Activation of MIP-2 and MCP-5 Expression in Methylmercury-Exposed Mice and Their Suppression by N-Acetyl-L-Cysteine.

Methylmercury (MeHg) is a well-known neurotoxin of the central nervous system (CNS). Neuroinflammation is one of the main pathways of MeHg-induced CNS impairment. This study aims to investigate the expressions of IL-6, MIP-2, and MCP-5, as biomarkers in relation with MeHg-induced CNS impairment and N-acetyl-L-cysteine (NAC) treatment in mice, as well as histopathological changes of brain tissue and clinical symptom such as ataxia. Twenty male Balb/c mice, aged 8-9 weeks, were divided into 4 groups and treated with saline (control), NAC [150 mg/kg body weight (BW) day], MeHg (4 mg Hg/kg BW), or a combination of MeHg and NAC for 17 days. MeHg induced the expression of IL-6, MIP-2, and MCP-5 in the serum, with median values (those in controls) of 55.06 (9.44), 15.94 (9.30), and 458.91 (239.91) mg/dl, respectively, and a statistical significance was observed only in IL-6 expression (p < 0.05). MIP-2 and MCP-5 expressions tended to increase in the cerebrum of MeHg-treated group compared with controls; however, the difference was not statistically significant. MeHg treatment also increased IL-6 expression in the cerebellum (7.73 and 4.81 mg/dl in MeHg-treated group and controls, respectively), with a marginal significance. NAC significantly suppressed MeHg-induced IL-6 and MIP-2 expressions in the serum (p < 0.05 for both), and slightly reduced MCP-5 expression in the cerebrum. Ataxia was observed in all MeHg-treated mice after 9-day exposure as well as the decrease of intact Purkinje cells in brain tissue (p < 0.05). These findings suggest that MeHg induced neurotoxicity by elevating the expression of IL-6, MIP-2, and MCP-5 and causing ataxia symptoms, and NAC reduced MeHg-mediated effects on the CNS.

Prostaglandin E2 and an EP4 receptor agonist inhibit LPS-Induced monocyte chemotactic protein 5 production and secretion in mouse cardiac fibroblasts via Akt and NF-κB signaling.

BACKGROUND: Prostaglandin E2 (PGE2) signals through 4 separate G-protein coupled receptor sub-types to elicit a variety of physiologic and pathophysiological effects. We have previously reported that mice lacking the EP4 receptor in the cardiomyocytes develop heart failure with a phenotype of dilated cardiomyopathy. Also, these mice have increased levels of chemokines, like MCP-5, in their left ventricles. We have recently reported that overexpression of the EP4 receptor could improve cardiac function in the myocardial infarction model. Furthermore, we showed that overexpression of EP4 had an anti-inflammatory effect in the whole left ventricle. It has also been shown that PGE2 can antagonize lipopolysaccharide-induced secretion of chemokines/cytokines in various cell types. We therefore hypothesized that PGE2 inhibits lipopolysaccharide (LPS)-induced MCP-5 secretion in adult mouse cardiac fibroblasts via its EP4 receptor. METHODS AND RESULTS: Our hypothesis was tested using isolated mouse adult ventricular fibroblasts (AVF) treated with LPS. Pre-treatment of the cells with PGE2 and the EP4 agonist CAY10598 resulted in reductions of the pro-inflammatory response induced by LPS. Specifically, we observed reductions in MCP-5 secretion. Western blot analysis showed reductions in phosphorylated Akt and IκBα indicating reduced NF-κB activation. The anti-inflammatory effects of PGE2 and EP4 agonist signaling appeared to be independent of cAMP, p-44/42, or p38 pathways. CONCLUSION: Exogenous treatment of PGE2 and the EP4 receptor agonist blocked the pro-inflammatory actions of LPS. Mechanistically, this was mediated via reduced Akt phosphorylation and inhibition of NF-κB.

Diets containing pomegranate polyphenol and soy isoflavone attenuate contact hypersensitivity in mice.

