the processes involved in the development of AD due to extrinsic allergen exposures.

METHODS

BALB/c mice were epicutaneously exposed to ovalbumin (OVA) for 3 weeks; each week was separated by a 2-week resting period. Mice were killed after each exposure week. Skin biopsies and blood were obtained for histological study, RNA isolation and antibody analysis.

RESULTS

There was a progressive and significant thickening of the epidermis and dermis in OVA-exposed mice. Significantly increased dermal cell infiltration of eosinophils, mast cells and total inflammatory cells, including CD3 and CD4 cells, was found after each OVA exposure week. Total IgE, IgG2a and OVA-specific IgE were significantly increased after the second and third exposure week, while OVA-specific IgG2a was significantly induced after the third exposure week. Gradual and/or significant increases in mRNA expression of IL-1beta, TNF-alpha, IL-4, IL-10, IL-13, IFN-gamma and IL-12p35 were found after each exposure week. Chemokines and their receptors involved in both T-helper type 1 (Th1)- and Th2-type cell recruitment (CCL1, CCL8, CCL11, CCL24, CXCL9, CXCL10, CCR1, CCR3, CCR5, CCR8 and CXCR3) were up-regulated significantly at different time-points.

CONCLUSIONS

This study provides an insight into the dynamic nature and time-dependent transition of skin inflammation and systemic immune responses in a murine AD model induced by repeated epicutaneous exposures to OVA.

BACKGROUND

Interleukin (IL)-33, a new member of the IL-1 cytokine family, has been recognized as a key cytokine that enhances T helper 2-balanced immune regulation through its receptor ST2; however, the function and relationship of the IL-33 and ST2 pathways in bronchial asthma are still unclear. We investigated the cellular origin and regulation of IL-33 and ST2 in allergic bronchial asthma in vivo and in vitro.

METHODS

BALB/c mice were sensitized by intraperitoneal injections of ovalbumin (OVA) with alum. Mice were exposed to aerosolized 1% OVA for 30 min a day for 7 days. These mice were then challenged with aerosolized 1% OVA 2 days after the last day of exposure. After the OVA challenge, the mice were sacrificed and their lung tissues were obtained. Mouse lung fibroblasts were cultured and treated with IL-33 or IL-13.

RESULTS

The levels of IL-33 mRNA and IL-33 protein in lung tissue increased after the OVA challenge. Most IL-33-expressing cells were CD11c+ cells and epithelial cells, and many ST2-expressing cells were stained lung fibroblasts and inflammatory cells. IL-33 induced eotaxin/CCL11 production in lung fibroblasts. IL-33 and IL-13 synergistically induced eotaxin expression.

CONCLUSIONS

IL-33 may contribute to the induction and maintenance of eosinophilic inflammation in the airways by acting on lung fibroblasts. IL-33 and ST2 may play important roles in allergic bronchial asthma.

The nature of the interactions between the intravascular parasite Schistosoma mansoni and the host pulmonary vasculature is critical in determining the outcome of infection. In this report, we show that lung schistosomula selectively induce the synthesis of IL-6 mRNA and protein in cultured human and mouse lung microvascular endothelial cells (EC) and that parasite excretory/secretory lipophilic compounds, particularly prostaglandin E(2), are responsible for this effect. In vivo, a striking increase of IL-6 expression is observed in the pulmonary microvasculature of S. mansoni-infected C57BL/6 mice suggesting that, in vivo, parasites also induce the synthesis of IL-6 in lung EC. In infected mice, IL-6 deficiency results in an accelerated mobilization of eosinophils into the lung tissue and in a dramatic increased number of recruited leukocytes, particularly eosinophils, in the airway. This effect is associated with an enhanced production of eotaxin (CCL11) and IL-5 in the lungs of IL-6 knockout (KO) animals. Finally, compared to wild-type mice, we detect a dramatic increased level of parasite mortality in the lungs of IL-6 KO mice. Taken together, we suggest that parasite larvae activate EC to produce IL-6 to escape the inflammatory reaction that develops in the lungs of infected hosts. Finally, we show that the parasite-induced IL-6 synthesis is mediated by a protein kinase A-dependent pathway that principally targets the cAMP-response element and the nuclear factor-kappaB sites from the -256/+20 region of the IL-6 promoter.

