Chemokine-like Factor 1 Research Articles

IMM-H004 protects against oxygen-glucose deprivation/reperfusion injury to BV2 microglia partly by modulating CKLF1 involved in microglia polarization

BackgroundIMM-H004 is a novel compound that has been shown to protect against cerebral ischemia/reperfusion injury in our previous works. Chemokine-like factor 1 (CKLF1) is a chemokine that exhibits increased expression in the ischemic brain. Dysregulation of microglia polarization dynamics is a mechanism of injury expansion poststroke. PurposesThe aim of present study was to investigate the effects of IMM-H004 on cell viability and microglia phenotypes in BV2 microglia suffering from oxygen-glucose deprivation/reperfusion and discussing the involvement of CKLF1 and possible mechanisms. ResultsIMM-H004 protected BV2 microglia from oxygen-glucose deprivation/reperfusion-induced toxicity. We found that the expression of CKLF1 was increased in BV2 microglia with oxygen-glucose deprivation/reperfusion, and IMM-H004 decreased this specially increased expression. Moreover, oxygen-glucose deprivation/reperfusion induced the BV2 microglia to polarize toward an M1 phenotype, and IMM-H004 modulated the polarization shift from the M1 phenotype and skewed toward the M2 phenotype, followed by suppressing the excessive inflammatory response and improving recovery. CKLF1 modulated BV2 microglia toward M1 polarization and induced an inflammatory response. By using receptor inhibitors, we found that OGD/R induced microglia polarization partly through CC chemokine receptor 4. Furthermore, the Co-IP assay showed that IMM-H004 decreased the amount of CKLF1 binding to CC chemokine receptor 4 in the BV2 microglia oxygen-glucose deprivation/reperfusion model. ConclusionsIMM-H004 protects BV2 microglia against oxygen-glucose deprivation/reperfusion injury partly by modulating microglia polarization and further regulating the inflammatory response. The CKLF1/CCR4 axis may be involved in the protective effects of IMM-H004 modulating microglia polarization.

C19, a C-terminal peptide of CKLF1, decreases inflammation and proliferation of dermal capillaries in psoriasis

Psoriasis is a chronic inflammatory autoimmune disease with undefined etiology. Chemokine-like factor 1 (CKLF1), a human cytokine that is a functional ligand for CCR4, displays chemotactic activities in a wide spectrum of leukocytes and plays an important role in psoriasis development. In previous study, our laboratory found that the expression of CKLF1 increased in psoriatic lesions. C19 as a CKLF1’s C-terminal peptide has been reported to exert inhibitory effects on a variety of diseases. However, the protective roles of C19 in endothelial cells proliferation and inflammatory cells chemotaxis remain elusive in psoriasis. In this study we examined the protective effect of C19 on both the cellular model and the animal model. The effects of C19 on endothelial cells proliferation and inflammatory cells chemotaxis were investigated in cultured human umbilical vein endothelial cells (HUVECs) and imiquimod-induced psoriasiform inflammation of BALB/c mice based on techniques including immunohistochemical analysis, quantitative real-time PCR (qRT-PCR), western blot, transwell, and EdU assay. This study shows that CKLF1-C19 significantly protects against psoriasis by inhibiting the infiltration of inflammatory cells and proliferation of microvascular cells, possibly via inhibiting MAPK pathways.

