Chemokine Receptors In Cells Research Articles

Chemokine receptors in primary and secondary lymphoid tissues.

Chemokine receptors are a complex superfamily of surface G protein-coupled receptors present mostly in leukocytes. In this chapter, we review the presence and functions of chemokine receptors in the immune cells of the primary and secondary lymphoid organs. Those include bone marrow, thymus, spleen, lymph nodes, and Peyer's patches as the main components of the gut-associated lymphoid tissue. There are general groups of chemokine receptors: conventional and atypical. We will mostly cover the role of conventional chemokine receptors, which are divided into four classes (CC, CXC, CX3C, and XC). Some relevant members are CXCR4, CXCR5, CCR4 and CCR7. For example, CXCR4 is a key chemokine receptor in the bone marrow controlling from the homing of progenitor cells into the bone marrow, the development of B cells, to the homing of long-lived plasma cells to this primary lymphoid organ. CCR7 and CCR4 are two of the main players in the thymus. CCR7 along with CCR9 control the traffic of thymic seed progenitors into the thymus, while CCR4 and CCR7 are critical for the entry of thymocytes into the medulla and as controllers of the central tolerance in the thymus. CXCR4 and CXCR5 have major roles in the spleen, guiding the maturation and class-switching of B cells in the different areas of the germinal center. In the T-cell zone, CCR7 guides the differentiation of naïve T cells. CCR7 also controls and directs the entry of T cells, B cells, and dendritic cells into secondary lymphoid tissues, including the spleen and lymph nodes. As new technologies emerge, techniques such as high dimensional spectral flow cytometry or single-cell sequencing allow a more comprehensive knowledge of the chemokine receptor network and their ligands, as well as the discovery of new interactions mediating unknown and overlooked mechanisms in health and disease.

Analysis of the associations of chemokine receptors expression on circulating Tfh2 and Th2 cells with sIgE level and disease severity in patients with AR

Objective: To clarify the chemotactic characteristics of type 2 helper T cells (Th2 cells) and type 2 follicular helper T cells (Tfh2 cells) in peripheral blood of patients with allergic rhinitis (AR), and to explore the associations between the chemokine receptors expression and the levels of antigen-specific IgE (sIgE) and the severity of the disease. Methods: The peripheral blood mononuclear cells of 41 patients with AR (20 males and 21 females, aged 35.0 (24.5, 47.0) years) and 42 healthy controls (24 males and 18 females, aged 35.0 (24.8, 46.5) years) treated in Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from April 2017 to February 2018 were isolated. The expressions of chemokine receptor (CCR)2, CCR3, CCR4, CCR5, CCR7, CCR8, chemokine C-X3-C-motif receptor 1 (CX3CR1) and C-X-C motif receptor 4 (CXCR4) in Th2 and Tfh2 cells were explored by fluorescence activated cell sorting (FACs). The relationship between the expression of these chemokine receptors in Th2 cells and Tfh2 cells and the levels of serum sIgE and the scores of visual analogue scale (VAS) was analyzed. Graphpad prism 7.0 software was used for statistical analysis. Results: The significant differences in chemotactic characteristics between Th2 cells and Tfh2 cells in the control group were found: Th2 cells highly expressed chemokine receptors CCR2, CCR3, CCR5, CCR8 and CX3CR1, while Tfh2 cells highly expressed immune cell homing chemokine receptors CCR7 and CXCR4. AR patients, compared to the control, expressed higher levels of CCR2, CCR5 and CX3CR1 on peripheral Th2 cells(all P<0.01). At the same time, the proportion of CCR2+and CCR5+Th2 cells was positively correlated with VAS score (r value was 0.58 and 0.61, respectively, both P<0.01). In AR patients, higher expression levels of CCR7 on Tfh2 cells were detected (P<0.01), and the proportion of CCR7+Tfh2 cells was positively correlated with the level of serum sIgE (r=0.51, P<0.01). Conclusion: The percentage of CCR2+ and CCR5+ Th2 cells in peripheral of AR patients can reflect the severity of AR to some extent, while the percentage of CCR7+ Tfh2 cells is positively correlated with the level of serum sIgE.