The transcription factor NF-kappaB plays a pivotal role in regulating inflammatory gene expression. Its effects are optimized by various coactivators, including histone acetyltransferases (HATs) such as CREB-binding protein/p300 and p300/CBP-associated factor (p/CAF). The molecular mechanisms regulating cofactor recruitment are poorly understood. In this study, we describe a novel role for protein kinase C (PKC) betaIotaIota in augmenting NF-kappaB-mediated TNF-alpha-induced transcription of the target gene CCL11 in human airway smooth muscle cells by phosphorylating the HAT p/CAF. Studies using reporters, overexpression strategies, kinase-dead and HAT-defective mutants, and chromatin immunoprecipitation showed that PKCbetaII activation was not involved in NF-kappaB translocation, but facilitated NF-kappaB-mediated CCL11 transcription by colocalizing with and phosphorylating p/CAF, and thereby acetylating histone H4 and promoting p65 association with the CCL11 promoter. The effect was dependent on p/CAF's HAT activity. Furthermore, mouse embryonic fibroblasts from PKCbeta knockout mice showed markedly reduced TNF-alpha-induced CCL11 expression and NF-kappaB reporter activity that was restored on PKCbetaII overexpression, suggesting a critical role for this pathway. These data suggest a novel important biological role for PKCbetaIotaIota in NF-kappaB-mediated CCL11 transcription by p/CAF activation and histone H4 acetylation.

Eosinophils are an important source of leukotriene (LT)C(4), which can be synthesized within lipid bodies-cytoplasmic organelles where eicosanoid formation may take place. Allergy-driven lipid body formation and function have never been investigated. Here, we studied the in vivo induction and role of lipid bodies within eosinophils recruited to sites of allergic inflammation. Using two murine models of allergic inflammation (asthma and pleurisy), we verified that parallel to the eosinophil influx, allergic challenge also induced lipid body formation within recruited eosinophils. Neutralizing antibodies to eotaxin/CCL11, RANTES/CCL5, or CCR3 partially inhibited lipid body formation within recruited eosinophils in the allergic pleurisy model. Likewise, intrapleural administration of RANTES or eotaxin also induced significant influx of eosinophils loaded with lipid bodies. By immunolabeling, we detected the presence of a key enzyme involved in the leukotriene metabolism-5-lipoxygenase-within eosinophil lipid bodies formed in vivo after allergen challenge. Furthermore, specific immunolocalization of newly formed LTC(4) demonstrated that lipid bodies were the sites of formation of this eicosanoid within infiltrating eosinophils. Therefore, allergic inflammation triggers in vivo formation of new lipid bodies within infiltrating eosinophils, a phenomenon largely mediated by eotaxin/RANTES acting via CCR3 receptors. Such in vivo allergen-driven lipid bodies function as intracellular compartments of LTC(4) synthesis.

Human blood eosinophils exhibit a hyperactive phenotype in response to chemotactic factors after cell "priming" with IL-5 family cytokines. Earlier work has identified ERK1/2 as molecular markers for IL-5 priming, and in this article, we show that IL-3, a member of the IL-5 family, also augments fMLP-stimulated ERK1/2 phosphorylation in primary eosinophils. Besides ERK1/2, we also observed an enhancement of chemotactic factor-induced Akt phosphorylation after IL-5 priming of human blood eosinophils. Administration of a peptide antagonist that targets the Src family member Lyn before cytokine (IL-5/IL-3) priming of blood eosinophils inhibited the synergistic increase of fMLP-induced activation of Ras, ERK1/2 and Akt, as well as the release of the proinflammatory factor leukotriene C(4). In this study, we also examined a human eosinophil-like cell line HL-60 clone-15 and observed that these cells exhibited significant surface expression of IL-3Rs and GM-CSFRs, as well as ERK1/2 phosphorylation in response to the addition of IL-5 family cytokines or the chemotactic factors fMLP, CCL5, and CCL11. Consistent with the surface profile of IL-5 family receptors, HL-60 clone-15 recapitulated the enhanced fMLP-induced ERK1/2 phosphorylation observed in primary blood eosinophils after priming with IL-3/GM-CSF, and small interfering RNA-mediated knockdown of Lyn expression completely abolished the synergistic effects of IL-3 priming on fMLP-induced ERK1/2 phosphorylation. Altogether, our data demonstrate a central role for Lyn in the mechanisms of IL-5 family priming and suggest that Lyn contributes to the upregulation of the Ras-ERK1/2 and PI3K-Akt cascades, as well as the increased leukotriene C(4) release observed in response to fMLP in "primed" eosinophils.

