CCL2 Research Articles

Correlation between pathological and functional changes of the retina and the expression of monocyte chemoattractant protein after retinal laser injury in mice

Objective To investigate the relationship between the pathological and functional changes of the retina and the expression of monocyte chemoattractant protein (MCP)-1 after retinal laser injury in mice.Methods A total of 116 C57BL/6 mice were randomly divided into the normal group (58 mice) and the injured group (58 mice).Retinal laser injuries were induced by Argon ion laser.At 1,3,7 days after laser injury,electroretinogram (ERG) responses were recorded to detect the function of the retina.Hematoxylin and eosin (HE) staining was performed to observe pathological changes.Quantitative real-time polymerase chain reaction (PCR) was performed to detect gene expression of MCP-1.Western blot was used to measure the protein expression of MCP-1.Results HE staining showed a progressive damage of the retinal structure.The results of ERG showed that the differences of dark-adaptive a wave (t =6.998,9.594,13.778) and b wave (t=12.089,13.310,21.989) amplitudes of 1,3 and 7 day post-injury between normal group and injured group were statistically significant (P =0.000).At 1 day post-injury,the differences of light adaptive b wave amplitudes between the two groups were statistically significant (t =8.844,P=0.000).While the differences of light-adaptive a wave amplitudes were not (t=2.659,P=0.200).At 3,7 days post-injury,the differences of a (t=3.076,7.544) and b wave amplitudes (t=10.418,8.485) between the two groups were statistically significant (P=0.000).In dark-adaptive ERG,the differences of a wave amplitudes between 1 day and 3 days (t=3.773),1 day and 7 days (t=5.070) and b wave amplitudes between 1 day and 7 days (t=4.762) were statistically significant (P＜0.01),while the differences of a wave amplitudes between the 3 days and 7 days (t=1.297) and b wave amplitudes between 1dayand 3 days (t=2.236),3 day and 7 days (t=2.526) were not significant (P=0.660,0.120,0.060).In light-adaptive ERG,the differences of a wave amplitudes between 1 day and 7 days (t=2.992)and b wave amplitudes between 1 day and 3 days (t=3.570),1day and 7 days (t=4.989) were statistically significant (P＜0.05),while the differences of a wave amplitudes between 1 day and 3 days (t=0.516),the 3 days and 7 days (t=2.475) and b wave amplitudes between 3 days and 7 days (t=1.419) were not significant (P=1.000,0.710,0.070).Quantitative real-time PCR showed that the differences of MCP-1 gene expression at 1,3 and 7 day post-injury between normal group and injured group were statistically significant (t=14.329,16.861,5.743; P＜0.05).Western blot showed that the differences of MCP-1 protein expression at 1,3 and 7 day post-injury between normal group and injured group were statistically significant (t=75.068,54.145,14.653; P＜0.05).Conclusion In the first 7 days after mice retinal laser injury,there are progressive pathological and functional damage of the retina,which might be correlated with MCP-1 expression. Key words: Retina diseases/etiology; Electroretinography; Chemokine CCL2; Animal experimentation

Let-7d suppresses growth, metastasis, and tumor macrophage infiltration in renal cell carcinoma by targeting COL3A1 and CCL7.

BackgroundMicroRNAs are endogenous small noncoding RNAs that are functionally involved in numerous critical cellular processes including tumorigenesis. Data mining using a microRNA array database suggested that let-7d microRNA may be associated with renal cell carcinoma (RCC) malignant progression. Here, we performed further analyses to determine whether let-7d is functionally linked to RCC malignancy.MethodsQuantitative real-time PCR was used to determine the level of mature let-7d in RCC clinical specimens and its correlation with clinicopathological data. Immunohistochemical staining was conducted to characterize the stroma of RCC. Let-7d overexpressing RCC cell lines combined with mouse models bearing cell-derived xenografts and patient-derived xenografts were used to assess the functional role of let-7d in vitro and in vivo.ResultsDownregulation of let-7d in clinical RCC samples was associated with advanced tumor grade and T stage and increased vascular invasion. An inverse relationship between let-7d expression and macrophage infiltration was found in clinical RCC samples. Functional studies indicated that ectopic expression of let-7d significantly inhibited RCC cell proliferation, migration, and peripheral blood monocyte (PBMC) recruitment in vitro, as well as tumor growth, metastasis, and tumor macrophage infiltration in vivo. In silico analysis and subsequent experimental validation confirmed collagen, type III, alpha 1 (COL3A1) and C-C subfamily chemokine member CCL7 as direct let-7d target genes. The addition of COL3A1 and CCL7 counteracted the inhibitory effects of let-7d on RCC cell proliferation, migration, and PBMC recruitment. The inhibition of let-7d increased cell proliferation, migration, and PBMC recruitment by the enhanced expression of COL3A1 and CCL7 genes in vitro. The mRNA levels of COL3A1 and CCL7 were inversely correlated with let-7d level in RCC clinical specimens.ConclusionsThese results suggest that let-7d may suppress RCC growth, metastasis, and tumor macrophage infiltration at least partially through targeting COL3A1 and CCL7.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-206) contains supplementary material, which is available to authorized users.

