Naringenin suppresses neutrophil infiltration into adipose tissue in high-fat diet-induced obese mice.

Recruitment of immune cells to adipose tissue is altered dramatically in obesity, which results in chronic inflammation of the adipose tissue that leads to metabolic disorders, such as insulin resistance and type 2 diabetes mellitus. The regulation of immune cell infiltration into adipose tissue has prophylactic and therapeutic implications for obesity-related diseases. We previously showed that naringenin, a citrus flavonoid, suppressed macrophage infiltration into adipose tissue by inhibiting monocyte chemoattractant protein-1 (MCP-1) expression in the progression phase to high-fat diet (HFD)-induced obesity. In the current study, we evaluated the effects of naringenin on neutrophil infiltration into adipose tissue, because neutrophils also infiltrate into adipose tissue in the progression phase to obesity. Naringenin suppressed neutrophil infiltration into adipose tissue induced by the short-term (2 weeks) feeding of a HFD to mice. Naringenin tended to inhibit the HFD-induced expression of several chemokines, including MCP-1 and MCP-3, in adipose tissue. Naringenin also inhibited MCP-3 expression in 3T3-L1 adipocytes and a co-culture of 3T3-L1 adipocytes and RAW264 macrophages. However, naringenin did not affect the expression of macrophage inflammatory protein-2 (MIP-2), an important chemokine for neutrophil migration and activation, in macrophages or in a co-culture of adipocytes and macrophages. Our results suggest that naringenin suppresses neutrophil infiltration into adipose tissue via the regulation of MCP-3 expression and macrophage infiltration.

The primary growth of laryngeal squamous cell carcinoma cells in vitro is effectively supported by paired cancer-associated fibroblasts alone.

Most primarily cultured laryngeal squamous cell carcinoma cells are difficult to propagate in vitro and have a low survival rate. However, in our previous work to establish a laryngeal squamous cell carcinoma cell line, we found that laryngeal cancer-associated fibroblasts appeared to strongly inhibit the apoptosis of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In this study, we investigated whether paired laryngeal cancer-associated fibroblasts alone can effectively support the growth of primarily cultured laryngeal squamous cell carcinoma cells in vitro. In all, 29 laryngeal squamous cell carcinoma specimens were collected and primarily cultured. The laryngeal squamous cell carcinoma cells were separated from cancer-associated fibroblasts by differential trypsinization and continuously subcultured. Morphological changes of the cultured laryngeal squamous cell carcinoma cells were observed. Immunocytofluorescence was used to authenticate the identity of the cancer-associated fibroblasts and laryngeal squamous cell carcinoma cells. Flow cytometry was used to quantify the proportion of apoptotic cells. Western blot was used to detect the protein levels of caspase-3. Enzyme-linked immunosorbent assay was used to detect the levels of chemokine (C-X-C motif) ligand 12, chemokine (C-X-C motif) ligand 7, hepatocyte growth factor, and fibroblast growth factor 1 in the supernatants of the laryngeal squamous cell carcinoma and control cells. AMD3100 (a chemokine (C-X-C motif) receptor 4 antagonist) and an anti-chemokine (C-X-C motif) ligand 7 antibody were used to block the tumor-supporting capacity of cancer-associated fibroblasts. Significant apoptotic changes were detected in the morphology of laryngeal squamous cell carcinoma cells detached from cancer-associated fibroblasts. The percentage of apoptotic laryngeal squamous cell carcinoma cells and the protein levels of caspase-3 increased gradually in subsequent subcultures. In contrast, no significant differences in the proliferation capacity of laryngeal squamous cell carcinoma cells cocultured with cancer-associated fibroblasts were detected during subculturing. High level of chemokine (C-X-C motif) ligand 12 was detected in the culture supernatant of cancer-associated fibroblasts. The tumor-supporting effect of cancer-associated fibroblasts was significantly inhibited by AMD3100. Our findings demonstrate that the paired laryngeal cancer-associated fibroblasts alone are sufficient to support the primary growth of laryngeal squamous cell carcinoma cells in vitro and that the chemokine (C-X-C motif) ligand 12/chemokine (C-X-C motif) receptor 4 axis is one of the major contributors.

