Spatially targeted chemokine exocytosis guides transmigration at lymphatic endothelial multicellular junctions.

Migrating cells preferentially breach and integrate epithelial and endothelial monolayers at multicellular vertices. These sites are amenable to forces produced by the migrating cell and subsequent opening of the junctions. However, the cues that guide migrating cells to these entry portals, and eventually drive the transmigration process, are poorly understood. Here, we show that lymphatic endothelium multicellular junctions are the preferred sites of dendritic cell transmigration in both primary cell co-cultures and in mouse dermal explants. Dendritic cell guidance to multicellular junctions was dependent on the dendritic cell receptor CCR7, whose ligand, lymphatic endothelial chemokine CCL21, was exocytosed at multicellular junctions. Characterization of lymphatic endothelial secretory routes indicated Golgi-derived RAB6+ vesicles and RAB3+/27+ dense core secretory granules as intracellular CCL21 storage vesicles. Of these, RAB6+ vesicles trafficked CCL21 to the multicellular junctions, which were enriched with RAB6 docking factor ELKS (ERC1). Importantly, inhibition of RAB6 vesicle exocytosis attenuated dendritic cell transmigration. These data exemplify how spatially-restricted exocytosis of guidance cues helps to determine where dendritic cells transmigrate.

Relationship between CCL21 and TREM2/DAP12 signaling pathways in spinal dorsal horn in remifentanil-induced hyperalgesia in mice with incisional pain / 中华麻醉学杂志

Objective:To evaluate the relationship between CCL21 and triggering receptor expressed on myeloid cells 2 (TREM2)/DNAX-activating protein of 12 kDa (DAP12) signaling pathways in the spinal dorsal horn in remifentanil-induced hyperalgesia in mice with incisional pain.Methods:Thirty-two SPF healthy male C57BL/6J mice, weighing 18-22 g, aged 8-10 weeks, were divided into 4 groups ( n=8 each) using a random number table method: control group (group C), CCL21 neutralizing antibody group (group anti-CCL21), remifentanil + incisional pain group (group R+ I), and CCL21 neutralizing antibody + remifentanil + incisional pain group (group anti-CCL21+ R+ I).A CCL21 neutralizing antibody 0.3 μg (diluted to 10 μl in normal saline) was intrathecally injected in anti-CCL21 and anti-CCL21+ R+ I groups twice a day.Normal saline 10 μl was intrathecally injected at the same time point twice a day in C and R+ I groups.Fifteen min after intrathecal injection, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at an interval of 15 min in C and anti-CCL21 groups, and remifentanil 10 μg/kg (diluted to 0.1 ml in normal saline) was injected via the caudal vein for 4 consecutive times at an interval of 15 min in R+ I and anti-CCL21+ R+ I groups.The tail-flick latency (TFL) and mechanical paw withdrawal threshold (MWT) were measured at 24 h before remifentanil or normal saline injection (T 0) and 3, 6, 24 and 48 h after stopping injection of remifentanil or normal saline (T 1-4).The mice were sacrificed after the last measurement of pain threshold, and L 4-6 segments of the spinal cord were removed for determination of the expression of TREM2 and DAP12 protein and mRNA (by Western blot or quantitative real-time polymerase chain reaction). Results:Compared with group C, TFL was significantly shortened and MWT was decreased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was up-regulated in group R+ I and R+ I+ anti-CCL21 ( P<0.05), and no significant change was found in the parameters mentioned above in group anti-CCL21 ( P>0.05).Compared with group R+ I, TFL was significantly prolonged and MWT was increased at T 1-4, and the expression of TREM2 and DAP12 protein and mRNA was down-regulated in group anti-CCL21+ R+ I ( P<0.05). Conclusions:CCL21 is involved in remifentanil-induced hyperalgesia by activating TREM2/DAP12 signaling pathways in the spinal dorsal horn of mice with incisional pain.

Shape and Function of Interstitial Chemokine CCL21 Gradients Are Independent of Heparan Sulfates Produced by Lymphatic Endothelium.

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.

Locally Triggered Release of the Chemokine CCL21 Promotes Dendritic Cell Transmigration across Lymphatic Endothelia.

Trafficking cells frequently transmigrate through epithelial and endothelial monolayers. How monolayers cooperate with the penetrating cells to support their transit is poorly understood. We studied dendritic cell (DC) entry into lymphatic capillaries as a model system for transendothelial migration. We find that the chemokine CCL21, which is the decisive guidance cue for intravasation, mainly localizes in the trans-Golgi network and intracellular vesicles of lymphatic endothelial cells. Upon DC transmigration, these Golgi deposits disperse and CCL21 becomes extracellularly enriched at the sites of endothelial cell-cell junctions. When we reconstitute the transmigration process in vitro, we find that secretion of CCL21-positive vesicles is triggered by a DC contact-induced calcium signal, and selective calcium chelation in lymphatic endothelium attenuates transmigration. Altogether, our data demonstrate a chemokine-mediated feedback between DCs and lymphatic endothelium, which facilitates transendothelial migration.

Insights into CCL21's roles in immunosurveillance and immunotherapy for gliomas.