Effects of hypergravity exposure after 30-days simulated weightlessness on chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque

To study the influence of hypergravity exposure after 30-days simulated weightlessness on the expression of chemokine CCL20 and its receptor CCR6 in lingual mucosa of rhesus macaque. Twenty-three male rhesus monkeys were divided according to the random number method into one control group (A, n = 3) and three experimental groups, including the weightlessness group (B, n = 3), the hypergravity group (C, n = 3) and hypergravity exposure after 30-days simulated weightlessness group (D, n = 14), which was further divided into four subgroups according to the values of overload as: +11 Gx/270s group (D1, n = 3), +13 Gx/230s group (D2, n = 4) and +15 Gx/200s group (D3, n = 4) and +13 Gx/230s with 9-days recovery group (D4, n = 3). Histopathological changes of lingual mucosa were observed by hematoxylin-eosin staining and the expressions of CCL20 and CCR6 were detected by immunohistochemistry (IHC) and quantitative real-time PCR (Q-PCR). Histological observation showed that no significant histopathological changes were found in the lingual mucosa in all experimental groups of the animals, however, there were more infiltrated lymphocyte and neutrophils in the experimental groups. The immunohistochemical scores of CCL20 in group C, group D2 and group D3 were 1.30 ± 0.11, 1.68 ± 0.62 and 2.26 ± 1.00, respectively, significantly higher than that in A group (0.47 ± 0.12, P < 0.05). The immunohistochemical scores of CCR6 expression in those three groups were 4.40 ± 1.48, 6.67 ± 2.04 and 7.02 ± 2.11, respectively, also remarkably higher than that in A group (1.33 ± 0.66, P < 0.05). As far as the mRNA expression was concerned, the expressions of CCL20 and CCR6 had similar change trend with their protein expressions. Hypergravity exposure after 30-days simulated weightlessness will not lead to significant pathological changes in lingual mucosa, but can induce the expression of chemokine CCL20 and its receptor CCR6.

CCL21/CCR7 axis activating chemotaxis accompanied with epithelial-mesenchymal transition in human breast carcinoma.

Secondary lymphoid tissue chemokine (SLC/CCL21) and its receptor CCR7 have been implicated in lymph node metastasis, whereas the mechanism of which remains unclear. Epithelial-mesenchymal transition (EMT) plays an important role in invasion and migration of cancer cells. We presumed that CCL21/CCR7 axis activates EMT process to induce cancer cell invasion and metastasis. Firstly, the expressions of CCR7 and EMT markers were examined by immunohistochemical staining in the primary breast carcinoma tissues from 60 patients who underwent radical mastectomy. Then, we investigated whether CCL21/CCR7 induces EMT process during mediating cancer cell invasion or migration in vitro. By immunohistolochemistry, high expressions of CCR7, Slug and N-cadherin were seen in 60, 65, and 76.67 % of tumors, respectively, and significantly associated with lymph node metastases as well as clinical pathological stage. Furthermore, the CCR7 expression was significantly correlated to Slug and N-cadherin. In vitro, stimulating breast cancer cell lines 1428, MCF-7 and MDA-MB-231 with CCL21, the invasion and migration of tumor cells were promoted, and simultaneously, EMT phenotype of tumor cells was enhanced, including down-regulation of E-cadherin, up-regulation of Slug, Vimentin and N-cadherin at both protein and mRNA levels. Inversely, knockdown of CCR7 by shRNA suppressed tumor cell invasion, migration and EMT phenotype induced by CCL21. These results indicated that CCL21/CCR7 axis could activate EMT process during chemotaxis of breast carcinoma cells.