Brain metastasis in renal cancer patients: metastatic pattern, tumour-associated macrophages and chemokine/chemoreceptor expression.

BACKGROUND: The mechanisms of brain metastasis in renal cell cancer (RCC) patients are poorly understood. Chemokine and chemokine receptor expression may contribute to the predilection of RCC for brain metastasis by recruitment of monocytes/macrophages and by control or induction of vascular permeability of the blood-brain barrier. METHODS: Frequency and patterns of brain metastasis were determined in 246 patients with metastatic RCC at autopsy. Expression of CXCR4, CCL7 (MCP-3), CCR2 and CD68(+) tumour-associated macrophages (TAMs) were analysed in a separate series of 333 primary RCC and in 48 brain metastases using immunohistochemistry. RESULTS: Fifteen percent of 246 patients with metastasising RCC had brain metastasis. High CXCR4 expression levels were found in primary RCC and brain metastases (85.7% and 91.7%, respectively). CCR2 (52.1%) and CCL7 expression (75%) in cancer cells of brain metastases was more frequent compared with primary tumours (15.5% and 16.7%, respectively; P<0.0001 each). The density of CD68(+) TAMs was similar in primary RCC and brain metastases. However, TAMs were more frequently CCR2-positive in brain metastases than in primary RCC (P<0.001). CONCLUSION: Our data demonstrate that the monocyte-specific chemokine CCL7 and its receptor CCR2 are expressed in tumour cells of RCC. We conclude that monocyte recruitment by CCR2 contributes to brain metastasis of RCC.

B lymphocytes trigger monocyte mobilization and impair heart function after acute myocardial infarction.

Acute myocardial infarction is a severe ischemic disease responsible for heart failure and sudden death. Here, we show that after acute myocardial infarction in mice, mature B lymphocytes selectively produce Ccl7 and induce Ly6C(hi) monocyte mobilization and recruitment to the heart, leading to enhanced tissue injury and deterioration of myocardial function. Genetic (Baff receptor deficiency) or antibody-mediated (CD20- or Baff-specific antibody) depletion of mature B lymphocytes impeded Ccl7 production and monocyte mobilization, limited myocardial injury and improved heart function. These effects were recapitulated in mice with B cell-selective Ccl7 deficiency. We also show that high circulating concentrations of CCL7 and BAFF in patients with acute myocardial infarction predict increased risk of death or recurrent myocardial infarction. This work identifies a crucial interaction between mature B lymphocytes and monocytes after acute myocardial ischemia and identifies new therapeutic targets for acute myocardial infarction.

Epigenetic transcriptional activation of monocyte chemotactic protein 3 contributes to long-lasting neuropathic pain.