Inhibition of chemokine-like factor 1 improves blood-brain barrier dysfunction in rats following focal cerebral ischemia

Disruption of blood-brain barrier (BBB) and subsequent edema are major contributors to the pathogenesis of ischemic stroke, and the current clinical therapy remains unsatisfied. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, plays important roles in immune response. The expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 activity showed neuroprotective effect by alleviating infiltration of neutrophil and neuron apoptosis in cerebral ischemia. However, few studies have focused on the role of CKLF1 on BBB integrity. The objective of present study was to investigate the role of CKLF1 on BBB integrity by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Brain water content, Evans blue leakage and the expression of aquaporin-4 (AQP-4), matrix metalloproteinase-9 (MMP-9), Zonula Occludens-1 (ZO-1) and Occludin were measured. After treatment with anti-CKLF1 antibody, brain water content and Evans blue leakage in ipsilateral hemisphere were decreased in a dose-dependent manner at 24h after reperfusion, but not changed in contralateral hemisphere. Anti-CKLF1 antibody reduced the expression of AQP-4 and MMP-9, and upregulated the expression of ZO-1 and Occludin. These results suggest that CKLF1 is involved in BBB disruption after reperfusion. Inhibition of CKLF1 protects against cerebral ischemia by maintaining BBB integrity, possibly via inhibiting the expression of AQP-4 and MMP-9, and increasing the expression of tight junction protein.

Chemokine-Like Factor 1 (CKLF-1) is Overexpressed in Keloid Patients: A Potential Indicating Factor for Keloid-Predisposed Individuals.

Chemokine-like factor 1 (CKLF-1) is a novel cytokine which have a crucial role in immune and inflammatory responses. In this study, the expression level of CKLF-1 was measured to assess the difference between keloid patients and people without keloid.Fifty samples were taken from 30 patients: 10 keloid patients; 10 scar patients; and 10 patients without obvious scarring. Patients were randomly selected from the hospitalized patients of Peking Union Medical College Hospital from September 2013 to July 2015. Five groups of samples were established: keloid samples from keloid patients (K); normal skin samples from keloid patients (KS); scar samples from scar patients (C); normal skin samples from scar patients (CS); and normal skin samples from patients without obvious scarring (S). Hematoxylin and eosin (H&E) staining was used to observe morphological changes. CKLF-1, IL-6, IL-8, IL-18, and TGF-β were detected by immunohistochemical and western blot technology. The expression of CKLF-1's mRNA was also measured by the real-time quantitative polymerase chain reaction (RT-qPCR).Compared to the K group, the other 4 groups presented significantly less inflammatory infiltration and lower expression levels of CKLF-1, IL-6, IL-8, IL-18, and TGF-β. Among the 3 normal skin groups, the expression level of CKLF-1 was significantly higher in the KS group than in the CS or S group. The mRNA expression was also obvious in the K and KS groups.CKLF-1 and other inflammatory factors were overexpressed in the samples from keloid patients, indicating that the formation of keloid may be related to inflammation and that CKLF-1 may play an important role in this process.

Distinct expression of chemokine-like factor 1 in synovium of osteoarthritis, rheumatoid arthritis and ankylosing spondylitis.

Chemokine-like factor 1 (CKLF1) is a newly cloned chemotactic cytokine with CCR4 being its functional receptor. Recent evidence demonstrates a role of CKLF1 in arthritis. The aim of this study was to quantify the expression of CKLF1 as well as assess the correlation between CKLF1 and plasma acute-phase markers. Synovium was obtained from 16 osteoarthritis (OA), 15 rheumatoid arthritis (RA) and 10 ankylosing spondylitis (AS) patients undergoing total joint arthroplasty, with other 11 patients treated for meniscal tears during sport accidents serving as normal controls. Levels of CKLF1 and CCR4 mRNA were detected by qRT-PCR, and the expression of CKLF1 was investigated by immunohistochemistry staining, subsequently analyzed with semiquantitative scores. Plasma acute-phase markers of inflammation were determined by ELISA. CKLF1 was found with a particularly up-regulated expression in synovim from AS and RA patients, and CCR4 mRNA levels increased in RA patients, not in OA or AS patients. Elevated levels of plasma markers of inflammation including CRP, ESR and D-dimer were observed in RA. Further, significantly positive correlations between relative expression levels of CKLF1 and CRP/ESR in RA patients and a positive correlation between CKLF1 and ESR in AS patients were found. There was no detectable correlation between CKLF1 and plasma D-dimer. This study confirms an increased but different level of CKLF1 in RA, OA and AS patients, all significantly higher than that in controls. Additionally, the significant positive correlations between CKLF1 levels and CRP/ESR in RA and between CKLF1 and ESR suggest that CKLF1 might contribute to the inflammation state and clinical symptoms in these rheumatic diseases. Further studies are required to investigate the utility of targeting specific CKLF1 for symptom control or disease modification in RA and AS.

