The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play an important role in lymphocyte homing and have also been associated with inflammatory, allergic and lung disorders. Cloning of the cynomolgus monkey genes encoding CCR7, CCL19 and CCL21 revealed 93-97% sequence identity of the deduced proteins with their respective human homologs. In chemotaxis assays, B300-19 cells transfected with the cynomolgus (c) CCR7 receptor migrated in response to cCCL19 and cCCL21 in a dose-dependent manner with EC(50) values of 324+/-188nM and 247+/-29nM, respectively. cCCL19 and cCCL21 also elicited calcium responses in stable cell CHO-K1 lines expressing the cCCR7 receptor with EC(50) values of 227+/-4nM and 484+/-163nM, respectively. Although both human (h) CCL19 and hCCL21 elicited increases in intracellular calcium at the cCCR7 receptor, hCCL19 almost completely inhibited subsequent stimulation by hCCL21 whilst hCCL21 failed to inhibit subsequent stimulation by hCCL19. These results identify novel cynomolgus monkey genes and provide a model system for pre-clinical studies of potential drug candidates.

The main goal of this study was to investigate the possibility of expressing recombinant protein of C-C chemokine ligand 21 (CCL21) in Solanum lycopersicum via agroinfiltration. CCL21 is a chemokine can be used for anti-metastatic of cancer cell lines. To examine the expression of CCL21 protein in S. lycopersicum, the construct of ccl21 was synthesized. This construct was cloned into pBI121 and the resulting CCL21 plasmid was agro-infiltrated into S. lycopersicum leaves. Within three days after infiltration, Expression of the foreign gene was confirmed by quantitative Real-time PCR. A recombinant CCL21 protein was immunogenically detected by western blot, dot blot and ELISA assay. And results showed that the foreign gene was expressed in the transformed leaves in high level. Also scratch assay was used to investigate the role of this protein in anti-metastatic function. The results demonstrated anti-metastatic of cancer cells in the presence of this protein.

Fibrocytes are bloodborne mesenchymal progenitor cells that are recruited to injured tissue sites and contribute to the repair process by acquiring a myofibroblast-like phenotype and producing extracellular matrix components and growth factors. Treatment with normal fibrocytes or their exosomes restores the ability of genetically diabetic mice to heal skin wounds, suggesting the existence of dysfunctional alterations in diabetic fibrocytes. This study compared the migratory, metabolic and functional characteristics of fibrocytes from patients with type 2 diabetes (T2DPs) and healthy controls (HCs). It was found that the frequency of these cells was abnormally low in the peripheral blood of T2DPs. Diabetic fibrocytes showed reduced expression of the C-X-C motif and C-C motif chemokine receptors (CXCR)4, (CCR)5, and CCR7, and demonstrated reduced migration in response to their ligands (CXCL)12, (CCL)5, and CCL21. They exhibited increased expression of the receptor for advanced glycation end product, suppression of the alternative AGE receptor 1, increased intracellular concentrations of AGEs, decreased expression of sirtuin-1 and elevated oxidative stress. In short-term cultures, fibrocytes from T2DPs released larger amounts of proinflammatory cytokines than those from HCs. Unlike normal fibrocytes, diabetic fibrocytes did not exhibit increased expression of type I collagen and α-smooth muscle actin on stimulation with transforming growth factor (TGF)-β1 and this abnormal response was associated with downregulation of TGF-β1 type II receptor on the cell surface. Study findings uncover multiple migratory and functional alterations of diabetic fibrocytes that may contribute to explain why T2DPs experience impaired wound healing and chronic ulcers. © 2018 IUBMB Life, 70(11):1122-1132, 2018.