Contact hypersensitivity (CHS) is frequently used as an animal model for human allergic contact dermatitis (ACD). Diets of pomegranate polyphenols (PPs) or soy isoflavones (SIs) each alleviated CHS symptoms; however, the effect of diets containing a mixture of PPs and SIs on CHS is unclear. We investigated the CHS-inhibitory effects of diets supplemented with a mixture of PPs and SIs at human physiologically relevant doses. Consuming the mixture of PPs and SIs attenuated ear swelling and reduced infiltration of Gr-1-positive cells. Ear swelling decreased in the PP and SI-treated mice compared to the SI-treated mice. The auricle tissues of the PP and SI-fed mice exhibited decreased production of CXCL2 and MCP-5 compared to the SI- and PP-treated mice, respectively. These results suggest that dietary supplementation with a mixture of PPs and SIs may have ACD-preventive effects and may prove more beneficial than supplementation with PPs or SIs alone.

Induction of the MCP chemokine cluster cascade in the periphery by cancer cell-derived Ccl3.

The induction of localized pro-inflammatory niches in the periphery is instrumental in metastasis. In order to better understand how tumors engage distal sites and activate a pro-inflammatory response we utilized syngeneic breast cancers as a model and showed that soluble factors from the neoplastic epithelium activate the expression of the monocyte chemoattractive protein (MCP) chemokines of the mouse 11C cluster that include Ccl1, Ccl2, Ccl7, Ccl8, Ccl11 and Ccl12. Tissues such as the lungs and the brain, that are more prone to colonization by breast cancer cells, were more sensitive to MCP cluster chemokine induction than others such as the liver. Subsequent analyses involving chemokine arrays in breast cancer cells and media followed by functional validation assays in in vitro and in vivo identified the cytokine Ccl3 as the principle mediator of the communication between the neoplastic epithelium and the peripheral tissues in terms of MCP cluster chemokine induction. Our results show that MCP chemokines are activated in peripheral tissues of breast cancer-bearing mice, by a mechanism that involves breast cancer cell-derived Ccl3. Interference with the expression of cancer cell-derived Ccl3 may find application in the management of breast cancer metastases.

The triterpenoid CDDO-imidazolide reduces immune cell infiltration and cytokine secretion in the KrasG12D;Pdx1-Cre (KC) mouse model of pancreatic cancer.

Because the 5-year survival rate for pancreatic cancer remains under 10%, new drugs are needed for the prevention and treatment of this devastating disease. Patients with chronic pancreatitis have a 12-fold higher risk of developing pancreatic cancer. LSL-KrasG12D/+;Pdx-1-Cre (KC) mice replicate the genetics, symptoms and histopathology found in human pancreatic cancer. Immune cells infiltrate into the pancreas of these mice and produce inflammatory cytokines that promote tumor growth. KC mice are particularly sensitive to the effects of lipopolysaccharide (LPS), as only 48% of KC mice survived an LPS challenge while 100% of wildtype (WT) mice survived. LPS also increased the percentage of CD45+ immune cells in the pancreas and immunosuppressive Gr1+ myeloid-derived suppressor cell in the spleen of these mice. The triterpenoid CDDO-imidazolide (CDDO-Im) not only reduced the lethal effects of LPS (71% survival) but also decreased the infiltration of CD45+ cells into the pancreas and the percentage of Gr1+ myeloid-derived suppressor cell in the spleen of KC mice 4-8 weeks after the initial LPS challenge. While the levels of inflammatory cytokine levels were markedly higher in KC mice versus WT mice challenged with LPS, CDDO-Im significantly decreased the production of IL-6, CCL-2, vascular endothelial growth factor and G-CSF in the KC mice. All of these cytokines are prognostic markers in pancreatic cancer or play important roles in the progression of this disease. Disrupting the inflammatory process with drugs such as CDDO-Im might be useful for preventing pancreatic cancer, especially in high-risk populations.

Contrasting mechanisms by which social isolation and restraint impair healing in male mice.