Chemokine-like factor 1-derived C-terminal peptides induce the proliferation of dermal microvascular endothelial cells in psoriasis.

Psoriasis is an inflammatory disease characterized by the abnormal proliferation of skin cells, including dermal microvascular endothelial cells. Recently, chemokine-like factor 1 (CKLF1) was found to participate in the local inflammation and cell proliferation. To explore its role in the pathogenesis of psoriasis, the expression of both CKLF1 and its receptor (CCR4) was determined in the psoriatic lesions. Also, the effect of the C-terminal peptides (C19 and C27) of CKLF1 on the proliferation of human umbilical vein endothelial cells was studied in vitro. By immunohistochemistry and immunofluorescence, the expression of both CKLF1 and CCR4 was determined in the psoriatic lesions. The effect of C-terminal peptides on human umbilical vein endothelial cells (HUVECs) was studied in vitro by the evaluation of cell proliferation and apoptosis. The in vivo assessment was performed accordingly through the subcutaneous injection peptides on BALB/c mice. The results showed that, by immunohistochemistry, both CKLF1 and CCR4 were increasingly expressed in psoriatic lesions as compared to normal skins. Moreover, the primary umbilical vein endothelial cells exhibited higher proliferation ratio under the C19 or C27 stimulation, which was even enhanced by the addition of psoriatic sera or TNF-α. Furthermore, the enhancement of peptide simulation was accompanied with the activation of ERK1/2-MAPKs pathway. In addition, such effect of C19 and C27 was mirrored by the hyperproliferation of cutaneous microvessels in BALB/c mice that were subcutaneously injected with the two peptides. Therefore, we concluded that CKLF1 plays a role in the pathogenesis of psoriasis by promoting the proliferation of microvascular endothelial cells that possibly correlates with ERK1/2-MAPKs activation.

Chemokine-like factor 1 promotes the migration of rat primary cortical neurons by the induction of actin polymerization.

Chemokine-like factor 1 (CKLF1), a newly cloned chemotactic cytokine, plays an essential role in immune cell migration. However, the potential role of CKLF1 in the cortical neuronal migration remains unclear. In the present research, by measuring the distance between the margin of brain slices and the leading population of migrating cells, we showed that the extent of cell migration was markedly enhanced in response to CKLF1 treatment, which was significantly inhibited by the simultaneous addition of anti-CKLF1 antibody. By immunofluorescence staining, it was found that CKLF1 induced actin polymerization in primary cerebral cortical neurons. By wound-healing assays, it was found that CKLF1 stimulated the migration of cortical neurons in a dose-dependent manner. In conclusion, our data suggest that CKLF1 promotes the migration of rat primary cerebral cortical neurons by the induction of actin polymerization.

Inhibition of chemokine-like factor 1 protects against focal cerebral ischemia through the promotion of energy metabolism and anti-apoptotic effect