CCL2/CCR2 signaling activation contributes to tooth movement pain

To test the hypothesis that the CCL2/CCR2 signaling pathway plays an important role in pain induced by experimental tooth movement. Male Sprague- Dawley rats weighing between 200 and 300 g were used in this study. Expression of CCL2/CCR2 in the trigeminal ganglion (TG) was determined by Western blotting 0 h, 4 h, 1 d, 3 d, 5 d, 7 d after tooth movement. Localization of the CCL2 was revealed by immunohistochemistry. Changes in body weight, nocifensive behaviors, and the effects of CCL2/CCR2 antagonists on these changes in pain behaviors were evaluated. Exogenous CCL2 was injected into periodontal tissues and added to TG neurons in culture and the resulting c-fos expression and pain responses were detected. In addition, the expression and cellular localization of CCL2 in the medullary dorsal horn (MDH) was determined by immunohistochemistry 3 d and 14 d after tooth movement. Experimental tooth movement led to a statistically significant increase in CCL2/CCR2 expression at the protein level from day 3 to 7 after application of force initiating tooth movement.When compared with control group (1.000 ± 0.000), CCL2 increased to (2.620 ± 0.128), (3.300 ± 0.197) and (1.740 ± 1.290) at day 3, 5 and 7 respectively, which were statistically significant (P < 0.05). CCR2 expression levels were (1.636 ± 0.061) and (1.766 ± 0.126) compared with that in control group (1.000 ± 0.000) at day 3 and 5 respectively with statistical significance (P < 0.05). Both of them peaked on day 5 (3.3 and 1.8 time compared to control group). Application of recombinant CCL2 led to the up-regulation of c-fos expression in vivo and in vitro, and triggered a corresponding nocifensive behavior in rats. The magnitude of the nocifensive behavior could be reduced by a CCR2 antagonist, and by CCL2 neutralizing antibody. Furthermore, we found a significant increase in the expression of CCL2, corresponding well to the up-regulation of the time spent on nocifensive behaviors after ETM. In addition, CCL2 was up-regulated in TG neurons and astrocytes in Vc. The CCL2/CCR2 axis was modulated by experimental tooth movement and involved in the development of tooth movement pain, and thus palyed an important role in orthodontic pain mechanism.

Interleukin-22 in the pathogenesis of psoriasis

As a critical cytokine expressed by the newly discovered CD4+ T cell subsets (including T helper type 17 (Th17) and Th22 cells),interleukin-22 (IL-22) acts as a molecular bridge between immunocytes and keratinocytes in the pathogenesis of psoriasis.The specific expression of IL-22 receptor determines the selective effect of IL-22 on keratinocytes.IL-22 may take part of the formation of lesions at the late stage by binding its receptor and activating the intracellular signal transducer and activator of transcription (STAT) pathway.Further more,IL-22 has a potential immunomodulatory effect by inducing the specific expression of cytokeratin 17 and CCL20 by keratinocytes. Key words: Psoriasis; Interleukin-22; Receptors, interleukin-22; Signaling pathway

Effect of dexamethasone on inflammatory response of thyrocytes

Objective To explore the impact of dexamethasone on inflammatory response of thyrocytes.Methods Primary thyrocytes were extracted from thyroid tissue of patients with Graves' disease.The cells were stimulated with interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α),and cultured in dexamethasone.Thyrocytes were divided into 4 groups:control group,dexamethasone group,TNF-α + IFN-γ group,and dexamethasone+TNF-α+IFN-γ group.Interferon-γ-induced protein 10 (CXCL10) and CCL2 in supernatant of cell cultured in 4 groups were detected by enzyme-linked immunosorbent assay.Cell protein in 4 groups was extracted and GSK-3β,P50,and P100 protein were detected by Western blotting.Results MTT assay demonstrated that 10-5 mmol/L concentration of dexamethasone was optimal for cell culture.The CXCL10 level in TNF-α+IFN-γ group was higher than that in the control group and dexamethasone group (P＜0.01),but no difference was found between dexamethasone+TNF-α+IFN-γgroup and TNF-α+IFN-γgroup(P＞0.05).The CCL2 level in TNF-α+IFN-γ group was higher than that in control group and dexamethasone group(P＜0.01).There was a significant lowering of CCL2 level in dexamethasone + TNF-α + IFN-γgroup compared with TNF-α + IFN-γ group (P ＜ 0.05).The expression of GSK-3β and P100 protein was increased in TNF-α + IFN-γgroup compared with control group.The expression of GSK-3β and P100 protein was lower in dexamethasone+TNF-α + IFN-γ group than that in TNF-α + IFN-γ group.Conclusion TNF-α + IFN-γ could stimulate the secretion of CXCL10 and CCL2 in thyrocytes and thus activate the inflammatory response.Dexamethasone could reduce CCL2 secretion.Dexamethasone had little effect on CXCL10.Dexamethasone could reduce GSK-3β and P100 expressions,and inhibit the activation of NF-κB signaling pathway and thus the inflammatory response. Key words: Thyrocytes; Inflammation; Graves' disease; Dexamethasone