There is an increasing biotechnological interest in the 'arming' of therapeutic antibodies with bioactive payloads. While many antibody-cytokine fusion proteins have been extensively investigated in preclinical and clinical studies, there are only few reports related to antibody-chemokine fusion proteins ('immunochemokines'). Here, we describe the cloning, expression, and characterization of 10 immunochemokines based on the monoclonal antibody F8, specific to the alternatively spliced extra domain A (EDA) of fibronectin, a marker of angiogenesis. Among the 10 murine chemokines tested in our study, only CCL19, CCL20, CCL21, and CXCL10 could be expressed and isolated at acceptable purity levels as F8-based fusion proteins. The immunochemokines retained the binding characteristics of the parental antibody, but could not be characterized by gel-filtration analysis, an analytical limitation which had previously been observed in our laboratory for the unconjugated chemokines. When radioiodinated preparations of CCL19-F8, CCL20-F8, CCL21-F8, and CXCL10-F8 were tested in quantitative biodistribution studies in tumor-bearing mice, the four fusion proteins failed to preferentially accumulate at the tumor site, while the unconjugated parental antibody displayed a tumor:blood ratio >20:1, 24 h after intravenous (i.v.) administration. The tumor-targeting ability of CCL19-F8 could be rescued only in part by preadministration of unlabeled CCL19-F8, indicating that a chemokine trapping mechanism may hinder pharmacodelivery strategies. While this article highlights expression, analytical, and biodistribution challenges associated with the antibody-based in vivo delivery of chemokines at sites of disease, it provides the first comprehensive report in this field and may facilitate future studies with immunochemokines.

Any vaginal product that alters the mucosal environment and impairs the immune barrier increases the risk of sexually transmitted infections, especially HIV infection, which thrives on mucosal damage and inflammation. The FDA-recommended rabbit vaginal irritation (RVI) model serves as a first line selection tool for vaginal products; however, for decades it has been limited to histopathology scoring, insufficient to select safe anti-HIV microbicides. In this study we incorporate to the RVI model a novel quantitative nuclease protection assay (qNPA) to quantify mRNA levels of 25 genes representing leukocyte differentiation markers, toll-like receptors (TLR), cytokines, chemokines, epithelial repair, microbicidal and vascular markers, by designing two multiplex arrays. Tissue sections were obtained from 36 rabbits (6 per treatment arm) after 14 daily applications of a placebo gel, saline, 4% nonoxynol-9 (N-9), and three combinations of the anti-HIV microbicides tenofovir (TFV) and UC781 in escalating concentrations (highest: 10% TFV+2.5%UC781). Results showed that increased expression levels of toll-like receptor (TLR)-4, interleukin (IL)-1β, CXCL8, epithelial membrane protein (EMP)-1 (P<0.05), and decreased levels of TLR2 (P<0.05), TLR3 and bactericidal permeability increasing protein (BPI) (P<0.001) were associated with cervicovaginal mucosal alteration (histopathology). Seven markers showed a significant linear trend predicting epithelial damage (up with CD4, IL-1β, CXCL8, CCL2, CCL21, EMP1 and down with BPI). Despite the low tissue damage RVI scores, the high-dose microbicide combination gel caused activation of HIV host cells (SLC and CD4) while N-9 caused proinflammatory gene upregulation (IL-8 and TLR4) suggesting a potential for increasing risk of HIV via different mechanisms depending on the chemical nature of the test product.

CCL19/MIP-3beta and CCL21/SLC are essential for chemotactic recruitment of mature dendritic cells (DC) to T-cell areas of secondary lymphoid tissue. Paucity of lymph node T-cells (plt/plt) mice lack CCL21-serine (ser) and CCL19 expression. We tested plt/plt and wild type (wt) BALB/c (H2d) mice as recipients of heart or skin allografts from C57BL/10J (H2b) donors. Donor DC trafficking to secondary lymphoid tissue was markedly reduced in plt heart but not skin allograft recipients. Heart, but not skin grafts survived significantly longer in plt recipients. Accordingly, T cells from plt heart transplant recipients demonstrated poor anti-donor responses in ex vivo MLR, compared to wt heart or wt and plt skin recipients. Moreover, donor-reactive T cells from plt heart recipients exhibited Th2-skewing in comparison to T cells from wt heart or skin graft recipients. Anti-CXCL9/Mig was administered for 2 weeks post-transplant to determine whether impairment of activated T-cell migration could further prolong cardiac allograft survival in plt recipients. CXCL9-antagonism extended graft survival significantly only in plt mice, likely due, in part, to retention of alloactivated T cells in secondary lymphoid tissue/reduction of graft-infiltrating T cells. Thus, targeting DC and activated T-cell migration concomitantly has additive effects in prolonging heart graft survival with potential for therapeutic application.