Little is known about mechanisms involved in skin-specific homing of cutaneous T-cell lymphoma (CTCL). Chemokine/chemokine receptor interactions have been implicated in the homing of lymphoma cells to various tissue sites. We investigated tissue samples and tumor cell suspensions of patients with CD30(+) CTCL (n = 8) and CD30(-) CTCL (mycosis fungoides, n = 6; Sézary syndrome, n = 6) for expression of the chemokine receptors CCR3, CCR4, and CCR8 and the CCR3 ligands eotaxin/CCL11, monocyte chemoattractant protein 3 (MCP-3)/CCL7, and RANTES (regulated on activation, normal T expressed and secreted)/CCL5. Of 8 CD30(+) CTCLs, 7 expressed CCR3, 4 CCR4, and none CCR8. CCR3 expression was not found in skin tissue samples from 12 CD30(-) CTCLs. Coexpression of CCR3 and CD30 was demonstrated by flow cytometry in tumor cell suspensions. Internalization experiments demonstrated functionality of CCR3 expressed by freshly isolated tumor cells. Actin polymerization as well as migration in response to eotaxin was demonstrated in a CD30(+) cutaneous lymphoma cell line. CCR3 ligand eotaxin/CCL11 was detected in lesional skin of CD30(+) CTCL by immunohistochemistry, preferentially in tumor cells. Eotaxin/CCL11 expression in tumor cells was confirmed by intracellular immunofluorescence. Analysis of cytokine expression pattern of CCR3-bearing infiltrating cells showed a predominance of interleukin-4 (IL-4) but not interferon-gamma (IFN-gamma) protein expression,1 consistent with a T-helper 2 (Th-2) profile. These results suggest that expression of CCR3 and its ligand eotaxin/CCL11 plays a role in the recruitment and retention of CD30(+) malignant T cells to the skin.

Substantial data show that infection with helminth parasites ameliorates colitis; however, oxazolone-induced colitis is exaggerated in mice infected with the tapeworm, Hymenolepis diminuta. We tested the hypothesis that the IL-5 response to helminth infection enhances the severity of oxazolone-induced colitis. Mice were infected with H. diminuta and 8 days later were treated with oxazolone ± anti-IL-5 antibodies. Colitis was assessed 72 hours postoxazolone treatment by disease activity scores, myeloperoxidase activity, and histopathology. Other mice received injections of a replication-deficient adenovirus that carried the IL-5 (Ad.IL-5) gene or a control adenovirus (Ad.delete) ± oxazolone. The effect of H. diminuta+oxazolone in CCL11/CCL22 (eotaxin-1 and 2) knockout (KO) mice was determined. Helminth infection and Ad.IL-5 treatment increased IL-5 and eosinophil numbers. In vivo neutralization of IL-5 significantly reduced the severity of colitis in H. diminuta+oxazolone-treated mice, and H. diminuta did not exaggerate oxazolone-induced colitis in CCL11/CCL22 KO mice. Mice receiving Ad.IL-5 only had no colitis, while oxazolone-induced colitis was more severe in animals cotreated with Ad.IL-5 (Ad.delete + oxazolone was not significantly different from oxazolone only). Thus, while there is much to be gleaned about antiinflammatory mechanisms from rodent-helminth model systems, these data illustrate the caveat that infection with helminth parasites as a therapy could be contraindicated in patients with eosinophilia or elevated IL-5 unless coupled to appropriate measures to block IL-5 and/or eosinophil activity.