Reduced local immune response with continuous positive airway pressure during one-lung ventilation for oesophagectomy

Transthoracic oesophagectomy requires prolonged one-lung ventilation causing systemic and local inflammatory responses. Application of continuous positive airway pressure (CPAP) to the collapsed lung potentially reduces pulmonary damage, hypoxia, and consequent inflammation. This randomized controlled trial studied the influence of CPAP applied to the collapsed right lung during thoracoscopic oesophagectomy on local and systemic inflammatory response. Broncho-alveolar lavage fluid (BALF) from the right collapsed and left ventilated lung and serum samples were obtained during surgery from 30 patients undergoing thoracolaparoscopic oesophagectomy for cancer who were randomized for one-lung ventilation with or without CPAP applied to the collapsed right lung. Concentrations of cytokines and chemokines, in BALF and serum, were determined with Luminex. Patients from the control (no CPAP) group had significantly increased concentrations of interleukin (IL)-1α, IL-1β, IL-10, tumour necrosis factor-alpha, macrophage inflammatory protein (MIP)-1α, pulmonary and activation-regulated chemokine (PARC), and IL-8 in the collapsed (right) lung when compared with patients from the CPAP group (P<0.05). The ventilated (left) lung of the control group showed increased concentrations of monocyte chemoattractant protein (MCP)-1 and MIP-1α (P<0.05). Serum concentrations of cytokines and chemokines increased during surgery, but did not differ between the control and CPAP groups. A significantly lower local immune response was observed during one-lung ventilation when CPAP was applied to the collapsed lung. The findings suggest a beneficial effect of CPAP on the collapsed lung during oesophagectomy with one-lung ventilation.

Role of JNK/MCP-1 signaling pathway in spinal cord in maintenance of bone cancer pain in rats

Objective To evaluate the role of c-Jun N-terminal kinase (JNK)/monocyte chemoattractant protein-1 (MCP-1) signaling pathway in the spinal cord in the maintenance of bone cancer pain (BCP) in rats.Methods Fifty female Sprague-Dawley rats,weighing 150-180 g,were randomly divided into 5 groups (n =10 each) using a random number table:sham operation group (group S),sham operation + JNK inhibitor SP600125 (group SP),BCP group,BCP + dimethyl sulfoxide (DMSO) group,and BCP+ SP600125 group (group BCP+ SP).BCP was induced by injecting Walker 256 mammary gland cancer cells into the bone marrow of the left tibia.On 10-12 days after BCP,SP600125 10 μg(10 μ1) was injected intrathecally once a day in SP and BCP + SP groups,and 5％ DMSO 10 μl was injected intrathecally once a day in BCP + DMSO group.Mechanical paw withdrawal threshold (MWT) was measured at 1 day before BCP and 3,6,9,10,11 and 12 days after BCP.After measurement of MWT at 12 days after BCP,the rats were sacrificed and L4-6 segments of the spinal cord were removed for determination of MCP-1 expression by using immuno-histochemistry and Western blot.Results Compared with S group,MWT was significantly decreased at 6-12 days after BCP,MCP-1 expression was upregulated in BCP,BCP + DMSO and BCP + SP groups (P ＜ 0.01),and no significant change was found in the parameters mentioned above in SP group (P ＞ 0.05).Compared with BCP group,MWT was significantly increased at 10-12 days after BCP,MCP-1 expression was down-regulated in BCP + SP group (P ＜ 0.01),and no significant change was found in the parameters mentioned above in BCP + DMSO group (P ＞ 0.05).Conclusion JNK/MCP-1 signaling pathway in the spinal cord may be involved in the maintenance of BCP in rats. Key words: Bone neoplasms ; Pain; Mitogen-activated protein kinases ; Chemokine CCL2 ; Spinal cord

The effect of inhaled glucocorticoid therapy on serum proteomics of asthmatic patients