Chemokine-like factor 1 (CKLF1) is a novel C-C chemokine, and plays important roles in immune response and brain development. In previous study, we have found that the expression of CKLF1 increased after focal cerebral ischemia and inhibition of CKLF1 using antagonist C19 peptide protected against cerebral ischemia. However, few studies have focused on the role of CKLF1 on neuronal apoptosis. The objective of present study is to investigate the role of CKLF1 on neuronal apoptosis by applying anti-CKLF1 antibodies in rat focal cerebral ischemia and reperfusion model. Antibodies against CKLF1 was applied to the right cerebral ventricle immediately after transient middle cerebral artery occlusion (MCAO), and infarct volume, neurological score, glucose metabolism and apoptosis-related protein were measured. Treatment with anti-CKLF1 antibody decreased infarct volume and neurological score, and inhibited neuronal apoptosis in a dose-dependent manner at 24h after reperfusion. Anti-CKLF1 antibody also reduced the level of phosphorylation of Akt (P-Akt), and led to decrease of pro-apoptotic protein Bcl-2 associated X protein (Bax) and increase of anti-apoptotic protein B cell lymphoma-2 protein (Bcl-2) and the ratio of Bcl-2/Bax, and inhibited caspase-3 at last. In addition, positron emission tomography (PET) indicated that anti-CKLF1 antibody increased glucose metabolism in ischemic hemisphere. These results suggest that CKLF1 is associated with neuronal apoptosis after cerebral ischemia and reperfusion. Neutralization of CKLF1 with antibodies shows neuroprotective effects against cerebral ischemia, which may be involved in inhibition of Akt pathway, regulation of apoptosis-related protein expression, and improvement glucose metabolism in ischemic hemisphere. Therefore, CKLF1 may be a novel target for the treatment of stroke.

Neutralization of chemokine-like factor 1, a novel C-C chemokine, protects against focal cerebral ischemia by inhibiting neutrophil infiltration via MAPK pathways in rats.

BackgroundInflammation plays a key role in the pathophysiology of ischemic stroke. Some proinflammatory mediators, such as cytokines and chemokines, are produced in stroke. Chemokine-like factor 1 (CKLF1), as a novel C-C chemokine, displays chemotactic activities in a wide spectrum of leukocytes and plays an important role in brain development. In previous studies, we have found that the expression of CKLF1 increased in rats after focal cerebral ischemia and treatment with the CKLF1 antagonist C19 peptide decreased the infarct size and water content. However, the role of CKLF1 in stroke is still unclear. The objective of the present study was to ascertain the possible roles and mechanism of CKLF1 in ischemic brain injury by applying anti-CKLF1 antibody.MethodsMale Sprague–Dawley rats were subjected to one-hour middle cerebral artery occlusion. Antibody to CKLF1 was applied to the right cerebral ventricle immediately after reperfusion; infarct volume and neurological score were measured at 24 and 72 hours after cerebral ischemia. RT-PCR, Western blotting and ELISA were utilized to characterize the expression of adhesion molecules, inflammatory factors and MAPK signal pathways. Immunohistochemical staining and myeloperoxidase activity was used to determine the extent of neutrophil infiltration.ResultsTreatment with anti-CKLF1 antibody significantly decreased neurological score and infarct volume in a dose-dependent manner at 24 and 72 hours after cerebral ischemia. Administration with anti-CKLF1 antibody lowered the level of inflammatory factors TNF-α, IL-1β, MIP-2 and IL-8, the expression of adhesion molecules ICAM-1 and VCAM-1 in a dose-dependent manner. The results of immunohistochemical staining and detection of MPO activity indicated that anti-CKLF1 antibody inhibited neutrophil infiltration. Further studies suggested MAPK pathways associated with neutrophil infiltration in cerebral ischemia.ConclusionsSelective inhibition of CKLF1 activity significantly protects against ischemia/reperfusion injury by decreasing production of inflammatory mediators and expression of adhesion molecules, thereby reducing neutrophils recruitment to the ischemic area, possibly via inhibiting MAPK pathways. Therefore, CKLF1 may be a novel target for the treatment of stroke.