Research has firmly established that infection by human immunodeficiency virus (HIV) leads to structural disruption in secondary lymph organs (SLOs) and that IL-7 expression by SLOs is downregulated in simian immunodeficiency virus (SIV)-infected rhesus macaques. However, the foregoing has not been demonstrated in HIV-infected patients. As well, SLO-produced chemokines and cytokines, other than IL-7, have not been tested. In this study, SLOs in HIV-infected patients exhibit decreased levels of lymphoid cytokines, such as IL-7 and C-C motif chemokine ligand 21 (CCL21), due to lower expression of lymphotoxin (LT)-β. Previous research has shown that LT-β is produced mainly by CD4⁺T cells in rhesus macaques, while our study found the same level of LT-β expressed by CD4⁺T and CD8⁺T cells in humans. CD8⁺T cells substitute for depleted CD4⁺T cells LT-β production. Only the total number of CD3⁺T cells can account for the majority of LT-β in human SLOs. This study indicates a possible mechanism and a potential target for improvement of SLO function in HIV-infected patients, a novel adjuvant therapy for AIDS.

In antigen trafficking from the skin, it has been postulated that Langerhans cells/dendritic cells are activated after capturing exogenous antigens, up-regulate the expression of the chemokine receptor, CCR7, and migrate into lymphoid organs in response to the signaling of a chemokine, CCL21, which is expressed in lymphatic vessels and T cell zone stromal cells. Here we demonstrate that there is a distinct pathway of antigen trafficking from skin in the steady state that is independent of CCL21-CCR7 signaling. Employing melanin granules as an endogenous traceable antigen, we developed a system for visualizing antigen trafficking using mice with melanocytosis in the skin. We found the abrogation of antigen trafficking into regional lymph nodes (LN) in CCL21-Ser-deficient paucity of lymph node T cells (plt) mice in the active state induced by lipopolysaccharide injection, corresponding with previous reports, but normal accumulation of antigen in regional LN under steady-state conditions. These findings suggest that self-antigen is trafficking constitutively using pathway(s) other than that of the active state and the constitutive trafficking might regulate self-reactivity of the immune system.

Human CCRL1 belongs to the family of silent chemokine receptors. This transmembrane protein plays a role in blunting function of chemokines through binding to them. This will attenuate immune responses. Interaction between CCRL1 and CCL21 determines this immune extinction. Thus inhibiting the action of this atypical chemokine seems to stimulate immune responses especially in the case of suppressed and immune deficient conditions. In this study we predicted 3D structure of CCRL1 using comparative modeling and Hiddebn Markov Model algorithm. Final predicted model optimized by Modeller v9.8 and minimized regarding energy level using UCSF chimera candidate version1.5.3. ClasPro webserver was used to find interacting residues between CCRL1 and CCL21. Interacting residues were used as target for chemical inhibitors by simulated docking study. For finding potential inhibitors, library of KEGG compounds screened and 97 obtained chemicals docked against interacting residues between CCRL1- CCL21 and MolDock was used as docking scoring function. Results indicated that Hexadecanal is a potential inhibitor of CCRL1- CCL21 interaction. Inhibition of this interaction will increase intercellular level of CCl21 and interaction between CCL21 and CCR7 causes immune potentiaiton.