Inhibitory effects of tolerogenic probiotics on migratory potential of lupus patient-derived DCs.

Objective(s):The present in vitro study aimed to evaluate whether Lactobacillus delbrueckii and Lactobacillus rhamnosus treatments can induce regulatory phenotype, together with modulating the expression of chemokine receptors (CRs) in dendritic cells (DCs). The CRs of DCs have been found to be involved in the pathogenesis of Systemic lupus erythematosus (SLE) through directing recruitment and migration of immune cells.Materials and Methods: In brief, monocytes of patients with SLE and healthy donors were isolated and differentiated to regulatory or inflammatory mature DCs through treatment with L. delbrueckii, L. rhamnosus, mixed probiotics, and LPS. Results:FACScan analysis showed that the expression of CRs including CXCR3, CCR5, CCR4, and CCR3, was significantly reduced in all probiotic-treated groups of SLE and healthy (control) donors when compared with the LPS treated group. Conclusion:The results demonstrated that tolerogenic probiotics could prevent or decrease the expression of inflammatory CRs on the surface of tolerogenic DCs during the maturation process.

Modulatory effect of chemokines on porcine endometrial stromal and endothelial cells

The endometrium undergoes cyclical changes during the estrous cycle and pregnancy. These alterations are controlled by various factors, including cytokines. The present study aimed to screen the effect of several chemokines (CCL2, CCL4, CCL5, CCL8, CXCL2, CXCL8, CXCL9, CXCL10, and CXCL12) on endometrial stromal and endothelial cells. Real-time PCR analysis revealed mRNA expression of all examined chemokines and their receptors in primary stromal cells and undetectable levels of CXCL9, CXCL10, and CXCR3 in endothelial cells. Immunocytochemical staining showed variable distribution of chemokine receptors in stromal and endothelial cells. All examined chemokines enhanced stromal cell proliferation, and CCL2 and CXCL12 also increased the migratory potential of these cells. The evaluation of a possible indirect effect of chemokines on angiogenesis and lymphangiogenesis demonstrated that CXCL12 may potentially negatively affect lymphatic vessel creation. Downregulation of VEGFC mRNA and protein expression was noticed after CXCL12 stimulation. Among all examined chemokines, CCL4 and CCL8 positively affected the proliferation and migration of endothelial cells. The number of capillary-like structures was significantly reduced after CXCL8, CXCL10, and CXCL12 stimulation. In conclusion, among all examined chemokines, CCL2 is thought to act as the modulator of stromal cell functions, whereas CCL4 and CCL8 are suggested to be potent factors directly stimulating blood vessel creation.

Role of chemokine receptors in thyroid cancer and immunotherapy.

Inflammation is currently regarded as an essential component of malignancies. It is now known that the tumor microenvironment may profoundly influence the biological behavior of cancer cells and ultimately the patient's outcome. Chemokine and their receptor play a major role in determining the immune phenotype of the cells infiltrating the thyroid tumor microenvironment. Experimental evidence shows that both normal and cancer thyroid cells express specific chemokine receptors. The expression of at least some of these receptors exerts several biological effects, which influence the course of the disease. The present review article will take into account the role of the most studied chemokine receptors (CXCR1, CXCR2, CXCR3, CXCR4, CXCR7, DARC, CCR3, CCR6 and CCR7) in the context of thyroid cancer. This review will focus on current knowledge provided by in vitro and in vivo studies specifically performed on thyroid cancer including (i) expression of chemokine receptors in normal and cancer thyroid cells; (ii) role of chemokine receptors in affecting the biological behavior of thyroid tumors including the metastatic process; (iii) current knowledge about immunotherapies through targeting of chemokine receptors in thyroid cancer.