A multiplex analysis for profiling the expression of candidate genes along with epigenetic modification may lead to a better understanding of the complex machinery of neuropathic pain. In the present study, we found that partial sciatic nerve ligation most remarkably increased the expression of monocyte chemotactic protein 3 (MCP-3, known as CCL7) a total of 33 541 genes in the spinal cord, which lasted for 4 weeks. This increase in MCP-3 gene transcription was accompanied by the decreased trimethylation of histone H3 at Lys27 at the MCP-3 promoter. The increased MCP-3 expression associated with its epigenetic modification observed in the spinal cord was almost abolished in interleukin 6 knockout mice with partial sciatic nerve ligation. Consistent with these findings, a single intrathecal injection of recombinant proteins of interleukin 6 significantly increased MCP-3 messenger RNA with a decrease in the level of Lys27 trimethylation of histone H3 at the MCP-3 promoter in the spinal cord of mice. Furthermore, deletion of the C-C chemokine receptor type 2 (CCR2) gene, which encodes a receptor for MCP-3, failed to affect the acceleration of MCP-3 expression in the spinal cord after partial sciatic nerve ligation. A robust increase in MCP-3 protein, which lasted for up to 2 weeks after surgery, in the dorsal horn of the spinal cord of mice with partial sciatic nerve ligation was seen mostly in astrocytes, but not microglia or neurons. On the other hand, the increases in both microglia and astrocytes in the spinal cord by partial sciatic nerve ligation were mostly abolished in interleukin 6 knockout mice. Moreover, this increase in microglia was almost abolished by CCR2 gene deletion, whereas the increase in astrocytes was not affected in nerve-ligated mice that lacked the CCR2 gene. We also found that either in vivo or in vitro treatment with MCP-3 caused robust microglia activation. Under these conditions, intrathecal administration of MCP-3 antibody suppressed the increase in microglia within the mouse spinal cord and neuropathic pain-like behaviours after nerve injury. With the use of a functional magnetic resonance imaging analysis, we demonstrated that a single intrathecal injection of MCP-3 induced dramatic increases in signal intensity in pain-related brain regions. These findings suggest that increased MCP-3 expression associated with interleukin 6 dependent epigenetic modification at the MCP-3 promoter after nerve injury, mostly in spinal astrocytes, may serve to facilitate astrocyte-microglia interaction in the spinal cord and could play a critical role in the neuropathic pain-like state.

CC chemokine ligand 7 expression in liver metastasis of colorectal cancer.

The main cause of death for colorectal cancer (CRC) patients is the development of metastatic lesions at sites distant from the primary tumor. Therefore, it is important to find biomarkers that are related to the metastasis and to study the possible mechanisms. Recent data have shown that soluble attractant molecules called chemokines support the metastasis of certain cancers to certain organs. To identify molecular regulators that are differentially expressed in liver metastasis of CRC, PCR array analysis was performed and CC chemokine ligand 7 (CCL7) showed remarkable overexpression in liver metastatic tumor tissues. To validate the results of the PCR array, 30 patients with primary CRC and liver metastases were selected. Immunohistochemistry and real-time PCR analysis showed that CCL7 was expressed in normal colonic epithelium and the expression was higher in liver metastases compared to primary CRC (p<0.001). Real-time PCR showed that the expression of CCR1, CCR2 and CCR3 was also higher in liver metastases compared to primary CRC (p=0.001, p=0.033 and p<0.001, respectively). In conclusion, correlation of CCL7 overexpression and its receptor expression with colon cancer liver metastasis suggests that CCL7 as a novel target in liver metastasis of CRC may be of potential clinical value for the prevention of hepatic recurrences.

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice.

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.

Expression of monocyte chemotactic protein 3 following simulated birth trauma in a murine model of obesity.

OBJECTIVE: To determine the effect of obesity on simulated birth trauma in leptin-deficient obese mice as measured by relative monocyte chemotactic protein 3 (MCP-3) expression. MATERIALS AND METHODS: A total of 25 wild-type and 25 obese C57BL/6 virgin female mice underwent 1 hour of vaginal distension (VD), sham VD, or anesthesia without VD. Pelvic organ tissues were then harvested either immediately or 24-hours post VD and subsequent real-time polymerase chain reaction analysis was performed. RESULTS: Urethral MCP-3 levels in wild-type mice were elevated from baseline at 0 hours with a return to baseline at 24 hours in both VD and sham VD groups. In obese mice, there was a 6-fold elevation in MCP-3 levels at 0 hours after sham VD vs control (P <.05), which then returned to baseline levels at 24 hours. After undergoing VD, MCP-3 levels increased to 6-fold baseline values (P = .002) at 0 hours, with continued elevation in MCP-3 levels to 15 times control levels (P = .0003) at 24 hours. CONCLUSIONS: MCP-3 is significantly over-expressed in the urethral tissues of both wild-type and obese mice immediately after any urethral manipulation. At 24 hours, the MCP-3 expression patterns become divergent between VD and sham VD in obese mice. With a greater degree of trauma, MCP-3 continued to rise at 24 hours, suggesting that the underlying obesity resulted in alterations in response to tissue injury, paralleling the degree of injury. Such associations warrant further investigation into the role of MCP-3 as a chemokine for stem cell migration, with implications for subsequent tissue repair mechanisms after birth trauma.