Stress modulates vital aspects of immune functioning in both human and non-human animals, including tissue repair. For example, dermal wounds heal more slowly and are associated with prolonged inflammation and increased bacterial load in mice that experience chronic physical restraint. Social stressors also negatively affect healing; however, previous studies suggest that the affected healing mechanisms may be stress model-specific. Here, the effects of either social isolation or physical restraint on dermal wound healing (3.5 mm wounds on the dorsum) were compared in hairless male mice. Social isolation beginning 3 weeks prior to wounding delayed healing comparably to physical restraint (12 h/day for eight days), in spite of marked differences in metabolic and hormonal consequences (i.e. body mass) between the two stress models. Additionally, isolated mice exhibited reductions in wound bacterial load and inflammatory gene expression (interleukin-1beta [IL-1ß], monocyte chemoattractant protein [MCP]), whereas restraint significantly increased both of these parameters relative to controls. Experimentally augmenting bacterial concentrations in wounds of isolated mice did not ameliorate healing, whereas this treatment accelerated healing in controls. This work indicates that social isolation and restraint stressors comparably impair healing, but do so through disparate mechanisms and at different phases of healing.

Hypoxia-reoxygenation affects whole-genome expression in the newborn eye.

PURPOSE: Resuscitation of newborns is one of the most frequent procedures in neonatal medicine. The use of supplementary oxygen during resuscitation of the asphyxiated newborn has been shown to be detrimental to vulnerable tissues. We wanted to assess transcriptional changes in ocular tissue after the acute use of oxygen in the delivery room in a hypoxia-reoxygenation model of the newborn mouse. METHODS: C57BL/6 mice (n = 57), postnatal day 7, were randomized to receive either 120 minutes of hypoxia, at 8% O2, followed by 30 minutes of reoxygenation with 21, 40, 60, or 100% O2 or to normoxia followed by 30 minutes of 21% or 100% O2. Whole ocular homogenates were analyzed by Affymetrix 750k expression array, and RT-PCR was performed for validation. Bayesian analysis of variance for microarray data (BAMarray) was used to identify single significant genes, and Gene Set Enrichment Analysis (GSEA) was applied to reveal significant pathway systems. RESULTS: In total, ∼ 92% of the gene expression changes were altered in response to reoxygenation with 60% or 100% O2 compared to expression at the lower percentages of 21% and 40%. After 100% O2 treatment, genes involved in inflammation (Ccl12), angiogenesis (Igfr1, Stat3), and metabolism (Hk2) were upregulated. Pathway analyses after hypoxia-reoxygenation revealed significant alterations of six pathways which included apoptosis, TGF-beta signaling, oxidative phosphorylation, voltage-gated calcium channel complex, mitochondrion, and regulation of RAS protein signal transduction. CONCLUSIONS: Hypoxia-reoxygenation can induce immediate transcriptional responses in ocular tissue involving inflammation, angiogenesis, energy failure, and Ras signaling.

Influence of 2 cryopreservation methods to induce CCL-13 from dental pulp cells.

INTRODUCTION: Cryopreservation preserves periodontal ligament cells but has a lower success rate with dental pulp cells (DPCs) because it causes inflammation. There are 2 well-known cryopreservation methods that reduce inflammation, slow freezing and rapid freezing, but the effects of the 2 methods on inflammation are not well-established. The purpose of this study was to compare the effects of the 2 different cryopreservation methods on CCL-13 induction from DPCs by using microarrays, real-time polymerase chain reaction (PCR), Western blotting, enzyme-linked immunosorbent assay, and confocal laser scanning microscopy (CLSM). METHODS: In this study, the concentration of cryoprotectant was fixed, and the methods compared differed with respect to freezing speed. Initially we screened the DPCs of cryopreserved teeth with expression microarrays, and CCL-13 was identified as a differentially expressed gene involved in generalized inflammation. We then compared the expression of CCL-13 after exposing teeth to the 2 cryopreservation methods by using real-time PCR, Western blot, enzyme-linked immunosorbent assay, and CLSM. RESULTS: Expression of CCL-13 was up-regulated significantly only in the rapid freezing group, except in measurements made by real-time PCR. CLSM analysis also confirmed this up-regulation visually. CONCLUSIONS: Rapid freezing increased the expression of CCL-13 in DPCs compared with slow freezing. Understanding the inflammatory effect of cryopreservation should help to establish an optimal cryoprofile to minimize inflammation of DPCs and reduce the need for endodontic treatment.