Protection by tyrosine kinase inhibitor, tyrphostin AG126, through the suppression of IL-17A, RORγt, and T-bet signaling, in the BTBR mouse model of autism

Autism spectrum disorder (ASD) is an extremely predominant neurodevelopmental disorder expressed as impairment in reciprocal social interaction along with repetitive, restricted, and stereotyped behaviors. The protein tyrosine kinase inhibitor, tyrphostin AG126 (AG126), regulates the expression of several genes that play an important role in the development of neuroinflammatory disorders. Here, we investigate the possible effects of AG126 (5 mg/kg daily through intraperitoneal injection) on self-grooming, marble burying, and hot plate test results in BTBR T + Itpr3tf/J mice (BTBR is a model of autism). We also explore the effects of AG126 administration on IL-17 A, RORγt, T-bet, and IFN-γ production in CD4+ T cells and on CCR6+ chemokine receptors in splenic cells. We further investigated the effect of AG126 administration on the mRNA and protein expression of IL-17 A, RORγt, T-bet, IFN-γ, and NF-κB in the brain tissue. Our results demonstrate that treatment of BTBR mice with AG126 reduced repetitive self-grooming scores and lowered hot plate sensitivity potentials. Furthermore, AG126 administration also caused a substantial reduction of IL-17 A, RORγt, T-bet, and IFN-γ production in CD4+ T cells and on CCR6+ chemokine receptors in splenic cells. BTBR mice treated with AG126 also show decreased mRNA and protein expression levels of IL-17 A, RORγt, T-bet, IFN-γ, and NF-κB activation in brain tissue. Our results indicate that treating BTBR mice with AG126 leads to protection against neuroimmune dysfunction/dysregulation through the inhibition of cytokines and transcription factor signaling. This mechanism may be useful in the development of future therapies for neuroimmune disorders.

A two-color fluorescence enhanced dot-blot assay for revealing co-operative expression of chemokine receptors in cells.

An ultra-high fluorescence enhancement for two dyes on photonic crystal films was achieved to construct a two-color immuno-dot blot assay. This assay was demonstrated to simultaneously detect chemokine receptor co-expressed in cancer cells and reveal their co-operative and subtle changes after binding with respective ligands and drugs.

Aberrant expression of chemokine receptors in lymphoma cells through flow cytometry-based segregation in patients with mycosis fungoides

Aberrant expression of chemokine receptors in lymphoma cells through flow cytometry-based segregation in patients with mycosis fungoides

Isolation and culture of corneal neovascular endothelial cells and expression of chemokine receptors

Background The pathogenesis and mechanism research of corneal neovascularization is of important significance for the prevention and management of corneal neovascularization. Some relative researches are being performed on non-corneal neovascularization-derived vascular endothelial cells, so the results are affected to a certain extent. Objective This study was to isolate and culture vascular endothelial cells from experimental corneal neovascularization tissue and detect the expression of chemokine receptors in vitro. Methods Corneal neovascularization models were established on 10 SPF male BALB/c mice with the age of 7-8 weeks by sticking the filter papers with NaOH on the central corneas, and then the immunofluorescence technique was use to assay the CD31 expression in corneal flatmount 2 weeks after modeling. Corneal pieces were made in 2 weeks after alkali burn and then were digested by collagenase type D. Vascular endothelial cells were isolated from neovascularized tissue by affinity purification using magnetic beads coated with anti-CD31. The cells were cultured on fibronectin-coated walls and then identified by immunocytochemistry. Reverse transcription-PCR was employed to detect the expressions of chemokine receptors in the cells. The use and care of the animals complied with ARVO Statement and this experimental procedure was approved by Soochow University Animal Care Committee. Results Corneal neovascularization occurred at 7 days and peaked at 2 weeks after modeling, and immunofluorescence exhibited the green network-like fluorescence for CD31 antibody in corneas. The cells grew against the wall 2 hours after culture with the polygon shape and large dimension, and the growth obviously quickened after passage. The cultured cells showed the positive response for CD31 antibody, showing the brown dye in cytoplasm, in contrast, the expression of CD31 was absent in corneal stromal cells. Chemokine receptors were positively expressed in the cells with the strongest expression levels in CCR1, CCR2, CCR3 and CCR4 mRNA and the weakest expression levels in CCR9, CXCR4 and CXCR5 mRNA, while CXCR3, CCR6, CCR10 and CX3CR1 mRNA were expressed with the moderate intensity. Conclusions Vascular endothelial cells can be obtained from experimental neovascularized corneas by affinity purification and express chemokine receptors, which facilitate the study of their biological properties. Key words: [Key words]Endothelial cells/corneal neovascularization-derived; Cells, cultured; Corneal neovascularization/chemically induced; Receptors, chemokine; Diseases models, animal; Mice, inbred BALB/c