Effects of caffeoylquinic acid derivatives and C-flavonoid from Lychnophora ericoides on in vitro inflammatory mediator production.

In this study we aimed at evaluating the effect of the major polar constituents of the medicinal plant Lychnophora ericoides on the production of inflammatory mediators produced by LPS-stimulated U-937 cells. The 6,8-di-C-beta-glucosylapigenin (vicenin-2) presented no effect on tumor necrosis factor (TNF)-alpha production, but inhibited, in a dose-dependent manner, the production of prostaglandin (PG) E2 without altering the expression of cyclooxygenase (COX)-2 protein. 3,5-Dicaffeoylquinic acid and 4,5-dicaffeoylquinic acid, at lower concentrations, had small but significant effects on reducing PGE2 levels; at higher doses these compounds stimulated PGE2 and also TNF-alpha production by the cells. All the caffeoylquinic acid derivatives, in a dose-dependent fashion, were able to inhibit monocyte chemoattractant protein-3 synthesis/release, with 4,5-DCQ being the most potent at the highest tested concentration. These results add important information on the effects of plant natural polyphenols, namely vicenin-2 and caffeoylquinic acid derivatives, on the production of inflammatory mediators by cultured cells.

Identification of potential genomic biomarkers for early detection of chemically induced cardiotoxicity in rats.

Cardiotoxicity represents one of the most serious side effects of new drugs. It is essential for pharmaceutical companies to detect potential cardiotoxicity of candidate drugs in non-clinical studies during the early stages of drug development. In this study, we aimed to detect potential genomic biomarkers of rat cardiotoxicity using a toxicogenomics approach. In order to achieve this, we induced cardiac lesions in rats following treatment with the three prototypical cardiotoxic compounds isoproterenol, doxorubicin and carbofuran. We then undertook histopathological examination and microarray analysis at 8 or 24h after single dosing. Using statistical and cluster analysis, we extracted 36 probe sets commonly up-regulated by the three cardiotoxic compounds. GO analysis revealed that these genes were functionally associated with either chemotaxis, tissue regeneration, positive regulation of cell proliferation, cellular organization and morphogenesis events in accordance with the degeneration of myocardium and inflammation observed in the histopathology analysis. Most of selected genes showed transient up-regulation at different time point for each compound. However, among these genes, Spp1, Fhl1, Timp1, Ccl7 and Reg3b revealed a sustained up-regulation with high expression levels at both time points for all three compounds. In conclusion, even though definitive validation studies are required for the establishment of their usefulness and reliability, these identified genes may prove to be the most promising candidate genomic biomarkers of cardiotoxicity in rats.

Paradoxical effects of PDGF-BB overexpression in endothelial cells on engineered blood vessels in vivo.

Therapeutic revascularization with either exogenous angiogenic growth factors or vascular cells has yet to demonstrate efficacy in the clinic. Injection of angiogenic growth factors often produces unstable and abnormal blood vessels. Blood vascular networks derived from implanted endothelial cells persist only transiently due to the insufficient recruitment of perivascular cells. We hypothesize that a combination of the two approaches may act synergistically to yield a better result. To enhance the recruitment of perivascular cells, human umbilical vein endothelial cells were genetically modified to overexpress platelet-derived growth factor (PDGF)-BB. PDGF-BB overexpression promoted both proliferation and migration of perivascular precursor cells (10T1/2 cells) in vitro. When mock-infected endothelial cells were implanted alone in vivo, they formed transient blood vascular networks that regressed by day 30. PDGF-BB overexpression enhanced the survival of endothelial cells in vivo. However, the PDGF-BB-expressing vessel network failed to establish patent blood flow. Co-implantation of PDGF-BB-overexpressing endothelial cells with 10T1/2 cells paradoxically resulted in the rapid regression of the vascular networks in vivo. PDGF-BB stimulated the expression of both chemokine (C-C motif) ligand 2 (CCL2) and CCL7 in 10T1/2 cells and led to the increased accumulation of macrophages in vivo. These results suggest a potential negative interaction between angiogenic growth factors and vascular cells; their use in combination should be carefully tested in vivo for such opposing effects.