Estrogen inhibits apoptosis and promotes CC motif chemokine ligand 13 expression on synovial fibroblasts in rheumatoid arthritis.

OBJECTIVE: Female patients have a higher prevalence of rheumatoid arthritis (RA) than male patients, suggesting that female sex hormones contribute to the disease pathogenesis. We herein report the findings of our study, which was conducted to clarify the role of estrogen in the pathogenesis of RA. METHODS: Cultured human synovial fibroblasts from a patient with RA were treated with 17ß-estradiol (E(2)). The effects of E(2) against cellular activation and apoptosis were evaluated. To identify the disease-related genes altered by E(2) treatment, the changes in the gene expression of the cells stimulated with and without E(2) were evaluated using a microarray analysis. RESULTS: We found that E(2)-mediated cellular activation signaling through extracellular signal-regulated kinase (ERK)-1/2. E(2) possessed a suppressive effect for apoptosis and a promotive effect for tumor necrosis factor (TNF)-α-induced matrix metalloproteinase (MMP)-3 production on the synovial fibroblasts. A microarray analysis revealed that E(2) profoundly upregulated CC motif chemokine ligand 13 (CCL13) gene expression. CONCLUSIONS: E(2) could mediate cellular activation signaling through ERK-1/2 on the synovial fibroblasts. The present data suggest that E(2) has adverse effects on the pathogenesis of RA as a result of unregulated cell death, increased TNF-α-induced MMP-3 production, and CCL13 overproduction, subsequently resulting in the disease progression of RA.

NADPH oxidase-derived reactive oxygen species increases expression of monocyte chemotactic factor genes in cultured adipocytes.

Excess glucose and free fatty acids delivered to adipose tissue causes local inflammation, which contributes to insulin resistance. Glucose and palmitate generate reactive oxygen species (ROS) in adipocytes, leading to monocyte chemotactic factor gene expression. Docosahexaenoate (DHA) has the opposite effect. In this study, we evaluated the potential sources of ROS in the presence of excess nutrients. Differentiated 3T3-L1 adipocytes were exposed to palmitate and DHA (250 µM) in either 5 or 25 mM glucose to evaluate the relative roles of mitochondrial electron transport and NADPH oxidases (NOX) as sources of ROS. Excess glucose and palmitate did not increase mitochondrial oxidative phosphorylation. However, glucose exposure increased glycolysis. Of the NOX family members, only NOX4 was expressed in adipocytes. Moreover, its activity was increased by excess glucose and palmitate and decreased by DHA. Silencing NOX4 inhibited palmitate- and glucose-stimulated ROS generation and monocyte chemotactic factor gene expression. NADPH, a substrate for NOX, and pentose phosphate pathway activity increased with glucose but not palmitate and decreased with DHA exposure. Inhibition of the pentose phosphate pathway by glucose-6-phosphate dehydrogenase inhibitors and siRNA suppressed ROS generation and monocyte chemotactic factor gene expression induced by both glucose and palmitate. Finally, both high glucose and palmitate induced NOX4 translocation into lipid rafts, effects that were blocked by DHA. Excess glucose and palmitate generate ROS via NOX4 rather than by mitochondrial oxidation in cultured adipocytes. NOX4 is regulated by both NADPH generated in the PPP and translocation of NOX4 into lipid rafts, leading to expression of monocyte chemotactic factors.

Latent infection by γherpesvirus stimulates profibrotic mediator release from multiple cell types.