BACKGROUND

Costimulatory blockade with anti-CD40L monoclonal antibody (mAb) plus donor-specific splenocyte transfusion (DST) induces alloantigen-specific tolerance. We previously showed that lymphotoxin signaling in the fibroblastic reticular cell (FRC) stromal subset was required for proper lymph node structure and function during tolerization in murine cardiac transplantation. Here we focused on FRC functions and hypothesized that DST and anti-CD40L mAb-modulated FRC interactions with CD4(+) T cells in mice.

METHODS

Mice were immunized or tolerized by DST or DST plus anti-CD40L mAb. Fibroblastic reticular cells were flow-sorted at different timepoints for characterization and in vitro proliferation and activation assays.

RESULTS

Fibroblastic reticular cells responded rapidly to DST by transcribing inflammatory cytokine and chemokine messenger RNAs, such as CXCL2, CXCL9, CXCL10, and CCL21. Conversely, anti-CD40L mAb inhibited FRC inflammatory responses. CD40 was expressed on FRC and agonistic anti-CD40 mAb activated FRC, which supported CD4(+) T-cell proliferation, whereas unstimulated FRC did not. Anti-CD3 mAb-activated CD4(+) T cells induced inflammatory cytokine and chemokine expressions by FRC, which were inhibited by anti-CD40L mAb. Thus, FRC phenotype was altered by interaction with CD4(+) T cells through CD40-CD40L, and activated FRC interacted directly with CD4(+) T cells to support T cell activation and proliferation in vitro.

CONCLUSIONS

Taken together, these results demonstrated that CD40 on FRC facilitated bidirectional communication between FRC and CD4(+) T cells via CD40-CD40L, thereby altering FRC gene expression of immune regulatory molecules. Because blockade of CD40-CD40L interactions results in tolerance in mice, identification of FRC-T cell interactions provides a new research target for tolerance induction.

The prevalence of health problems in the offspring of pregnant diabetic mothers has recently been verified. Therefore, the present study was designed to investigate the influence of dietary camel whey protein (CWP), administered as a supplement to streptozotocin (STZ)-induced diabetic pregnant mice, on the efficiency of the immune system of the offspring. Three groups of female mice (n = 10) were used: non-diabetic control mice, diabetic mice, and diabetic mice orally administered CWP during the pregnancy and lactation periods. We then tested the immune response of B and T cells in adult male offspring (n = 15 in each group) by using flow cytometry, western blotting, and ELISAs. Our data demonstrated that the offspring of diabetic dams exhibited several postpartum complications, such as significant aberrant overexpression of activating transcription factor-3 (ATF-3), significant elevation of the plasma levels of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) and reactive oxygen species (ROS), marked decreases in the plasma levels of IL-2 and IL-7, significant inhibition of CCL21- and CXCL12-mediated chemotaxis of B- and T-lymphocytes, and a marked decrease in the proliferative capacity of antigen-stimulated B- and T-lymphocytes. Interestingly, administration of CWP to diabetic dams substantially restored the expression of ATF-3 and the levels of ROS, pro-inflammatory cytokines, IL-2, and IL-7 in the offspring. Furthermore, the chemotaxis of B- and T-lymphocytes toward CCL21 and CXCL12 and the proliferative capacities of these lymphocytes were restored in the male offspring of diabetic mice administered CWP. Our data provide evidence of a protective role of CWP in decreasing the tendency of the offspring of diabetic mothers to develop diabetes and related complications.