Oncostatin M-induced and constitutive activation of the JAK2/STAT5/CIS pathway suppresses CCL1, but not CCL7 and CCL8, chemokine expression.

The recruitment of leukocytes to injured tissue is crucial for the initiation of inflammatory responses as well as for immune surveillance to fight tumor progression. In this study, we show that oncostatin M, a member of the IL-6-type cytokine family and potent proinflammatory cytokine stimulates the expression of the chemokines CCL1, CCL7, and CCL8 in primary human dermal fibroblasts at a faster kinetic than IL-1beta or TNF-alpha. The production of CCL1 and CCL8 is important for migration of monocytes, while specific Abs against CCL1 additionally inhibit the migration of T lymphocytes. We identify the mitogen-activated protein kinases ERK1/2 and p38 as crucial factors for the enhanced expression of CCL1 and CCL8. Depletion of the ERK1/2 target genes c-Jun or c-Fos strongly decrease CCL1 and CCL8 expression, while p38 MAPK prolongs the half-life of CCL1, CCL7, and CCL8 mRNA through inhibition of tristetraprolin. None of the STAT transcription factors STAT1, STAT3, or STAT5 stimulate transcription of CCL1 or CCL8. However, we identify a negative regulatory function of activated STAT5 for the gene expression of CCL1. Importantly, not STAT5 itself, but its target gene cytokine inducible SH2-domain containing protein is required for the STAT5 inhibitory effect on CCL1 expression. Finally, we show that constitutive activation of STAT5 through a mutated form of JAK2 (JAK2 V617F) occurring in patients with myeloproliferative disorders similarly suppresses CCL1 expression. Taken together, we identify novel important inflammatory target genes of OSM which are independent of STAT signaling per se, but depend on MAPK activation and are partly repressed through STAT5-dependent expression of cytokine inducible SH2-domain containing protein.

Cigarette smoke combined with Toll-like receptor 3 signaling triggers exaggerated epithelial regulated upon activation, normal T-cell expressed and secreted/CCL5 expression in chronic rhinosinusitis.

BACKGROUND: Chronic rhinosinusitis (CRS) is characterized by persistent mucosal inflammation and frequent exacerbations. OBJECTIVE: To determine whether innate epithelial responses to cigarette smoke or bacterial or viral pathogens may be abnormal in CRS leading to an inappropriate inflammatory response. METHODS: Primary nasal epithelial cells (PNECs) were grown from middle turbinate biopsies of 9 healthy controls and 11 patients with CRS. After reaching 80% to 90% confluence, PNECs were exposed to medium or cigarette smoke extract (CSE) 5% (vol/vol) for 1 hour, washed, then stimulated with staphylococcal lipoteichoic acid, LPS, or double-stranded RNA (dsRNA). After 24 hours, gene expression was quantified by QRT-PCR. RESULTS: At baseline, PNECs revealed elevated TNF-alpha and growth-related oncogene-alpha (a C-X-C chemokine)/CXCL1 (GRO-alpha) (4-fold increase, P = .02; and 16-fold increase, P = .004, respectively) in subjects with CRS compared with controls with normal levels of IL-1beta, IL-6, IL-8/CXCL8, human beta-defensin-2, monocyte chemoattractant protein 2/CCL8, monocyte chemoattractant protein 3/CCL7, and regulated upon activation, normal T-cell expressed and secreted (RANTES)/CCL5. Immunostaining of nasal biopsies, however, revealed comparable epithelial staining for TNF-alpha, GRO-alpha, and RANTES. There were no differences in mRNA induction by CSE, TNF-alpha, lipoteichoic acid, LPS, or dsRNA alone. The combination of CSE+dsRNA induced exaggerated RANTES (12,115-fold vs 1500-fold; P = .03) and human beta-defensin-2 (1120-fold vs 12.5-fold; P = .05) in subjects with CRS. No other genes were differentially induced. Furthermore, CSE+dsRNA induced normal levels of IFN-beta, IFN-lambda1, and IFN-lambda2/3 mRNA in subjects with CRS. CONCLUSION: Cigarette smoke extract plus dsRNA induces exaggerated epithelial RANTES expression in patients with CRS. We propose that an analogous response to cigarette smoke plus viral infection may contribute to acute exacerbations and eosinophilic mucosal inflammation in CRS.