To screen the biomarkers which may play important roles in the pathogenesis and therapy of asthma by using serum comparative proteomics. From June 2011 to September 2012, 30 chronic persistent asthmatic patients (asthma group) and 30 healthy controls (control group) were selected for study in our hospital. All the asthmatic patients were given 8 week-treatment with inhaled glucocorticoids (ICS). Then comparative proteomics were employed to identify differential proteins in serum samples from the control group, the asthma pre-treatment group and the asthma post-treatment group. The differential proteins which had significant differences before and after ICS therapy were selected for Western blot analysis and ELISA detection. Besides, the correlation between the differential proteins and IgE, eosinophils (EOS), neutrophil percentage(NEUT%), and FEV1% were analyzed. Eleven differential proteins were identified. Among them, heat shock protein 70 (HSP70), eosinophil chemotactic protein (Eotaxin) and vitamin D binding protein (VDBP) had significant differences in protein abundance before and after treatment. The differential expression of the 3 proteins in each group was confirmed by Western blot. ELISA data showed that the serum levels of HSP70 and Eotaxin were significantly higher in the asthma pre-treatment group [(439 ± 103)ng/L, (183 ± 79)ng/L] than those in the control group [(209 ± 58)ng/L, (91 ± 46)ng/L] (t = 5.281, 4.972, all P < 0.01), but significantly lower in the asthma post-treatment group[(247 ± 96) ng/L, (105 ± 58)ng/L] than those in the pre-treatment group (t = 4.157, 3.892, all P < 0.01). However, the serum level of VDBP was significantly lower in the asthmatics pre-treatment group [(318 ± 115)mg/L] than that in the control group [(541 ± 98)mg/L] (t = 3.878, P < 0.01), but significantly higher in the asthma post-treatment group[(479 ± 132)mg/L] than that in the pre-treatment group(t = 3.572, P < 0.01). The correlation analysis showed that HSP70 was correlated positively with IgE and NEUT%, but negatively with FEV1% (r = 0.568, 0.613, -0.516, all P < 0.01). Eotaxin was correlated positively with IgE and EOS, but negatively with FEV1% (r = 0.752, 0.826, -0.618, all P < 0.01). VDBP was correlated negatively with NEUT%, but positively with FEV1% (r = -0.537, 0.426, all P < 0.05). HSP70, Eotaxin, and VDBP may participate in the pathogenesis of asthma, and may become the potential targets for ICS therapy.

Inhibited experimental mouse corneal neovascularization by CCR3 antagonist

To explore the effect of CCR3 antagonist on the development of experimental corneal neovascularization. Mouse corneas were burned by NaOH to induce corneal neovascularization.Fifty four clean male BABL/c mice aged 7-8 weeks were divided into control group, CCR3 antagonist group and VEGF antibody positive group according to randomized number table. The gene expression of CCR3 and its ligand eotaxin in burned corneas was examined by Real-time PCR. CCR3 antagonist was locally administrated after alkali injury and the formation of corneal neovascular 2 weeks after injury was examined using a digital camera linked to a slit lamp microscope and corneal whole mount staining with CD31. The mRNA and protein expression of chemokines in burned corneas was detected by Real-time PCR and western blot. Compared to control group, CCR3 antagonist treated mice resulted in significantly decreased corneal neovascularization. The related CNV area was 0.51 ± 0.03 in the CCR3 antagonist group, and that in the control group was 0.77 ± 0.15, with significant difference between them (t = 12.91, P = 0.00).Western blot detection did not show significant difference of VEGF protein expression between two groups.Expression level of VEGF in the CCR3 antagonist group was 0.91 ± 0.24, and that in the control group was 1.15 ± 0.30, showing no significant difference (t = 1.08, P = 0.34). Alkali-induced corneal neovascularization was inhibited by CCR3 antagonist. The mechanism that CCR3 pathway plays an important role in corneal neovascularization needs further exploration.

Toll like receptors signaling pathways directly increase the expression of functional IL-17RA in neu-roglial cells