Infection with respiratory syncytial virus (RSV) in neonatal mice leads to exacerbated disease if mice are reinfected with the same virus as adults. Both T cells and the host major histocompatibility complex genotype contribute to this phenomenon, but the part played by innate immunity has not been defined. Since macrophages and natural killer (NK) cells play key roles in regulating inflammation during RSV infection of adult mice, we studied the role of these cells in exacerbated inflammation following neonatal RSV sensitization/adult reinfection. Compared to mice undergoing primary infection as adults, neonatally sensitized mice showed enhanced airway fluid levels of interleukin-6 (IL-6), alpha interferon (IFN-α), CXCL1 (keratinocyte chemoattractant/KC), and tumor necrosis factor alpha (TNF-α) at 12 to 24 h after reinfection and IL-4, IL-5, IFN-γ, and CCL11 (eotaxin) at day 4 after reinfection. Weight loss during reinfection was accompanied by an initial influx of NK cells and granulocytes into the airways and lungs, followed by T cells. NK cell depletion during reinfection attenuated weight loss but did not alter T cell responses. Depletion of alveolar macrophages with inhaled clodronate liposomes reduced both NK and T cell numbers and attenuated weight loss. These findings indicate a hitherto unappreciated role for the innate immune response in governing the pathogenic recall responses to RSV infection.

Systemic inflammation shifts the brain microenvironment towards a proinflammatory state. However, how peripheral inflammation mediates changes in the brain remains to be clarified. We aimed to identify hippocampal cells and cytokines that respond to endotoxemia. Mice were intraperitoneally injected with lipopolysaccharide (LPS) or saline, and examined 1, 4, and 24 h after injection. Tissue cytokine concentrations in the spleens and hippocampi were determined by multiplex assays. Another group of mice were studied immunohistologically. Fourteen cytokines showed an increased concentration in the spleen, and 10 showed an increase in the hippocampus after LPS injection. Cytokines increased at 4 h (CCL2, CXCL1, CXCL2, and interleukin-6) were expressed by leptomeningeal stromal cells, choroid plexus stromal cells, choroid plexus epithelial cells, and hippocampal vascular endothelial cells, all of which were located at the brain-immune interface. Receptors for these cytokines were expressed by astrocytic endfeet. Cytokines increased at 24 h (CCL11, CXCL10, and granulocyte-colony stimulating factor) were expressed by astrocytes. Cells of the brain-immune interface therefore respond to endotoxemia with cytokine signals earlier than hippocampal parenchymal cells. In the parenchyma, astrocytes play a key role in responding to signals by using endfeet located in close apposition to the interface cells via cytokine receptors.

BACKGROUND

We reported the emergence of a distinct myelitis in patients with atopic diathesis (atopic myelitis [AM]) by a nationwide survey throughout Japan. Similar cases have recently been reported in Caucasians. Pathologic studies of biopsied spinal cord specimens revealed chronic active inflammation with eosinophilic infiltration.

OBJECTIVE

To clarify the cytokine/chemokine alterations in CSF from patients with AM in comparison to other causes of myelitis.

METHODS

We measured 27 cytokines, chemokines, and growth factors simultaneously in CSF from 22 patients with AM, 20 with opticospinal multiple sclerosis (OSMS), 11 with HTLV-1-associated myelopathy (HAM), 9 with Sjögren syndrome-related myelitis (SM), and 20 with other noninflammatory neurologic diseases (OND), using a fluorescent bead-based immunoassay.

RESULTS

In patients with AM, CCL11 and interleukin (IL)-9 were significantly increased as compared with patients with OND and other myelitis while in patients with OSMS interferon-gamma and granulocyte-colony stimulating factor levels were significantly higher than in patients with OND and other causes of myelitis. Significant increase of IL-17 in comparison to patients with OND was found only in patients with OSMS, irrespective of presence or absence of anti-aquaporin-4 (AQP4) antibody. In patients with HAM, CXCL10 and CCL5 were higher than in patients with OND and other myelitis. In patients with SM, CCL3 and CCL4 were higher than in patients with OND. In patients with AM, CCL11, IL-9, and IL-1 receptor antagonist (IL-1ra) showed positive correlations with the final Kurtzke Expanded Disability Status Scale scores while IL-1ra and IL-12(p70) had positive correlations with disease duration.

CONCLUSIONS

Intrathecal upregulation of CCL11 and Th2 cytokines is characteristic of atopic myelitis, which is distinct from interleukin-17/interferon-gamma-related autoimmune condition of opticospinal multiple sclerosis.