Although γherpesvirus infections are associated with enhanced lung fibrosis in both clinical and animal studies, there is limited understanding about fibrotic effects of γherpesviruses on cell types present in the lung, particularly during latent infection. Wild-type mice were intranasally infected with a murine γherpesvirus (γHV-68) or mock-infected with saline. Twenty-eight days postinfection (dpi), ∼14 days following clearance of the lytic infection, alveolar macrophages (AMs), mesenchymal cells, and CD19-enriched cell populations from the lung and spleen express M(3) and/or glycoprotein B (gB) viral mRNA and harbor viral genome. AMs from infected mice express more transforming growth factor (TGF)-ß(1), CCL2, CCL12, TNF-α, and IFN-γ than AMs from mock-infected mice. Mesenchymal cells express more total TGF-ß(1), CCL12, and TNF-α than mesenchymal cells from mock-infected mice. Lung and spleen CD19-enriched cells express more total TGF-ß(1) 28 dpi compared with controls. The CD19-negative fraction of the spleen overexpresses TGF-ß(1) and harbors viral genome, but this likely represents infection of monocytes. Purified T cells from the lung harbor almost no viral genome. Purified T cells overexpress IL-10 but not TGF-ß(1). Intracellular cytokine staining demonstrated that lung T cells at 28 dpi produce IFN-γ but not IL-4. Thus infection with a murine γherpesvirus is sufficient to upregulate profibrotic and proinflammatory factors in a variety of lung resident and circulating cell types 28 dpi. Our results provide new information about possible contributions of these cells to fibrogenesis in the lungs of individuals harboring a γherpesvirus infection and may help explain why γHV-68 infection can augment or exacerbate fibrotic responses in mice.

Protease-activated receptor 1 activation is necessary for monocyte chemoattractant protein 1-dependent leukocyte recruitment in vivo.

Thrombin, acting through a family of protease-activated receptors (PARs), is known to amplify inflammatory responses, but the in vivo importance of PARs in inflammation is not fully appreciated. In a mouse heart-to-rat transplant model, where it is possible to distinguish graft (mouse) from systemic (rat) chemokines, we show that donor PAR-1 is required to generate the local monocyte chemoattractant protein (MCP)-1 needed to recruit rat natural killer cells and macrophages into the hearts. We have confirmed the importance of this mechanism in a second model of thioglycollate-induced peritonitis and also show that PAR-1 is important for the production of MCP-3 and MCP-5. Despite the presence of multiple other mediators capable of stimulating chemokine production in these models, these data provide the first evidence that thrombin and PAR activation are required in vivo to initiate inflammatory cell recruitment.

A role of monocyte chemoattractant protein-4 (MCP-4)/CCL13 from chondrocytes in rheumatoid arthritis.

We studied the role of monocyte chemoattractant (MCP)-4/CCL13 in the pathogenesis of rheumatoid arthritis (RA). MCP-4 was highly expressed in cartilage from RA patients. Interferon-gamma significantly stimulated MCP-4/CCL13 production in human chondrocytes, and this effect was enhanced in combination with interleukin-1beta or tumor necrosis factor-alpha. MCP-4/CCL13 induces the phosphorylation of extracellular signal-regulated kinase in fibroblast-like synoviocytes and activates cell proliferation, and PD98059 completely inhibits these effects. These data suggest that interferon-gamma in combination with interleukin-1beta/tumor necrosis factor-alpha activates the production of MCP-4/CCL13 from chondrocytes in RA joints, and that secreted MCP-4/CCL13 enhances fibroblast-like synoviocyte proliferation by activating the extracellular signal-regulated kinase mitogen-activated protein kinase cascade.

Differential regulation of chemokine expression by Th1 and Th2 cytokines and mechanisms of eotaxin/CCL-11 expression in human airway smooth muscle cells.