Objective To investigate whether toll like receptor （ TLR） signaling pathways can in-crease the expression of IL-17 R in neuralglial cells , and if they can whether the increased IL-17 R is func-tional.Methods Experimental autoimmune encephalomyelitis （EAE） was induced in B6 mice by immuni-zation with an emulsion of myelin oligodendrocyte glycoprotein 35-55 （ MOG35-55 ） in complete Freund’s adju-vant （CFA）.The expression of Il17ra and Il17rc in the brains and spinal cords of mice with EAE were de-tected by real-time PCR.Luxol fast blue （ LFB） staining was performed to the spinal cord sections to detect tissue demyelination.Immunohistological staining against IL-17RA and CD3 were undertook to visualize IL-17RA＋and CD3 ＋ cells.Same approaches were also applied to immunized Rag1 -/- mice to figure out whether T cells infiltration is necessary for increasing IL-17RA expression in the central nervous system （ CNS） .Then B6 mice were immunized with incomplete Freund′s adjuvant （ IFA） plus different TLRs ago-nists to measure the expression of Il17ra in the brains and spinal cords by qPCR .The purified astrocytes , microglia and oligodendrocytes isolated from neonatal mice brains were cultured in vitro for two weeks , and then treated with different TLRs agonists .The expression of Il17ra at mRNA and protein levels in the cells were determined by qPCR and Western blot respectively .The astrocytes were treated with IL-17A and LPS individually or in the combination to detect the level of CCL 2, CXCL8 and IP-10 in the supernatant by ELISA.Results B6 mice with induced EAE showed significantly increased Il17ra expressions in the brain＆amp;nbsp;and spinal cord , which was also detected in immunized Rag1 -/-mice.Although no spinal cord demyeliza-tion and CD3 cells infiltration were detected in Rag1 -/-mice, significantly increased number of IL-17RA positive cells could still be visualized .In vivo TLRs agonist participated immunization and in vitro treatment of purified neuroglial cells demonstrated that TLRs agonists could directly evoke IL -17RA expression in the CNS or cultured astrocytes , microglia and oligodendrocytes with high efficiency .Both IL-17 A and LPS could stimulate astrocytes to secrete CCL2, CXCL8 and IP-10, however, a combined use of IL-17A and LPS fur-ther augmented the production of these chemokines to a large extend .Conclusion Taken together , we con-cluded that TLRs agonists could directly stimulate neuroglial cells to express IL -17RA which functionally re-spond to IL-17A by secreting chemokines . Key words: Toll-like receptor; Neuroglial cells; IL-17 receptor; Expression

Research progress in chemokine receptor 3 and its ligands in ophthalmology

Chemokine receptor 3 (CCR3) and its ligands eotaxin are inflammatory factors,and they were thought to be related to asthma and allergic diseases before.Recent studies showed that CCR3 plays significant roles in many eye diseases,such as choroidal noevascularization (CNV),allergic cunjunctivititis,age-related macular degeneration (AMD),central retinal vein occlusion and retinal degeneration etc.The biological function study suggested that CCR3 and eotaxin participate in not only promoting the transportation of eosinophilic granulocyte and mastocyte but also inflammatory response.To understand the roles of CCR3 and eotaxin in eye diseases has a very important significance for the recognition and treatment of eye diseases.The structure and function of CCR3 and its ligands,and the relationship between CCR3 and some eye diseases were reviewed. Key words: Chemokine receptor 3 ; Eotaxin ; Choroidal noevascularization ; Ophthalmology

The study of anti-inflammatory on Eotaxin and CCR3 of asthmatic Rats

Objective To observe the effects of Dexamethasone and Montelukast on Eotaxin and CCR3 of asthmatic rats.Methods Fourty-eight SD rats were sensitized and challenged by 1％ ovabumin to establish asthmatic model.Results The number of Eotaxin mRNA and Eotaxin protein positive cells as well as the number of CCR3 protein positive cells form group B,C and D were down-regulated significintly,compared to those form group A (P＜0.01).The number of CCR3 protein positive cells in lung tissue and bone marrow form groups B and C,as compared with group D,the difference was significant.Conclusion Dexamethasone probably inhibited the expression of Eotaxin and CCR3; and decreased eosinophil number in the level of bone marrow,Montelukast probably down-regulated them.Montelukast showed apparently synergistic effect with Dexamethasone to inhibit the expression of Eotaxin and CCR3. Key words: Asthma; Eotaxin; CCR3; Dexamethasone; Montelukast

Effect of secondary lymphoid tissue chemokine suppression on experimental ulcerative colitis in mice.

The secondary lymphoid tissue chemokine (CCL21) is closely associated with lymphoid homing and anti-tumor immune responses. CCL21 also has a chemotactic effect on intestinal lymphocytes. This study mainly focused on CCL21 expression in experimental ulcerative colitis and on the effects of CCL21 suppression on this disease in mice. The mouse colitis model was induced by dextran sulfate sodium (DSS) in 40 female BALB/c mice that were equally distributed into five groups: control, DSS, propylene glycol, triptolide (TL), and dexamethasone treatment groups. The disease activity index, general morphology score of the colon, and histological pathology score of colon tissues were evaluated. CCL21 expression was examined in colons of mice by immunohistochemistry, reverse transcription-polymerase chain reaction, and Western blotting analysis. CCL21 was upregulated in the mouse model of ulcerative colitis (control group vs DSS group/propylene glycol group, P<0.01). The TL and dexamethasone treatments improved colitis symptoms and decreased CCL21 expression (TL group/dexamethasone group vs DSS group/propylene glycol group, P<0.05). In conclusion, CCL21 was shown to be involved in the induction of ulcerative colitis. Suppression of CCL21 expression decreased damage induced from ulcerative colitis, indicating that CCL21 targeted therapy might be an effective treatment for this disease.