We have already shown that metallophilic macrophages, which represent an important component in the thymus physiology, are lacking in lymphotoxin-β receptor-deficient mice. However, further molecular requirements for the development and correct tissue positioning of these cells are unknown. To this end, we studied a panel of mice deficient in different chemokine ligand or receptor genes. In contrast to normal mice, which have these cells localized in the thymic cortico-medullary zone (CMZ) as a distinct row positioned between the cortex and medulla, in plt/plt (paucity of lymph node T cells) mice lacking the functional CCL19/CCL21 chemokines, metallophilic macrophages are not present in the thymic tissue. Interestingly, in contrast to the CCL19/21-deficient thymus, metallophilic macrophages are present in the CCR7-deficient thymus. However, these cells are not appropriately located in the CMZ, but are mostly crowded in central parts of thymic medulla. The double staining revealed that these metallophilic macrophages are CCR7-negative and CXCR3-positive. In the CXCL13-deficient thymus the number, morphology and localization of metallophilic macrophages are normal. Thus, our study shows that CCL19/21 and its possible signaling through CXCR3 are required for the development of thymic metallophilic macrophages, whereas the CXCL13-CXCR5 signaling is not necessary.

Immune recovery after profound lymphopenia is a major challenge in many clinical situations, such as allogeneic hematopoietic stem cell transplantation (allo-HSCT). Recovery depends, in a first step, on hematopoietic lymphoid progenitors production in the bone marrow (BM). In this study, we characterized CD34+Lin-CD10+ lymphoid progenitors in the peripheral blood of allo-HSCT patients. Our data demonstrate a strong recovery of this population 3 months after transplantation. This rebound was abolished in patients who developed acute graft-versus-host disease (aGVHD). A similar recovery profile was found for both CD24+ and CD24- progenitor subpopulations. CD34+lin-CD10+CD24- lymphoid progenitors sorted from allo-HSCT patients preserved their T cell potentiel according to in vitro T-cell differentiation assay and the expression profile of 22 genes involved in T-cell differentiation and homing. CD34+lin-CD10+CD24- cells from patients without aGVHD had reduced CXCR4 gene expression, consistent with an enhanced egress from the BM. CCR7 gene expression was reduced in patients after allo-HSCT, as were its ligands CCL21 and CCL19. This reduction was particularly marked in patients with aGVHD, suggesting a possible impact on thymic homing. Thus, the data presented here identify this population as an important early step in T cell reconstitution in humans and so, an important target when seeking to enhance immune reconstitution.

Sublingual immunotherapy (SLIT) has been considered to be a painless and efficacious therapeutic treatment of allergic rhinitis which is known as type I allergy of nasal mucosa. Nevertheless, its mechanisms need to be further investigated. In this study, we constructed an effective murine model of sublingual immunotherapy in allergic rhinitis, in which mice were sublingually administered with ovalbumin (OVA) followed by intraperitoneal sensitization and nasal challenge of OVA. Sublingually treated mice showed significantly decreased specific IgE responses as well as suppressed Th2 immune responses. Sublingual administration of OVA did not alter the frequency of CD4(+)CD25(+) regulatory T cells (Tregs), but led to upregulation of Foxp3- and IL-10-specific mRNAs in the Tregs of cervical lymph nodes (CLN), which strongly suppressed Th2 cytokine production from CD4(+)CD25(-) effector T cells in vitro. Furthermore, sublingual administration of plasmids encoding the lymphoid chemokines CCL19 and CCL21-Ser DNA together with OVA suppressed allergic responses. These results suggest that IL-10-expressing CD4(+)CD25(+)Foxp3(+) Tregs in CLN are involved in the suppression of allergic responses and that CCL19/CCL21 may contribute to it in mice that received SLIT.