BACKGROUND: Airway smooth muscle (ASM) cells may contribute to the pathogenesis of asthma including airway inflammation and remodeling. We focused our study on the regulation of chemokine expression by cytokines and analyzed the mechanisms of eotaxin/CCL-11 expression in ASM cells. METHODS: Human ASM cells were cultured in vitro and treated with IL-4, interferon-gamma (IFNgamma), and tumor necrosis factor-alpha (TNFalpha). Secretion of chemokines into the culture medium was analyzed by ELISA. Expression of eotaxin mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Binding of transcription factor signal transducer activator of transcription (STAT) 6 to the eotaxin promoter-derived DNA was analyzed by pull-down Western blot. To assess transcriptional regulation of eotaxin, cells were transfected with eotaxin promoter-luciferase reporter plasmids, and activity was determined by dual luciferase assay. RESULTS: The Th2 cytokine IL-4 preferentially stimulated the expression of the CC chemokine receptor (CCR) 3-ligand chemokines eotaxin, eotaxin-3, and MCP-4. The Th1 cytokine IFNgamma stimulated the expression of chemokines IP-10 and RANTES. IL-4 stimulated nuclear translocation of signal transducer activator of transcription 6 (STAT6) and its binding to the eotaxin promoter region. IL-4 activated the eotaxin promoter and its activity was inhibited by mutation of the binding site for STAT6 in the promoter. CONCLUSIONS: The Th2 cytokine IL-4 preferentially stimulated the expression of CCR3 ligand chemokines including eotaxin in ASM cells. The transcription factor STAT6 may play a pivotal role in the activation of eotaxin transcription in response to IL-4.

Hypoxia-inducible factor-1 (HIF-1) is involved in the regulation of hypoxia-stimulated expression of monocyte chemoattractant protein-1 (MCP-1/CCL2) and MCP-5 (Ccl12) in astrocytes.

BACKGROUND: Neuroinflammation has been implicated in various brain pathologies characterized by hypoxia and ischemia. Astroglia play an important role in the initiation and propagation of hypoxia/ischemia-induced inflammation by secreting inflammatory chemokines that attract neutrophils and monocytes into the brain. However, triggers of chemokine up-regulation by hypoxia/ischemia in these cells are poorly understood. Hypoxia-inducible factor-1 (HIF-1) is a dimeric transcriptional factor consisting of HIF-1alpha and HIF-1beta subunits. HIF-1 binds to HIF-1-binding sites in the target genes and activates their transcription. We have recently shown that hypoxia-induced expression of IL-1beta in astrocytes is mediated by HIF-1alpha. In this study, we demonstrate the role of HIF-1alpha in hypoxia-induced up-regulation of inflammatory chemokines, human monocyte chemoattractant protein-1 (MCP-1/CCL2) and mouse MCP-5 (Ccl12), in human and mouse astrocytes, respectively. METHODS: Primary fetal human astrocytes or mouse astrocytes generated from HIF-1alpha+/+ and HIF-1alpha+/- mice were subjected to hypoxia (<2% oxygen) or 125 muM CoCl2 for 4 h and 6 h, respectively. The expression of HIF-1alpha, MCP-1 and MCP-5 was determined by semi-quantitative RT-PCR, western blot or ELISA. The interaction of HIF-1alpha with a HIF-1-binding DNA sequence was examined by EMSA and supershift assay. HIF-1-binding sequence in the promoter of MCP-1 gene was cloned and transcriptional activation of MCP-1 by HIF-1alpha was analyzed by reporter gene assay. RESULTS: Sequence analyses identified HIF-1-binding sites in the promoters of MCP-1 and MCP-5 genes. Both hypoxia and HIF-1alpha inducer, CoCl2, strongly up-regulated HIF-1alpha expression in astrocytes. Mouse HIF-1alpha+/- astrocytes had lower basal levels of HIF-1alpha and MCP-5 expression. The up-regulation of MCP-5 by hypoxia or CoCl2 in HIF-1alpha+/+ and HIF-1alpha+/- astrocytes was correlated with the levels of HIF-1alpha in cells. Both hypoxia and CoCl2 also up-regulated HIF-1alpha and MCP-1 expression in human astrocytes. EMSA assay demonstrated that HIF-1 activated by either hypoxia or CoCl2 binds to wild-type HIF-1-binding DNA sequence, but not the mutant sequence. Furthermore, reporter gene assay demonstrated that hypoxia markedly activated MCP-1 transcription but not the mutated MCP-1 promoter in transfected astrocytes. CONCLUSION: These findings suggest that both MCP-1 and MCP-5 are HIF-1 target genes and that HIF-1alpha is involved in transcriptional induction of these two chemokines in astrocytes by hypoxia.