MSCs and inflammation: not only a guardian role

The literature on the relation between mesenchymal stem cells (MSCs) and inflammation is continuing to expand at a rapid rate with over 600 entries in PubMed under “MSCs and inflammation” starting from 2002. Inflammation is an essential part of the malignant microenvironment. Chemokines, leukocyte infiltration and cytokines are crucial elements, which contribute to cancer-related inflammation. Attracted by chemokines, MSCs are recruited at injury sites. After exposure to inflammatory factors in the local microenvironment, MSCs secrete several cytokines and vascular endothelial growth factor, which promote immunosuppression, angiogenesis and tumor growth. Here we compare by RT-PCR the expression of selected genes, related to inflammation, on MSCs derived from control (C-MSCs) and inflamed tissues (I-MSCs). First of all, an immunohistochemistry using anti-CD43 antibody was performed to better test the status of inflammation at the moment of tissues’ collection. CD43 is known as marker of inflammation, since it is expressed by most T cells, activated B cells, basophils, macrophages, monocytes and NK cells. Its expression was absent in “control” tissues, while it was strong in the “inflamed”. Subsequently, RNA was extracted, retro-transcribed and used for quantitative PCR. The genes were selected according to their role in inflammation: IL6 and IL8 (known as pro-inflammatory interleukins), TNFα (involved in systemic inflammation), CXCL2 (secreted by monocytes and macrophages and is chemotactic for polymorphonuclear leukocytes), CCL20 (strongly chemotactic for lymphocytes, its expression is induced by inflammatory cytokines), IFNγ (an important activator of macrophages) and TGFβ1 (promoting immunosuppression). Quantification of mRNA expression was calculated with the 2−ΔΔCt method, where ΔCt = Ct (gene of interest) − Ct (control gene) and Δ(ΔCt) = ΔCt (I-MSCs) − ΔCt (C-MSCs). The results revealed that the expression of all tested genes was higher in MSCs derived from inflamed tissues than in MSCs from control tissues (expressed as 1). This study underlines how MSCs are not inert guardians on inflammation, but as they play an active role.

Mechanical Stretch Upregulates SDF-1α in Skin Tissue and Induces Migration of Circulating Bone Marrow-Derived Stem Cells into the Expanded Skin

Skin and soft tissue expansion is a procedure that stimulates skin regeneration by applying continuous mechanical stretching of normal donor skin for reconstruction purposes. We have reported that topical transplantation of bone marrow-derived mesenchymal stem cells (MSCs) can accelerate mechanical stretch induced skin regeneration. However, it is unclear how circulating MSCs respond to mechanical stretch in skin tissue. MSCs from luciferase-Tg Lewis rats were transplanted into a rat tissue expansion model and tracked in vivo by luminescence imaging. Expression levels of chemokines including macrophage inflammatory protein-1α, thymus and activation-regulated chemokine, secondary lymphoid tissue chemokine, cutaneous T-cell attracting chemokine, and stromal-derived factor-1α (SDF-1α) were elevated in mechanically stretched tissues, as were their related chemokine receptors in MSCs. Chemotactic assays were conducted in vitro and in vivo to assess the impact of chemokine expression on MSC migration. MSC migration was observed in mechanically stretched skin. Mechanical stretching induced temporal upregulation of chemokine expression. Among all the tested chemokines, SDF-1α showed the most significant increase in stretched skin, suggesting a strong connection to migration of MSCs. The in vitro chemotactic assay showed that conditioned medium from mechanically stretched cells induced MSC migration, which could be blocked with the CXCR4 antagonist AMD3100, as effectively as medium containing 50 ng/ml rat recombinant SDF-1α. Results from in vivo study also showed that MSC migration to mechanically stretched skin was significantly blocked by AMD3100. Moreover, migrating MSCs expressed differentiation markers, suggesting a contribution of MSCs to skin regeneration through differentiation. Mechanical stretching can upregulate SDF-1α in skin and recruit circulating MSCs through the SDF-1α/CXCR4 pathway.