ACKR4 also called CCX-CKR, CCRL1 as a member of atypical chemokine receptors, regulates the biological responses by clearance or transporting homeostatic chemokines such as CCL19, CCL21, CCL25, and CXCL13. Since these chemokines are involved in cancer development and metastasis, ACKR4 could have inhibition roles in cancer cell proliferation and invasion. Forming complexes with chemokine receptors by ACKR4 as in the case of hCXCR3 which lead to chemotaxis prevention is the other function of this protein is. However, as an atypical chemokine receptor, ACKR4 is less well-characterized compared to other members. Here, as the first step in understanding the molecular mechanisms of ACKR4 action, transfectants in HEK293T cell, was generated. In this study, ACKR4 coding sequence was cloned and human embryonic kidney 293T cells were used for recombinant production of ACKR4 protein. The liposome-mediated transfection with ACKR4 CDs, were detected in ACKR4 positive cells as early as 48 h post-transfection. The production of ACKR4 protein was confirmed using RT-PCR, dot blot, western blot, and flow cytometry. ACKR4 may represent a novel molecular target in cancer therapy, which might provide a chance for new therapeutic strategy. Therefore, the first step in the understanding of the molecular mechanisms of ACKR4 action is generation ACKR4-HEK293T recombinant cells.

Chemokines are secreted proteins that direct the migration of immune cells and are involved in numerous disease states. For example, CCL21 (CC chemokine ligand 21) and CCL19 (CC chemokine ligand 19) recruit antigen-presenting dendritic cells and naïve T-cells to the lymph nodes and are thought to play a role in lymph node metastasis of CCR7 (CC chemokine receptor 7)-expressing cancer cells. For many chemokine receptors, N-terminal posttranslational modifications, particularly the sulfation of tyrosine residues, increases the affinity for chemokine ligands and may contribute to receptor ligand bias. Chemokine sulfotyrosine (sY) binding sites are also potential targets for drug development. In light of the structural similarity between sulfotyrosine and phosphotyrosine (pY), the interactions of CCL21 with peptide fragments of CCR7 containing tyrosine, pY, or sY were compared using protein NMR (nuclear magnetic resonance) spectroscopy in this study. Various N-terminal CCR7 peptides maintain binding site specificity with Y8-, pY8-, or sY8-containing peptides binding near the α-helix, while Y17-, pY17-, and sY17-containing peptides bind near the N-loop and β3-stand of CCL21. All modified CCR7 peptides showed enhanced binding affinity to CCL21, with sY having the largest effect.

The cys-cys (C-C) chemokine ligand 21 is a member of the C-C chemokines that constitute a group of heparin-binding cytokines with a pattern of four or six conserved cysteines. The CCL21 is known to be expressed in secondary lymphoid tissues, however it has rarely been reported for the expression on peripheral lymphatic vessels in somatic tissue. Here we investigated the expression of CCL21 on lymphatic vessels identified by anti-desmoplakin in uninflamed and inflamed human gingiva. In uninflamed tissue the expression of CCL21 was detected on lymphatic vessels in gingiva. In uninflamed gingiva the expression of CCL21 was detected on all lymphatic capillaries of the mucosal connective tissue papillae. There were two types of collecting lymphatic vessels in the lamina propria mucosae expressing CCL21 strongly or very weakly. In inflamed gingiva no expression of CCL21 was detected on lymphatic vessels. In all tissue sections no blood vessels expressing CCL21 were observed. These results may suggest that the expression of CCL21 is predominantly induced in the peripheral lymphatic endothelium of the uninflamed mucosal microcirculation, and that under inflamed conditions a reduction of CCL21 occurs in lymphatic endothelium.

During normal pregnancy, the thymus undergoes a severe reduction in size and thymocyte output, which may contribute to maternal-fetal tolerance. It is presently unknown whether the pregnancy-induced thymic involution also affects nonlymphoid thymic cell populations and whether these changes in stromal cells play a role in the reduction in thymocyte numbers. Here, we characterize the changes in thymic lymphoid and nonlymphoid cells and show that pregnancy results in a reduction of all major thymic lymphoid cell populations, including the early T-lymphoid progenitors (TLPs) and thymic regulatory T cells. In addition to the thymocytes, the thymic involution also includes all major nonlymphoid cell populations, which show a profound reduction in cell numbers. We also show that during pregnancy, the thymic nonlymphoid cells exhibit decreased expression of chemokines that are essential for TLP homing: CCL25, CXCL12, CCL21, and CCL19. In addition, the expression of these chemokines was substantially downregulated by short-term treatment with progesterone but not estrogen. Collectively, these findings suggest a novel mechanism for the pregnancy-induced reduction in TLP homing and the resulting thymic involution.