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

Understanding the role of circulating chemokine (C-C motif) ligand 2 in patients with chronic ischemia threatening the lower extremities

The role of chemokine (C-C motif) ligand 2 (CCL2) in peripheral artery disease is unclear. We measured the difference between serum and plasma levels of CCL2 in patients with chronic ischemia threatening the lower extremities following the observation that atypical chemokine receptors in blood and tissue cells may prevent CCL2 from entering the circulation and consequently modulate its function of attracting monocytes to the site of lesion. To identify the influence of CCL2, we compared the patients' values to those in bio-banked samples from a control population. Further, we explored the association with the Asp42Gly polymorphism (rs12075) in Duffy antigen chemokine receptor; one of these atypical chemokine receptors. When possible, we evaluated in surgically excised normal and affected arteries the calcium burden as well as the expression of CCL2 and related receptors reflecting the inflammatory status. Our findings indicate that circulating CCL2 was significantly associated with the severity and presence of the disease (OR 0.966, 95% CI 0.944 to 0.988, p = 0.003). Circulating CCL2 was dependent on the rs12075 genotype (AA>AG>GG), which, probably, indicates a higher expression of chemokine receptor in the arteries of AA subjects. The associations with genetic variants and the over-expression of atypical chemokine receptors in diseased arteries may have potential implications and our data indicate that CCL2 may represent a previously unrecognized factor that needs to be considered in the screening of patients with risk factors for peripheral artery disease.

Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury

Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury

Quantitative Storm using Organic Dyes

Quantitative super-resolution imaging is important for understanding and determining the exact stoichiometry of molecular complexes or oligomerization states of proteins within a cell. This has been done previously using fluorescence proteins, however these proteins require extensive computational correction due to their blinking. Organic dyes are commonly used for super-resolution imaging because of their high photon numbers, unfortunately blinking corrections for these dyes are very difficult. Here, we develop a technique for quantitative super resolution microscopy that allows us to count the absolute number of molecules using organic “caged” dyes, which do not blink. We calibrate our system and find that caged-fluorescein and caged-carborhodamine, when conjugated with SNAP-tag labeling, allow for quantitative counting of targets. We apply this technique to the characterization of the oligomerization states of chemokine receptors in mammalian cells.

Effect of coupling factor 6 on chemokine receptors in vascular endothelial cells

Effect of coupling factor 6 on chemokine receptors in vascular endothelial cells

Effect of CO2 pneumoperitoneum on the expression of the chemokine receptors CXCR4 and CCR7 in colorectal carcinoma cells in vitro