Chemokine (C-C) motif ligand 21 (CCL21) is involved in immunosurveillance and has recently garnered the attention of neuro-oncologists and neuroscientists. CCL21 contains an extended C-terminus, which increases binding to lymphatic glycosaminoglycans and provides a mechanism for cell trafficking by forming a stationary chemokine concentration gradient that allows cell migration via haptotaxis. CCL21 is expressed by endothelial cells of the blood-brain barrier in physiologic and pathologic conditions. CCL21 has also been implicated in leukocyte extravasation into the central nervous system. In this review, we summarize the role of CCL21 in immunosurveillance and explore its potential as an immunotherapeutic agent for the treatment of gliomas.

Clinical prediction value of chemokine CCL21 level for acute coronary syndrome / 中华老年医学杂志

Objective To investigate the clinical prediction value of chemokine CCL21 level for acute coronary syndrome.Methods Totally 212 patients receiving coronary arteriography were divided into acute myocardial infarction group(AMI,n=72),unstable angina pectoris group(UAP,n=76),and stable angina pectoris group(SAP,n=64).The serum level of chemokine CCL21 was detected by double-antibody sandwich enzyme-linked immunosorbent assay (ELISA)and the pathological changes of coronary artery were measured by Gensini scoring system.All patients were followed up for six months and the cardiovascular adverse events were recorded.Results The serum level of CCL21 was(169.72±14.64)ng/L in AMI group,(154.42±16.50)ng/L in UAP group,and(143.87±9.80) ng/L in SAP group,with statistically significant differences (F =99.818,P =0.000).Serum levels of CCL21 in ACS group and SAP group were positively correlated with Gensini score(r=0.474,P =0.000;r=0.350,P=0.049).Binary Logistic regression analysis revealed that chemokine CCL21 was an independent risk factor for predicting acute coronary syndrome (OR =1.049,P =0.022).The CCL21-judged area under the ROC curve in acute coronary syndrome group was 0.887 ± 0.028 (P =0.000),with diagnostic point of serum level of chemokine CCL21 at 159.15 ng/L,sensitivity of 0.635,specificity of 0.981.Serum level of CCL21 was higher in the patients with cardiovascular adverse events than in the patients without cardiovascular adverse events[(168.57±7.24)ng/L vs.(156.92± 6.53) ng/L],with statistically significant difference (t =16.100,P =0.000).Conclusions Serum level of chemokine CCL21 reflects the severity degree of coronary artery disease.The chemokine CCL21,as an independent and effective marker,can predict acute coronary syndrome.

Role of cemokine ligand 21 in spinal cord in tibia bone cancer pain in rats / 中华麻醉学杂志

Objective To investigate the role of chemokine ligand 21 (CCL21) in the spinal cord in tibia bone cancer pain (BCP) in rats.Methods Forty adult female SD rats weighing 160-180 g were randomly divided into 5 groups (n=8 each):sham operation group (group Ⅰ ); sham operation + CCL21 neutralizing antibody group (groupⅡ); BCP group (group [); BCP + PBS group (group Ⅳ); BCP + control IgG group (groupⅤ)and BCP + CCL21 neutralizing antibody group (group Ⅵ).BCP was induced by inoculating Walker-256 mammary gland carcinoma cells into the rat tibia medullary cavity in groups Ⅲ-Ⅵ.PBS 15 μl,IgG 10 μg and CCL21 neutralizing antibody 10 μg were injected intrathecally (IT) at 14 days after intra-tibial injection of Walker-256 mammary gland cancer cells in groups Ⅳ- Ⅵ respectively.Mechanical withdrawal threshold to yon Frey filament stimulation (MWT) was measured at 1 day before (To,baseline) ; 7 and 14 d after Walker-256 cell injection (T1,T2)and at 0.5,1,2,4,8,12,24 and 48 h after intrathecal injection (T3-10 ).Results Intra-tibial injection of Walker-256 mammary gland cancer cells significantly decreased MWT as compared with the baseline values in administration of CCL21 neutralizing antibody at T5-8 as compared with MWT before intrathecal administration at T2 in group Ⅵ.MWT was significantly lower in groups Ⅲ- Ⅳ than in groups Ⅰ and Ⅱ.MWT was significantly higher at T5-8 in group Ⅵ than in groups Ⅲ - Ⅴ.Conclu]sion CCL21 in the spinal cord is involved in the maintenance of tibia BCP in rats.

CCL21/CCR7 axis and mechanisms of tumor invasion and metastasis / 国际肿瘤学杂志

Secondary lymphoid tissue chemokine (CCL21) is a double-edged sword, which exerts antitumor, anti-infection immune response by binding to the receptor CCR7 on the surface of the multiple immune cells. However, a variety of tumor cells also express the receptor CCR7, the combination of CCL21 with CCR7promotes the invasion and metastasis of tumor cells, leading to the facilitation of tumor development. Therefore,exploring the mechanism(s) of tumor invasion and metastasis might be helpful for use of CCL21 as tumor gene therapy through blocking of CCL21's promotion of tumor invasion and metastasis.