BACKGROUND

Dendritic cell (DC)-based vaccination has been investigated as immunotherapy for several types of cancer. A potential drawback to vaccination with autologous monocyte-derived DCs (MoDCs) could be that MoDCs from patients are functionally impaired. In case of androgen-independent prostate cancer (CaP), the cancer itself, diverse prior therapies, and the hormone manipulation may affect the immune system.

METHODS

MoDCs from patients suffering from androgen-independent CaP were generated according to a clinically applicable protocol to evaluate the phenotype, maturation capacity, migration, and T-cell stimulation of these cells compared with those generated from tumor-free donors.

RESULTS

MoDCs generated from CaP patients could be fully matured and efficiently migrated towards the chemokine CCL21. They had a strong potency to activate allogeneic CD4+ and CD8+ T-cells and to present antigens to specific CTL.

CONCLUSIONS

Our data suggest that MoDCs from patients with androgen-independent CaP are functionally intact and hence qualify as cellular vaccines for immunotherapy of advanced stage CaP.

To determine the extent to which splenic T cells were affected by Schistosoma mansoni infection, we investigated the ability of the T cells to produce interferon (IFN)-gamma, as well as their chemotactic ability 7 wk PI. In this study, we report that splenic T cells from Balb/c mice with S. mansoni infections were capable of producing levels of IFN-gamma comparable with splenic T cells from naive mice. However, the T cells exhibited altered chemotactic activity, as evidenced by an inability to respond to secondary lymphoid-tissue chemokine (SLC/CCL21). Although no difference in chemokine expression was found between the spleens of infected versus control mice, chemokine production was greater in the livers of infected versus control mice. Collectively, these data indicate that Balb/c mice with 7-wk S. mansoni infection possess splenic T cells with altered chemotactic activity and that the alterations may be a consequence of the granulomatous response in the liver.

BACKGROUND

Triptolide is an immunosuppressive fraction purified from a Chinese medicinal plant. In an effort to develop a new immunosuppressive strategy for vascularized composite allotransplantation, the authors investigated the effects of combined treatment with cyclosporin A and triptolide on the survival of rat groin flap allotransplants.

METHODS

Groin flap transplantation was performed from Brown Norway rats to Fischer 344 recipients, which were then treated with triptolide, cyclosporin A, or both. Flap biopsy specimens were harvested, stained, and submitted to histopathologic evaluation. Levels of CCR5, CCR7, CCL19, CCL21, and Foxp3 in spleen were examined by real-time polymerase chain reaction, and the percentage of CD4+CD25+ regulatory T cells was detected by flow cytometry.

RESULTS

The mean survival time for allografts in recipients receiving triptolide and cyclosporin A was 57 ± 7.7 days compared with 20.5 ± 2.3 days for cyclosporin A alone, 23.3 ± 3.6 days for triptolide alone, and 7.8 ± 0.8 days for no treatment. Histologic examination also showed that inflammatory cell infiltration was reduced in grafts with combination treatment. Down-regulation of CCR5, CCR7, and CCL19 in the combination treatment was accompanied by increased expression of Foxp3. Flow cytometric analysis also revealed that the percentage of CD4+CD25+ regulatory T cells in the combination treatment was higher than in the monotherapy groups.

CONCLUSIONS

Combination therapy with triptolide and cyclosporin A substantially prolonged allograft survival, which means calcineurin inhibitor-related drug-toxicity may be alleviated and treatment cost reduced. This immunosuppressive effect is mediated by inhibition of dendritic cells maturation and the expansion of regulatory T cells.