Background The ability of pneumoperitoneum in laparoscopic surgery to promote proliferation and metastasis of colorectal cancer has become a focus of research in the field of minimally invasive surgery. The aim of this research was to investigate the effect of CO2 pneumoperitoneum under different pressures and exposed times on the expression of chemokine receptors in colorectal carcinoma cells. Methods We constructed an in vitro pneumoperitoneum model. SW480 colon carcinoma cells were exposed to CO2 pneumoperitoneum under different pressures (6, 9, 12, and 15 mmHg) for 1, 2, and 4 hours. These cells were then cultivated under the same conditions as normal SW480 colon carcinoma cells without CO2 pneumoperitoneum (control group), treated at 37°C, and 5% CO2. The expression of the chemokine receptors CXC receptor 4 (CXCR4) and chemokine C receptor 7 (CCR7) was detected by immunocytochemistry and reverse transcriptase polymerase chain reaction after being cultivated for 0, 24, 48, and 72 hours. Results Immunocytochemistry showed that CXCR4 expression in SW480 cells was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups for the same exposure times compared with controls (P &lt;0.05). CCR7 expression in SW480 cells was significantly decreased in the 12 and 15 mmHg CO2 pneumoperitoneum-treated groups compared with controls (P &lt;0.05). CXCR4 and CCR7 expression increased up to the level of the control group after 24 and 48 hours (P &gt;0.05). If the CO2 pneumoperitoneum pressure increased, CXCR4 and CCR7 expression decreased at all exposure times. If the CO2 pneumoperitoneum exposure time prolonged, there were no significant differences in CXCR4 and CCR7 expression under the same pressure. Under all exposure times, CXCR4 and CCR7 mRNA expression was significantly decreased in the 6, 9, 12, and 15 mmHg CO2 pneumoperitoneum-treated groups (P &lt;0.05) compared with controls, and it increased up to the level of controls after being cultivated for 48 hours (P &gt;0.05). If the CO2 pneumoperitoneum pressure increased (with all exposure times) and exposure time prolonged (under the same pressure), there were no significant differences in CXCR4 and CCR7 expression. Conclusions CXCR4 and CCR7 expression is temporarily affected after continuous CO2 pneumoperitoneum treatment. The high pressure of CO2 pneumoperitoneum plays an important role in suppressing the expression of these chemokine receptors. Different lengths of time of exposure to a CO2 pneumoperitoneum-like environment do not change CXCR4 and CCR7 expression.

Expression of a Distinct Set of Chemokine Receptors in Adipose Tissue-Derived Stem Cells is Responsible for In Vitro Migration Toward Chemokines Appearing in the Major Pelvic Ganglion Following Cavernous Nerve Injury.

IntroductionAdipose tissue-derived stem cells (ADSCs) herald tremendous promise for clinical application in a wide range of injuries and diseases. Several preclinical reports demonstrate their efficacy in the treatment of cavernous nerve (CN) injury-induced erectile dysfunction in rats. It was recently illustrated that these effects were established as a result of ADSC migration to the major pelvic ganglion (MPG) where these cells induced neuroregeneration in loco.AimsThe study aims to identify chemotactic factors in the MPG following injury and to match upregulated chemokines to their respective receptors in human ADSC on the genomic, structural, and functional levels.MethodsQuantitative real-time polymerase chain reaction, fluorescence-activated cell sorting (FACS), intracellular FACS, immunofluorescence microscopy, migration assays, and calcium imaging were used in this study.Main Outcome MeasuresThe main outcomes are chemokine expression in the MPG following CN injury, and the functional and structural presence of chemokine receptors in ADSC.ResultsCCR4, CX3CR1, and XCR1 are functionally and structurally present in human ADSC, and are activated by the chemokines CCL2, CX3CL1, and XCL1 respectively, which are upregulated in the MPG following CN injury. CXCR4 and its ligand CXCL12 (SDF1) are likely no major homing factors for ADSC. Expression of chemokine receptor mRNA in ADSC did not necessarily translate into receptor presence at the cell surface and/or functional activation of these receptors. Most of the expressed chemokine receptors were detected in the intracellular compartment of these cells.ConclusionsWe identified the ligand/chemokine receptor pairs CCL2/CCR4, CX3CL1/CX3CR1, and XCL1/XCR1 as potentially responsible for ADSC homing toward the MPG following CN injury. The intracellular localization of various chemokine receptors likely indicates redirecting of chemokine receptors to the cell surface under specific cellular conditions. Furthermore, modification of expression of these receptors at the genomic level may potentially lead to improved migration toward injury sites and thus enhancement of treatment efficacy. Albersen M, Berkers J, Dekoninck P, Deprest J, Lue TF, Hedlund P, Lin C-S, Bivalacqua TJ, Van Poppel H, De Ridder D, and Van der Aa F. Expression of a distinct set of chemokine receptors in adipose tissue-derived stem cells is responsible for in vitro migration toward chemokines appearing in the major pelvic ganglion following cavernous nerve injury. Sex Med 2013;1:3–15.