Chemokine-Like Factor 1 (CLFK1) Is Over-Expressed in Patients with Atopic Dermatitis

Background: Human chemokine-like factor 1 (CKLF1), a recently discovered chemokine, has a broad spectrum of biological functions in immune-mediated diseases. It is highly expressed on Th2 lymphocytes and is a functional ligand for human CCR4. CKLF1 has a major role in the recruitment and activation of leucocytes, which plays an important role in the pathogenesis of allergic diseases. The present study was designed to determine the expression of CKLF1 in skin and serum in patients with atopic dermatitis (AD).Methods: The CKLF1 protein expression in skin lesion was analyzed by immunohistochemistry and ELISA. The mRNA expression of CKLF1 in skin lesion was detected by Real-time PCR. The serum levels of CKLF1, IgE, eotaxin, IL-4, IL-5, and IL-13 were measured by ELISA.Results: Histopathological changes in the skin of AD patients showed local inflammation with epidermal thickening and significant inflammatory cellular infiltration. Immunohistochemistry results demonstrated that CKLF1-staining positive cells were located in the epidermal and dermis, and that the CKLF1 expression in AD patients was significantly higher than that in normal control. The CKLF1 mRNA expression in AD patients was significantly higher than that in healthy controls. Serum CKLF1 and IgE levels were significantly increased in AD patients, as were the serum levels of IL-4, IL-5, IL-13 and eotaxin.Conclusions: Both CKLF1 protien and mRNA levels are overexpressed in the skin lesion of AD patients, along with an increase in serum CKLF1 level, indicating that CKLF1 may play an important role in the development of atopic dermatitis.

Antagonistic effect of C19 on migration of vascular smooth muscle cells and intimal hyperplasia induced by chemokine-like factor 1

A chemokine-like factor 1 (CKLF1) is a recently discovered chemokine with broad-spectrum biological functions in inflammation and autoimmune diseases. C19 as a CKLF1's C-terminal peptide has been reported to exert inhibitory effects in a variety of diseases. However, the roles of CKLF1 and C19 on vascular smooth muscle cell (VSMC) migration and neointima formation still remain elusive. The effects of CKLF1 and C19 on VSMC migration and neointimal formation were investigated in cultured VSMCs and balloon-injured rat carotid arteries based on techniques including adenovirus-induced CKLF1 overexpression, gel based perivascular administration of C19, Boyden chamber, scratch-wound assay, real-time PCR, western blot and immunohistochemical analysis. CKLF1 was noticed to accumulate preferentially in neointima after the injury and colocalize with VSMCs. Luminal delivery of CKLF1 adenovirus to arteries exacerbated intimal thickening while perivascular administration of C19 to injured arteries attenuated this problem. In cultured primary VSMCs, CKLF1 overexpression up-regulated VSMC migration, which was down-regulated by C19. These data suggest that CKLF1 has a pivotal role in intimal hyperplasia by mediating VSMC migration. C19 was demonstrated to inhibit CKLF1-mediatated chemotaxis and restenosis. Thus further studies on C19 may provide a new treatment perspective for atherosclerosis and post-angioplasty restenosis.

Effects of chemokine-like factor 1 on vascular smooth muscle cell migration and proliferation in vascular inflammation

ObjectiveVascular smooth muscle cell (VSMC) migration and proliferation are key components of vascular inflammation that may lead to atherosclerosis and restenosis, in which cytokines are considered as pivotal factors regarding recruitment of VSMC. A member of recently described family of chemokines, chemokine-like factor 1 (CKLF1), displays a wide spectrum of chemotaxis. This study investigated the role of CKLF1 in VSMC migration and proliferation during the process of vascular inflammation. Methods and results: The effects of CKLF1 on migration, proliferation and neointimal formation were investigated in cultured VSMCs, rat balloon injured arteries and human atherosclerotic plaques. CKLF1 overexpression greatly enhanced, whereas shRNA knockdown markedly retarded, VSMC migration and proliferation in vitro. In addition, CKLF1 protein accumulated preferentially in neointima of the injured rat arteries in vivo. CKLF1 overexpression resulted in a 2.5-fold increase in intimal thickness. In contrast, shRNA-mediated CKLF1 knockdown significantly suppressed neointima formation by 70% compared that in control group. Intriguingly, besides animal model, higher level of CKLF1 was observed in human atherosclerotic plaques than that in normal arteries. ConclusionCKLF1 plays an essential role in migration and proliferation of VSMCs, which in turn facilitated neointimal hyperplasia and atherosclerosis. Inhibition of CKLF1 activity provides a potential target for the prevention of atherosclerosis and restenosis.