Synergy between coproduced CC and CXC chemokines in monocyte chemotaxis through receptor-mediated events.

CC and CXC chemokines coinduced in fibroblasts and leukocytes by cytokines and microbial agents determine the number of phagocytes infiltrating into inflamed tissues. Interleukin-8/CXCL8 and stromal cell-derived factor-1/CXCL12 significantly and dose-dependently increased the migration of monocytes, expressing the corresponding CXC chemokine receptors CXCR2 and CXCR4, toward suboptimal concentrations of the monocyte chemotactic proteins CCL2 or CCL7. These findings were confirmed using different chemotaxis assays and monocytic THP-1 cells. In contrast, the combination of two CC chemokines (CCL2 plus CCL7) or two CXC chemokines (CXCL8 plus CXCL12) did not provide synergy in monocyte chemotaxis. These data show that chemokines competing for related receptors and using similar signaling pathways do not synergize. Receptor heterodimerization is probably not essential for chemokine synergy as shown in CXCR4/CCR2 cotransfectants. It is noteworthy that CCL2 mediated extracellular signal-regulated kinase 1/2 phosphorylation and calcium mobilization was significantly enhanced by CXCL8 in monocytes, indicating cooperative downstream signaling pathways during enhanced chemotaxis. Moreover, in contrast to intact CXCL12, truncated CXCL12(3-68), which has impaired receptor signaling capacity but can still desensitize CXCR4, was unable to synergize with CCL2 in monocytic cell migration. Furthermore, AMD3100 and RS102895, specific CXCR4 and CCR2 inhibitors, respectively, reduced the synergistic effect between CCL2 and CXCL12 significantly. These data indicate that for synergistic interaction between chemokines binding and signaling of the two chemokines via their proper receptors is necessary.

[Construction and expression of a prokaryotic expression plasmid of idiotypic vaccine against B cell lymphoma: encoding the fusion genes of single-chain variable fragment and MCP-3].

The aim was to construct a prokaryotic expression plasmid encoding the fusion gene of single-chain variable fragment and monocyte chemotactic protein-3 (MCP-3). The cDNAs of immunoglobulin (Ig) VH and Ig VL were amplified by RT-PCR and assembled into the single-chain variable fragment (scFv) by recombinant PCR method. The cDNAs of Ig VH and Ig VL were connected by a (Gly4Ser)3 linker. Then, the fragments of scFv and MCP-3 were connected with a NDAQAPKS spacer, using recombinant PCR method again. The results indicated that the fusion gene of scFv-MCP-3 were constructed correctly and cloned into the prokaryotic expression plasmid successfully identified by sequencing and restriction endonucleases examination. Finally, the fusion protein was expressed in E coli DH5alpha under induction by arabinose. And the fusion protein was 65 kD and account for 30% of the total protein of the bacteria. In conclusion, a prokaryotic plasmid, encoding the fusion gene of single-chain variable fragment with MCP-3 and expressing idiotype protein vaccination against B cell lymphoma, was constructed correctly.