Campylobacter jejuni-Mediated Induction of CC and CXC Chemokines and Chemokine Receptors in Human Dendritic Cells

Campylobacter jejuni is a leading worldwide bacterial cause of human diarrheal disease. Although the specific molecular mechanisms of C. jejuni pathogenesis have not been characterized in detail, host inflammatory responses are thought to be major contributing factors to the resulting typical acute colitis. The intestinal mucosal chemokine response is particularly important in the initial stages of bacterium-induced gut inflammation. Chemokines attract blood phagocytes and lymphocytes to the site of infection and regulate immune cell maturation and the development of localized lymphoid tissues. The production of chemokines by dendritic cells (DCs) following Campylobacter infection has not yet been analyzed. In the current study, we infected human monocyte-derived DCs with C. jejuni to examine the production of key proinflammatory chemokines and chemokine receptors. The chemokines, including CC families (macrophage inflammatory protein 1α [MIP-1α], MIP-1β, RANTES) and CXC families (growth-related oncogene α [GRO-α], IP-10, and monokine induced by gamma interferon [MIG]), were upregulated in Campylobacter-infected DCs. Chemokine receptors CCR6 and CCR7, with roles in DC trafficking, were also induced in Campylobacter-infected DCs. Further, Campylobacter infection stimulated the phosphorylation of P38, P44/42, and stress-activated protein kinase/Jun N-terminal kinase (SAPK/JNK) mitogen-activated protein kinases (MAPKs) in DCs. NF-κB activation was specifically involved in chemokine induction in DCs infected with C. jejuni. Additionally, STAT3 was significantly increased in Campylobacter-infected DCs compared to that in uninfected DCs. These results suggest that DCs play a significant role in the initiation and modulation of the inflammatory response by enlisting monocytes, neutrophils, and T lymphocytes during human intestinal infection with Campylobacter.

IFN-γ 874A&gt;T Genotype Is Associated With Higher CCR5 Expression in Peripheral Blood Mononuclear Cells From HIV+ Patients

We previously showed that certain cytokines impact on the expression of drug transporters and chemokine receptors in peripheral blood mononuclear cells. Known single-nucleotide polymorphism genes in the genes encoding interferon γ (874A>T, rs62559044) and interleukin 2 (-330T>G, rs2069763) were genotyped in 66 HIV+ patients, and the impact of single-nucleotide polymorphisms on expression of ABCB1, ABCC1, ABCC2, CXCR4, and CCR5 in peripheral blood mononuclear cells from HIV+ patients was assessed. The IFN-γ 874A>T allele and viral load were independently associated with CCR5 expression in patients. These associations have potential implications for HIV disease progression and treatment response that now warrant further study.

Alteration of CXCR7 Expression Mediated by TLR4 Promotes Tumor Cell Proliferation and Migration in Human Colorectal Carcinoma

The link between inflammation and colorectal carcinoma has been acknowledged. However, the impact of bacterial lipopolysaccharide (LPS) binding to Toll-like receptor 4 (TLR4) on chemokine receptors in human colorectal carcinoma cells still remains to be elucidated. The present study shows that exposure to LPS elevated CXC chemokine receptor 7 (CXCR7) expression in colorectal carcinoma SW480 and Colo 205 cell lines expressing TLR4/myeloid differential protein (MD-2). CXCR7 is associated with SW480 cell proliferation and migration. However, exposure of SW480 and Colo 205 cells to LPS had no effect on CXCR4 expression. To further support the above results, the expression of TLR4, MD-2, and CXCR7 was analyzed in human colorectal carcinoma tissues. Higher rates of TLR4 (53%), MD-2 (70%), and CXCR7 (29%) expression were found in colorectal carcinoma tissues than in normal tissues. We demonstrated that the recombination of TLR4, MD-2 and CXCR7 strongly correlated with tumor size, lymph node metastasis and distant metastasis in colorectal carcinoma tissue samples (p = 0.037, p = 0.002, p = 0.042, resp.). Accordingly, simultaneous examination of the expression of TLR4, MD-2 and CXCR7 in cancer tissues of colorectal carcinoma may provide valuable prognostic diagnosis of carcinoma growth and metastasis. Interplay of TLR4, MD-2 and CXCR7 may be of interest in the context of novel immunomodulatory therapies for colorectal carcinoma.