Expression of vasoactive molecules in aqueous humor of patients with macular edema secondary to central retinal vein occlusion

Objective To observe the levels of vascular endothelial growth factor (VEGF),interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) in aqueous humor of patients with macular edema secondary to central retinal vein occlusion (CRVO).Methods Forty eyes of 40 consecutive patients with macular edema secondary to CRVO (CRVO group) were enrolled in this study.The patients included 25 males and 15 females.The patient age ranged from 38 to 76 years.The control group was 20 patients with senile cataract who underwent phacoemulsification,including 10 males and 10 females.The levels of VEGF165,VEGF165 b,IL-6 and MCP-1 in aqueous humor were determined by enzyme-linked immunosorbent assay.The correlation of VEGF,and IL-6,and MCP-1 were analyzed.Results The median aqueous level of VEGF165,IL-6 and MCP-1 were 1089.0,165.6,1253.0 pg/ml respectively in CRVO group,which were higher than the control group's results (168.2,4.7,216.4 pg/ml respectively),the differences were statistically significant (Z=-4.549,-6.008,-5.343; P＜0.001).The VEGF165b in CRVO group and control group were 834.0,915.9 pg/ml respectively,the difference was not statistically significant (Z=-0.207,P＞0.05).The ratio of VEGF165 b to VEGF165 in CRVO group and control group were 2.71,7.28 respectively,the difference was statistically significant (t=-3.007,P＜0.05).There was a highly positive correlation between IL-6 and VEGF in CRVO group (r=0.526,P=0.001) and also mild positive correlation in control group (r=0.425,P=0.070).No correlation between MCP-1 and VEGF was observed in both groups (CRVO group:r=0.211,P＞0.05.Control group:r=-0.019,P＞0.05).Conclusions VEGF165,IL-6 and MCP-1 levels were increased in CRVO patients while the VEGF165 b was normal.The ratio between VEGF165 b and VEGF165 in aqueous humor of patients with macular edema secondary to CRVO was decreased. Key words: Retinal vein occlusion/complications; Macular edema; Aqueous humor; Vascular endothelial growth factor A; Interleukin-6; Chemokine CCL2

Altered profile of chemokines in fibromyalgia patients

Fibromyalgia (FM) is a syndrome characterized by widespread chronic pain. Its aetiology is still poorly understood, and there are no haematochemical or instrumental tests on which to base a diagnosis. Recent studies suggest that its pathogenesis may involve cytokines, in particular, chemokines - cytokines that regulate cell traffic under both physiological and pathological conditions. The aim of this study was to determine possible differences in the profile of systemic concentrations of chemokines between FM patients and healthy women (HW; controls). The study participants were women diagnosed with FM (n = 17) and a control group of HW (n = 10). Serum concentrations of thymus and activation-regulated chemokine (TARC)/(CCL17), monokine induced by gamma-interferon (MIG)/(CXCL9), macrophage-derived chemokine (MDC)/(CCL22), interferon-inducible T-cell alpha chemoattractant (I-TAC)/(CXCL11), eotaxin (CCL11), pulmonary and activation-regulated chemokine (PARC)/(CCL18) and hemofiltrate CC-chemokine-4 (HCC-4)/(CCL16) were determined by enzyme-linked immunosorbent assay and compared between the FM and HW groups. FM patients had elevated serum levels of the following inflammatory chemokines: TARC (P < 0.001), MIG (P < 0.001), MDC (P < 0.01), I-TAC (P < 0.01) and eotaxin (P < 0.05). No differences were found in the circulating concentrations of PARC and HCC-4 (homoeostatic chemokines). Since FM patients present higher serum concentrations of inflammatory chemokines than HW, the evaluation of these biomarkers could help in the diagnosis of this syndrome.

The anti-tumor effect and increased tregs infiltration mediated by rAAV-SLC vector.

To explore the anti-tumor effect and immune mechanism mediated by a new recombinant adeno-associated virus (rAAV) encoding secondary lymphoid tissue chemokine (SLC) mature peptide gene. AAV Helper-Free system was used for rAAV-SLC package. The anti-tumor effect of SLC was detected by bearing tumor established from Hepal-6 cells both in C57BL/6J and nude mice. Flow cytometry analysis and IHC for Tumor-infiltrating T cells and CD11c+DCs were also investigated to explore the immunological mechanism. rAAV-SLC was successfully packaged in AAV293 cells and transfected Hepal-6 tumor cells at high efficiency. The anti-tumor effect was demonstrated by less tumor weight and longer survival outcome. Coincident with the anti-tumor response, local elaboration of SLC within the tumor bed elicited a heavy infiltration of CD4+, CD8+T cells and CD11c+ dendritic cells into the tumor sites. More importantly, there was higher infiltration of Foxp3+ regulatory T cells (Tregs). Local elaboration of SLC mediated by rAAV-SLC has strong T cell mediated anti-tumor effect. The study also suggested that Tregs in the tumor microenvironment tampered the anti-tumor effect.