Impaired lymphoid chemokine-mediated migration due to a block on the chemokine receptor switch in human cytomegalovirus-infected dendritic cells

Dendritic cell (DC) migration from the site of infection to the site of T-cell priming is a crucial event in the generation of antiviral T-cell responses. Here we present to our knowledge the first functional evidence that human cytomegalovirus (HCMV) blocks the migration of infected monocyte-derived DCs toward lymphoid chemokines CCL19 and CCL21. DC migration is blocked by viral impairment of the chemokine receptor switch at the level of the expression of CCR7 molecules. The inhibition occurs with immediate-early-early kinetics, and viral interference with NF-kappaB signaling is likely to be at least partially responsible for the lack of CCR7 expression. DCs which migrate from the infected cultures are HCMV antigen negative, and consequently they do not stimulate HCMV-specific CD8(+) T cells, while CD4(+)-T-cell activation is not impaired. Although CD8(+) T cells can also be activated by alternative antigen presentation mechanisms, the spatial segregation of naive T cells and infected DCs seems a potent mechanism of delaying the generation of primary CD8(+)-T-cell responses and aiding early viral spread.     

Virus stimulation of human mast cells results in the recruitment of CD56? T cells by a mechanism dependent on CCR5 ligands

The trafficking of effector cells to sites of infection is crucial for antiviral responses. However, the mechanisms of recruitment of the interferon-γ-producing and cytotoxic CD56(+) T cells are poorly understood. Human mast cells are sentinel cells found in the skin and airway and produce selected proinflammatory mediators in response to multiple pathogen-associated signals. The role of human mast cell-derived chemokines in T-cell recruitment to virus infection was examined. Supernatants from primary human cord blood-derived mast cells (CBMCs) infected with mammalian reovirus were examined for chemokine production and utilized in chemotaxis assays. Virus-infected CBMCs produced several chemokines, including CCL3, CCL4, and CCL5. Supernatants from reovirus-infected CBMCs selectively induced the chemotaxis of CD8(+) T cells (10±1%) and CD3(+)CD56(+) T cells (19±5%). CD56(+) T-cell migration was inhibited by pertussis toxin (65±9%) and met-RANTES (56±7%), a CCR1/CCR5 antagonist. CD56(+) T cells expressed CCR5, but little CCR1. The depletion of CCL3, CCL4, and CCL5 from reovirus-infected CBMC supernatants significantly (41±10%) inhibited CD56(+) T-cell chemotaxis. This study demonstrates a novel role for mast cells and CCR5 in CD56(+) T-cell trafficking and suggests that human mast cells enhance immunity to viruses through the selective recruitment of cytotoxic effector cells to virus infection sites. These findings could be exploited to enhance local T-cell responses in chronic viral infection and malignancies at mast cell-rich sites.     

Phenotypic and functional characterisation of CCR7+ and CCR7- CD4+ memory T cells homing to the joints in juvenile idiopathic arthritis

The aim of the study was to characterise CCR7+ and CCR7- memory T cells infiltrating the inflamed joints of patients with juvenile idiopathic arthritis (JIA) and to investigate the functional and anatomical heterogeneity of these cell subsets in relation to the expression of the inflammatory chemokine receptors CXCR3 and CCR5. Memory T cells freshly isolated from the peripheral blood and synovial fluid (SF) of 25 patients with JIA were tested for the expression of CCR7, CCR5, CXCR3 and interferon-gamma by flow cytometry. The chemotactic activity of CD4 SF memory T cells from eight patients with JIA to inflammatory (CXCL11 and CCL3) and homeostatic (CCL19, CCL21) chemokines was also evaluated. Paired serum and SF samples from 28 patients with JIA were tested for CCL21 concentrations. CCR7, CXCR3, CCR5 and CCL21 expression in synovial tissue from six patients with JIA was investigated by immunohistochemistry. Enrichment of CD4+, CCR7- memory T cells was demonstrated in SF in comparison with paired blood from patients with JIA. SF CD4+CCR7- memory T cells were enriched for CCR5+ and interferon-gamma+ cells, whereas CD4+CCR7+ memory T cells showed higher coexpression of CXCR3. Expression of CCL21 was detected in both SF and synovial membranes. SF CD4+ memory T cells displayed significant migration to both inflammatory and homeostatic chemokines. CCR7+ T cells were detected in the synovial tissue in either diffuse perivascular lymphocytic infiltrates or organised lymphoid aggregates. In synovial tissue, a large fraction of CCR7+ cells co-localised with CXCR3, especially inside lymphoid aggregates, whereas CCR5+ cells were enriched in the sublining of the superficial subintima. In conclusion, CCR7 may have a role in the synovial recruitment of memory T cells in JIA, irrespective of the pattern of lymphoid organisation. Moreover, discrete patterns of chemokine receptor expression are detected in the synovial tissue.     