Developmental expression of chemokine-like factor 1, a novel member of chemokines family, in postnatal rat cerebral cortex

Chemokine-like factor 1 (CKLF1) has been implicated to induce the migration of neuroblastoma cells and is abundant in fetal brain but scarce in adult brain. Given the importance of neural cell migration in brain development, it is possible that the chemotaxis of CKLF1 is required during brain development. Therefore, it is essential to know the detailed expression profiles of CKLF1 during brain development first. However, the developmental expression patterns of CKLF1 still remain unclear. We aimed to investigate the temporal and spatial expressions of CKLF1 during cerebral cortex postnatal development in rats. By reverse-transcription PCR/immunoblotting at multiple time points, the mRNA/protein expressions of CKLF1 were in abundance at birth, then decreased progressively within the next two weeks and almost disappeared in adulthood. By immunohistochemistry staining, an obvious expression of CKLF1 was observed in the cerebral cortex, hippocampus, olfactory bulb, some specific nuclei and commissural fibers. Concluding, the temporal expression pattern of CKLF1 was coincident with the postnatal developmental stages and the spatial locations of CKLF1 were some destinations of neural cell migration or regions where myelination normally occurs during cerebrum postnatal development.

Progress in Chemokine-Like Factor 1 Research over the Past 10 Years: From Promoting Inflammatory Cell Trafficking to Inhibiting Allergic Airway Inflammation

Chemokine-like factor 1 (CKLF1) is a cytokine, first described in 2001. CKLF1 is highly expressed in the lung and leukocytes. Recombinant CKLF1 has chemotactic activity on leukocytes, and stimulates the proliferation of murine skeletal muscle cells. Administration of CKLF1 in mice caused dramatic pathological changes in the lungs, including peribronchial leukocyte infiltration, epithelial shedding, collagen deposition, and proliferation of bronchial smooth muscle cells. Expression of CKLF1 mRNA in PBMCs and CKLF1 immunoreactivity in bronchial mucosa were found to be higher in an asthmatic group than in controls. A CKLF1-specific monoclonal antibody was generated using an intramuscular injection of pCDI-CKLF1 followed by electroporation in vivo, instead of a conventional protein immunization strategy. CCR4 is a functional receptor for CKLF1; this was confirmed using a chemotaxis assay, calcium flux assays, and receptor internalization. Two peptides from secreted CKLF1 in cell supernatants were obtained when recombinant CKLF1 was stably expressed in Drosophila S2 cells, termed CKLF1-C27 (C27) and CKLF1-C19 (C19). C27 and C19 had the same effect through CCR4 as the recombinant CKLF1 protein. Although with weaker chemotactic activity, C19 can inhibit chemotaxis induced by other chemokines, such as CKLF1 and TARC/CCL17. Chemically synthesized C19 peptide was injected intraperitoneally to inhibit allergic inflammation associated with asthma in mice, resulting in a significant reduction in AHR, airway eosinophilia, and the number of lymphocytes in BALF. Most recently, C19 peptide was used to treat murine allergic rhinitis. Intranasal administration of C19 reduced allergic symptoms, such as sneezing and rubbing, and serum concentrations of IgE. Mice treated with C19 or budesonide, a intranasal glucocorticoid steroid for the treatment of non-infectious rhinitis, for the treatment of non-infectious rhinitis, showed fewer eosinophils in the submucosa or peribronchiolar zone, while the nasal mucosa of untreated allergic mice displayed significant increases in eosinophils and conspicuous hyperplasia of the mucous glands