Differential Expression of Adhesion Molecules and Chemokine Receptors Confers Increased Migrative Capacity to ZAP-70-Positive Subclones in CLL Primary Cells,

Abstract 3889Chronic Lymphocytic Leukemia (CLL) is a lymphoproliferative disease characterized by the accumulation and proliferation of mature B-lymphocytes. CLL clinical course is extremely heterogeneous; patients with worse prognosis can be identified by the presence of high ZAP-70 expression. Increasing evidence indicates that the microenvironment plays a critical role providing survival and proliferative signals to CLL cells. In this sense, ZAP-70 protein expression has been related to increased capability of the cells to respond to several survival and migration signals provided by the cellular microenvironment through chemokines and cell-to-cell direct contact.We aimed to analyze the expression levels of several adhesion molecules and chemokine receptors potentially involved in CLL pathogenesis and progression in subclones of CLL cells with high or low ZAP-70 expression within the same patient. For this we obtained peripheral blood mononuclear cells from 40 patients diagnosed with CLL at our institution after informed consent. We then performed a flow cytometry analysis with 7 parameters that allowed for the measurement of the expression of different molecules in CD19+/CD5+ CLL cells with high or low ZAP-70 expression. The expression level of ZAP-70 protein in CD3+ T lymphocytes was used to set the threshold between CLL cells with low or high ZAP-70 expression (Figure 1). Using this approach we analyzed the differential expression of CCR7, CXCR4, CXCR5, CD44, CD49d and CD62L. Interestingly, we found that the expression levels of all the adhesion molecules and cytokine receptors analyzed were significantly higher in those CLL subclones with high ZAP-70 expression compared to CLL cells with low ZAP-70 expression within the same patient (Table 1), suggesting that the relationship with the microenvironment is not uniform across the CLL clone, but those CLL cells with higher expression of ZAP-70 have increased potential to receive signals from other cells. In order to analyze if this could translate into an increased capacity of the CLL cells with high ZAP-70 expression to migrate towards chemokines, we performed an in vitro transmigration assay across bare polycarbonate filters using primary CLL cells from 7 of the patients. We allowed the cells to migrate for 6 hours towards a media containing 1 μg/ml of CCL21 (the ligand of CCR7) and proceed to measure the percentage of CD19+/CD5+ CLL cells with ZAP-70 expression among those cells that had transmigrated and to compare it with the percentage of ZAP-70-positive cells within the ones that did not migrate. Of note, for all the cases analyzed, we observed that the percentage of ZAP-70-positive cells was significantly higher in the cells that had migrated compared to the cells present in the upper chamber (p=0.018) indicating that ZAP-70-positive CLL cells have an enhanced ability to respond to and to migrate towards CCL21. In conclusion, the differential expression of adhesion molecules and chemokine receptors in primary CLL cells with higher expression of ZAP-70 can influence their relationship with the microenvironment and confers them a higher migratory potential towards CCL21. [Display omitted] Table 1:MFI: mean fluorescence intensity SEM: standard error of the meanMean MFI ± SEM Low ZAP-70 CLL cellsMean MFI ± SEM High ZAP-70 CLL cellsPaired samples t-test p valueCCR7163.56 ± 11.3178.20 ± 13.40.007CXCR4263.01 ± 36308.95 ± 45.30.004CXCR5604.02 ± 62.6652.89 ± 67.50.001CD441081.11 ± 76.91210.8 ± 82.5>0.001CD49d58.43 ± 16.169.6 ± 17.6>0.001CD62L33.81 ± 8.947.63 ± 12.1>0.001 Disclosures:No relevant conflicts of interest to declare.