Cytokine LIGHT is a type II transmembrane protein belonging to the TNF family that was originally identified as a weak inducer of apoptosis. It plays a role in inducing maturation of dendritic cells, such as upregulating CD80, CD86 expression on dendritic cells. However, whether LIGHT induces CC chemokine expression in DC and promotes their migration remains unknown. In this study, we found that esDC express CCR7 and CCR10 (the receptor of CCL27) upon the LIGHT stimulation. LIGHT also upregulates CCL27, but not CCL19 and CCL21 expression in esDC. The esDC migration potential has been increased in LIGHT activated DCs compared with control cells. LIGHT activated DCs autocrine CCL27 which regulate their migration as Blockage of CCL27 on esDC using neutralizing antibody reduces migration potential. In signaling study, we identified that LIGHT activated NF-kappaB in esDC and inhibition of NF-kappaB activation by specific inhibitor can partly attenuate the effect of LIGHT in regulation of CCL27 expression. Moreover, Shp-2 is required in LIGHT activated NF-kappaB because Knockdown of Shp-2 affects the NF-kappaB activation induced by LIGHT and consequently influences LIGHT mediated CCL27 expression. TRAF6 is critical in DC maturation in recent reports; however, knockdown of TRAF6 expression using siRNA did not alter CCL27 expression in LIGHT matured DCs. Our study demonstrates that LIGHT stimulation enhances CCL27 expression through activation of NF-kappaB in DCs.

Treatment with high iodine concentrations can delay oncogenic activation effects, reduce cell growth and return thyroid-specific gene and protein expression levels to normal. During rearranged during transfection (RET)/papillary thyroid carcinoma (PTC) 3 activation, excess iodine can act as a protective agent in thyroid follicular cells. The chemokine receptor CCR7 serves a critical role in lymphocyte trafficking into and within lymph nodes, the preferential metastatic site for PTC. However, the potential associations between chemokine (C‑C motif) ligand 21 (CCL21)/C‑C chemokine receptor type 7 (CCR7) interaction and iodine concentrations in primary cultures of PTC with RET/PTC expression remain unclear. Proliferation assays of primary cultures of PTC cells with RET/PTC1 and RET/PTC3 expression indicated that CCR7 activation by its specific ligand, CCL21, was associated with significantly increased cell proliferation. Flow cytometry data indicated that CCL21/CCR7 interaction significantly increased the fraction of cells in the G2/M phase of the cell cycle. Western blotting indicated that CCL21/CCR7 interaction significantly upregulated cyclin A, cyclin B1 and cyclin‑dependent kinase 1 (CDK1) expression. Western blotting determined that CCL21/CCR7 interaction significantly enhanced the levels of phosphorylated extracellular signal‑regulated kinase (P‑ERK). Co-immunoprecipitation confirmed that there was interaction between P‑ERK and cyclin A, cyclin B1 or CDK1, particularly in the presence of CCL21. Sodium iodide (NaI, 10-5 M) significantly abolished the effects of exogenous CCL21. These results suggest that CCL21/CCR7 interaction contributes to G2/M progression of RET/PTC‑expressing cells via the ERK pathway in association with 10‑5 M NaI.

In multicellular organisms, cell migration is a complex process. Examples of this are observed during cell motility in the interstitial space, full of extracellular matrix fibers, or when cells pass through endothelial layers to colonize or exit specific tissues. A common parameter for both situations is the fast adaptation of the cellular shape to their irregular landscape. In this chapter, we describe two methods to study cell migration in complex environments. The first one consists in a multichamber device for the visualization of cell haptotaxis toward the collagen-binding chemokine CCL21. This method is used to study cell migration as well as deformations during directed motility, as in the interstitial space. The second one consists in microfabricated channels connected to small constrictions. This procedure allows the study of cell deformations when single cells migrate through small holes and it is analogous to passage of cells through endothelial layers, resulting in a simplified system to study the mechanisms operating during transvasation. Both methods combined provide a powerful hub for the study of cell plasticity during migration in complex environments.