Alleviation of murine allergic rhinitis by C19, a C-terminal peptide of chemokine-like factor 1 (CKLF1)

Human chemokine-like factor (CKLF1) is a human cytokine that exhibits chemotactic activities on a wide spectrum of leukocytes. One of CKLF1's C-terminal peptides, C19, exerts inhibitory effects on chemotaxis mediated by mouse Ccr3 and Ccr4 and human CCR3 and CCR4. Mouse models of asthma show that C19 can also inhibit the Th2 response. CCR3 and CCR4 are chemokine receptors important to allergic rhinitis, a condition whose pathogenesis is similar to that of asthma. Here, we established a mouse model of allergic rhinitis by repetitive sensitization and intranasal challenge with OVA and assessed whether C19 has therapeutic effects on this model. In this study, both intranasal and intraperitoneal administration of C19 reduced allergic symptoms such as sneezing and rubbing and serum concentration of IgE. C19 showed a strong ability to suppress eosinophil accumulation in nasal mucosa and lung tissues. C19 was able to suppress the Th2 cytokine IL-4 without augmenting the Th1 cytokine IFN-γ in BAL and IL-4+ cells in the local nasal tissue. In terms of symptom amelioration, IgE reduction, and eosinophilia suppression, C19 was found to be as effective against allergic rhinitis as Budesonide. Moreover, intranasal treatment has a stronger therapeutic effect than other types of administration, and it may be more convenient and safe. For these reasons, C19 may have potential in the treatment of allergic rhinitis.

Expression of chemokine-like factor 1 after focal cerebral ischemia in the rat

Cerebral ischemic injury is associated with the induction of a series of pro-inflammatory mediators such as cytokines and chemokines. The chemokine-like factor 1(CKLF1), as a novel human cytokine, displays chemotactic activities in a wide spectrum of leukocytes. The present study was conducted to determine if CKLF1 was produced in the brain of rats subjected to transient middle cerebral artery occlusion (TMCAO). Therefore, RT-PCR, Western blot and immunohistochemistry were utilized to characterize the expression of CKLF1 at different times after TMCAO. The result showed that almost no expression of CKLF1 was found in the sham-operated or contralateral cerebral cortex and hippocampus. CKLF1 expression significantly increased in the ischemic cerebral cortex and hippocampus, elevating at 12 h and peaking at 2 days after reperfusion. CKLF1 positive staining was mainly present in the cerebral cortex, hippocampus, thalamus, and hypothalamus. These results demonstrate that the expression of CKLF1 increases after focal cerebral ischemia in rat brain. Thus, CKLF1 may be a potential therapeutic target for cerebral ischemia.

The CKLF1-C19 peptide attenuates allergic lung inflammation by inhibiting CCR3- and CCR4-mediated chemotaxis in a mouse model of asthma

Human chemokine-like factor 1 (CKLF1) is a functional ligand for human CCR4, which is highly expressed on Th2 lymphocytes and plays an important role in the pathogenesis of asthma. The expression and function of CKLF1 are associated with asthma. The CKLF1 C-terminal peptides C19 and C27 also interact with human CCR4. Albeit with weaker chemotactic activity, C19 can inhibit chemotaxis induced by both CKLF1 and CCL17. Here, we explore whether C19 can act as an antagonist in the development of asthma. A mouse model of asthma and in vitro and in vivo chemotaxis assays were used. Using a mouse model of asthma, we demonstrate here that C19 reduces airway eosinophilia, lung inflammation and airway hyperresponsiveness; in contrast, C27 has little effect on these parameters. The inhibitory effects of C19 on CCR4-mediated chemotaxis could be observed in human Th2 lymphocytes and in the splenocytes from ovalbumin-sensitized mice. Furthermore, we show that C19 can inhibit CCL11-induced chemotaxis of mouse eosinophils and human CCR3-transfected or mouse Ccr3-transfected HEK293 cells. In vivo chemotaxis assays revealed that C19 and C27 can reduce CCL11-mediated recruitment of eosinophils into the peritoneal cavity and that this inhibitory effect is stronger for C19 than for C27. Thus, C19 can attenuate airway eosinophilia and lung inflammation by inhibiting CCR3- and CCR4-mediated chemotaxis in a mouse model of asthma. Given its ability to inhibit human CCR3- and CCR4-meditated chemotaxis, C19 has great therapeutic potential for use in the treatment and control of allergic asthma.