West Nile virus encephalitis: sequential histopathological and immunological events in a murine model of infection.

West Nile virus (WNV) has emerged as an important cause of encephalitis in humans and horses in North America. Although there is significant knowledge about the pathogenesis of disease caused by this flavivirus and about the immunity against it, no reports exist describing the sequence of pathological changes and their correlation to the immune response in the brain following infection with WNV. In this report the authors describe the major histopathological changes, as well as changes in cytokine and chemokine expression, in brains from WNV-infected C57Bl/6 mice. During the course of infection skin, spleen and kidney were all sites of WNV replication before virus reached the brain. In brain, increased expression of the chemokines monocyte chemoattractant protein (MCP)-5 (CCL12), interferon gamma inducible protein 10 (IP-10; CXCL10), and monokine induced by gamma interferon (MIG; CXCL9) preceded the expression of interferon gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha), which have previously been considered to be key early cytokines in the pathogenesis and immune response of WNV encephalitis. These results suggest that the chemokines MCP-5, IP-10, and MIG are important triggers of inflammation in brain due to their early up-regulation following WNV infection.

Ligand up-regulation does not correlate with a role for CCR1 in pathogenesis in a mouse model of non-lymphocyte-mediated neurological disease.

CCR1 ligands, including CCL3, CCL5, and CCL7, are up-regulated in a number of neurological disorders in humans and animal models. CCR1 is expressed by multiple cell types in the central nervous system (CNS), suggesting that receptor signaling by neuronal cell types may influence pathogenesis. In the current study, the authors used a mouse model of retrovirus infection to study the contribution of CCR1 to neuropathogenesis in the absence of lymphocyte recruitment to the CNS. In this model, infection of neonatal mice with the neurovirulent retrovirus Fr98 results in increased expression of proinflammatory chemokines in the CNS, activation of glial cells, and development of severe neurological disease. Surprisingly, no difference in neuropathogenesis was observed between CCR1-sufficient and CCR1-deficient mice following infection with the neuropathogenic virus Fr98. CCR1 was also not necessary for control of virus replication in the brain or virus-induced activation of astroglia. Additionally, CCR1 deficiency did not affect the up-regulation of its ligands, CCL3, CCL5, or CCL7. Thus, CCR1 did not appear to have a notable role in Fr98-induced pathogenesis, despite the correlation between ligand expression and disease development. This suggests that in the absence of inflammation, CCR1 may have a very limited role in neuropathogenesis.

Protein expression pattern in experimental pneumococcal meningitis.

In this study, we investigated cytokine expression during experimental pneumococcal meningitis. Mice were intracisternally infected with Streptococcus pneumoniae and treated with ceftriaxone starting at 24 h after infection. At different time points before and after antibiotic therapy, the cytokine expression pattern was determined in mouse brains using protein arrays. Underlining the power of this method, the meningitis-relevant cytokines interleukin-1beta (IL-1beta), IL-6, KC, macrophage inflammatory protein-2 (MIP-2), and monocyte chemoattractant protein-1 (MCP-1/CCL2) were markedly elevated in infected animals. Newly identified proteins during the acute stage of the disease (until 30 h after infection) included lymphotactin (XCL-1), MIP-1gamma (CCL9) and MCP-5 (CCL12), cytokine responsive gene- 2 (CRG-2/CXCL10) and CXCL16, and insulin-like growth factor binding protein 3 (IGFBP3). During later stages, an induction of T-cell activation-3 (TCA-3/CCL1), platelet factor-4 (PF-4/CXCL4) and stromal derived factor-1alpha (SDF-1alpha/CXCL13), and IL-4 was observed. The validity of this method was supported by an additional ELISA analysis of the expression profile of CXCL16 and IGFBP3, which was identical to that observed by protein array. In conclusion, the use of protein array technology led to an extension of the current picture of protein expression in pneumococcal meningitis. Most important, new factors that might play a role in pneumococcal meningitis were identified.