Discovery and Optimization of Novel 3-Piperazinylcoumarin Antagonist of Chemokine-like Factor 1 with Oral Antiasthma Activity in Mice

Chemokine-like factor 1 (CKLF1) is a novel functional cytokine that acts through its receptor CC chemokine receptor 4 (CCR4). Activation of CCR4 by CKLF1 plays an important role in diseases such as asthma and multiple sclerosis. This article describes a cell-based screening assay using an FITC-labeled CCR4 agonist (CKLF1-C27), a CKLF1 peptide fragment. Screening of our in-stock small-molecule library identified a 3-piperazinylcoumarin analogue 1 (IC(50) = 4.36 x 10(-6) M) that led to the discovery of orally active compound 41 (IC(50) = 2.12 x 10(-8) M) through systematic optimization. Compound 41 blocked the calcium mobilization and chemotaxis induced by CKLF1-C27 and reduced the asthmatic pathologic changes in lung tissue of human CKLF1-transfected mice. Further studies indicated that compound 41 ameliorated pathological changes via inhibition of the NF-kappaB signal pathway.

Expressional and functional studies of CKLF1 during dendritic cell maturation

Chemokine-like factor 1 (CKLF1) was the first member of the CKLF-like MARVEL transmembrane domain containing member (CMTM) family to be discovered. Its expression level is increased clearly in peripheral blood lymphocytes upon phytohemagglutinin stimulation, but little is known about the expression and function of CKLF1 in dendritic cells (DCs), which are the most potent antigen-presenting cells. In the present study, we showed that CKLF1 was highly expressed in monocytes. During differentiation from monocytes to immature DCs, CKLF1 was increased dramatically on day 2 and then decreased from day 3 to 5. Upon maturation with different stimuli, CKLF1 was down-regulated. Two peptides of CKLF1, C19 and C27, stimulated the effect of immature DCs (imDCs) on T-cell proliferation and IFN-γ production. Further study on DC maturation showed that C19 and C27 up-regulated HLA-DR expression and IL-12 secretion, with no obvious effects on CD80, CD83 or CD86. Thus, CKLF1-C19 and -C27 can stimulate antigen-presenting capability of imDCs.

The expression of chemokine-like factor 1 in human atherosclerotic plaques

To evaluate the difference of the expression level of chemokine-like factor 1 (CKLF1) mRNA and protein between human atherosclerotic plaques and normal arteries. Real-time quantitative PCR was performed with experimental group (24 cases of atherosclerotic plaques) specimens and control group (25 cases of normal arteries) specimens. Serial microscopic sections were used for immunohistochemistry with CKLF1 antibody in 17 experimental group specimens and 10 control group specimens. The endothelial cell, vascular smooth muscle cell (VSMC) and macrophage were CKLF1 positive expression cells in atherosclerotic plaques. There was a foundational expression of CKLF1 mRNA in control group (RQ(Median) = 2.4), and the expression of CKLF1 mRNA in experimental group is up-regulated significantly (RQ(Median) = 132.2, P < 0.05). The VSMC positive expression rate of CKLF1 protein was 100% in experimental group and 70% in control group, the former was up-regulated significantly. The expression of CKLF1 is up-regulated in atherosclerotic plaques. It may play an important role in the process of immune and inflammatory responses of atherogenesis.