Inflammatory responses to pneumovirus infection in IFN-alpha beta R gene-deleted mice.

Pneumonia virus of mice (PVM; family Paramyxoviridae) is a natural pathogen of rodents that reproduces important clinical features of severe respiratory syncytial virus infection in humans. As anticipated, PVM infection induces transcription of IFN antiviral response genes preferentially in wild-type over IFN-alphabetaR gene-deleted (IFN-alphabetaR-/-) mice. However, we demonstrate that PVM infection results in enhanced expression of eotaxin-2 (CCL24), thymus and activation-regulated chemokine (CCL17), and the proinflammatory RNase mouse eosinophil-associated RNase (mEar) 11, and decreased expression of monocyte chemotactic protein-5, IFN-gamma-inducible protein-10, and TLR-3 in lung tissue of IFN-alphabetaR-/- mice when compared with wild type. No differential expression of chemokines MIP-1alpha or MIP-2 or Th2 cytokines IL-4 or IL-5 was observed. Differential expression of proinflammatory mediators was associated with distinct patterns of lung pathology. The widespread granulocytic infiltration and intra-alveolar edema observed in PVM-infected, wild-type mice are replaced with patchy, dense inflammatory foci localized to the periphery of the larger blood vessels. Bronchoalveolar lavage fluid from IFN-alphabetaR-/- mice yielded 7- to 8-fold fewer leukocytes overall, with increased percentages of eosinophils, monocytes, and CD4+ T cells, and decreased percentage of CD8+ T cells. Differential pathology is associated with prolonged survival of the IFN-alphabetaR-/- mice (50% survival at 10.8 +/- 0.6 days vs the wild type at 9.0 +/- 0.3 days; p < 0.02) despite increased virus titers. Overall, our findings serve to identify novel transcripts that are differentially expressed in the presence or absence of IFN-alphabetaR-mediated signaling, further elucidating interactions between the IFN and antiviral inflammatory responses in vivo.

Synthesis and biological evaluation of 1,2,4-triazinylphenylalkylthiazolecarboxylic acid esters as cytokine-inhibiting antedrugs with strong bronchodilating effects in an animal model of asthma.

The influx of leukocytes (eosinophils, lymphocytes, and monocytes) into the airways and their production of proinflammatory cytokines contribute to the severity of allergic asthma. We describe here the synthesis and pharmacological evaluation of a series of triazinylphenylalkylthiazolecarboxylic acid esters that were designed to act as lung-specific antedrugs and inhibitors of the production of interleukin (IL)-5, a primary eosinophil-activating and proinflammatory cytokine. Closer examination of the hydroxypropyl ester, 15, indicated its high metabolic stability (t(1/2) > 240 min) in human lung S9 fraction but rapid conversion (t(1/2) = 15 min) into the pharmacologically inactive carboxylic acid by human liver preparations. In stimulated human whole blood cultures, 15 reduced not only the production of IL-5 (IC(50) = 78 nM) but also the biosynthesis of the monocyte chemotactic proteins MCP-1 (IC(50) = 220 nM), MCP-2 (IC(50) = 580 nM), and MCP-3 (IC(50) = 80 nM). In vivo, intratracheal administration of 15 (6 mg/animal) to allergic sheep, either before (-4 h) or after (+1.5 h) the pulmonary allergen challenge, completely abrogated the late-phase airway response and reduced the bronchial hyperreactivity to inhaled carbachol.