In vitro infection of human dendritic cells by Aspergillus fumigatus conidia triggers the secretion of chemokines for neutrophil and Th1 lymphocyte recruitment

Given the role played by chemokines in the selective homing of immune cells, we sought to characterize the profile of chemokines produced by human dendritic cells (DC) following in vitro Aspergillus fumigatus infection and their ability to recruit cells involved in the antifungal defense. At the onset of A. fumigatus infection, DC released elevated amounts of CXCL8 that promote the migration of polymorphonuclear cells (PMN). Moreover, soluble factors released from A. fumigatus-infected DC increased also the surface expression of two activation markers, CD11b and CD18, on PMN. A. fumigatus infection resulted also in CCL3, CCL4, CXCL10 and CCL20 productions that induce the migration of effector memory Th1 cells. Moreover, the late expression of CCL19 suggests that A. fumigatus-infected DC could be implicated in the migration of CCR7⁺ naïve T cells and mature DC in lymph nodes. Together these results suggested the involvement of human DC in the regulation of innate and adaptive immunity against A. fumigatus through the recruitment of cells active in the fungal destruction.     

Dendritic cells express CCR7 and migrate in response to CCL19 (MIP-3beta) after exposure to Helicobacter pylori

Helicobacter pylori infection induces chronic inflammation in the gastric mucosa with a marked increase in the number of lymphoid follicles consisting of infiltrating B and T cells, neutrophils, dendritic cells (DC) and macrophages. It has been suggested that an accumulation of mature DC in the tissue, resulting from a failure of DC to migrate to lymph nodes, may contribute to this chronic inflammation. Migration of DC to lymph nodes is regulated by chemokine receptor CCR7, expressed on mature DC, and the CCR7 ligands CCL19 and CCL21. In this study we analysed the maturation, in vitro migration and cytokine production of human DC after stimulation with live H. pylori. For comparison, DC responses to non-pathogenic Escherichia coli bacteria were also evaluated. Stimulation with H. pylori induced maturation of DC, i.e. up-regulation of the chemokine receptors CCR7 and CXCR4 and the maturation markers HLA-DR, CD80 and CD86. The H. pylori-stimulated DC also induced CD4(+) T-cell proliferation. DC stimulated with H. pylori secreted significantly more interleukin (IL)-12 compared to DC stimulated with E. coli, while E. coli-stimulated DC secreted more IL-10. Despite low surface expression of CCR7 protein following stimulation with H. pylori compared to E. coli, the DC migrated equally well towards CCL19 after stimulation with both bacteria. Thus, we could not detect any failure in the migration of H. pylori stimulated DC in vitro that may contribute to chronic gastritis in vivo, and our results suggest that H. pylori induces maturation and migration of DC to lymph nodes where they promote T cell responses.     

Regulation of trafficking receptor expression in human forkhead box P3+ regulatory T cells

Forkhead Box P3(+) (FOXP3(+)) T cells are regulatory cells important for maintaining immune tolerance. While chemokine- and other homing-receptors are important for T cell migration, it has been unclear how they are regulated in FOXP3(+) T cells. We thoroughly investigated, ex vivo and in vitro, the regulation of chemokine receptor expression on human FOXP3(+) T cells in neonatal cord blood, adult peripheral blood, and tonsils. We found that human FOXP3(+) T cells undergo changes in trafficking receptors according to their stages of activation and differentiation. FOXP3(+) T cells are divided into CD45RA(+) (naive type) and CD45RO(+) (memory type) FOXP3(+) T cells in neonatal blood, adult blood, and tonsils. CD45RA(+)FOXP3(+) T cells mainly express lymphoid tissue homing receptors (CD62L, CCR7, and CXCR4), while CD45RO(+)FOXP3(+) T cells highly express both Th1 and Th2-associated trafficking receptors along with the lymphoid tissue homing receptors at reduced frequencies. Up-regulation of Th1/Th2-associated trafficking receptors begins with activation of CD45RA(+)FOXP3(+) T cells and is completed after their differentiation to CD45RO(+) cells. Some chemokine receptors such as CXCR5 and CXCR6 are preferentially expressed by many FOXP3(+) cells at a specific stage (CD69(+)CD45RO(+)) in tonsils. Our in vitro differentiation study demonstrated that CD45RA(+)FOXP3(+) T cells indeed undergo chemokine receptor switch from CD45RA(+) (secondary lymphoid tissue homing) to CD45RO(+) type (lymphoid and nonlymphoid tissue homing). The orderly regulation of trafficking receptors in FOXP3(+) T cells according to stages of differentiation and activation is potentially important for their tissue-specific migration and regulation of immune responses in humans.     

CCR7 directs the migration of thymocytes into the thymic medulla

Developing thymocytes migrate from the cortex to the medulla of the thymus as a consequence of positive selection. This migration is likely to be essential for tolerance because it allows the developing cells to move into an environment that is optimal for negative selection. Guidance mechanisms that draw positively selected thymocytes into the medulla have not been clarified, but several studies have implicated chemokines in the process. CCR7, the receptor for the medullary chemokines CCL19 and CCL21, is induced on thymocytes during their positive selection. In this study we show that premature expression of CCR7 repositions CD4(+)CD8(+) double-positive cells into the medulla of transgenic mice. This repositioning of the thymocytes is accompanied by impairment of their development. The data show the involvement of CCR7 in medullary migration and emphasize the importance of proper thymocyte positioning for efficient T cell development.     

Cutting edge: nonproliferating mature immune cells form a novel type of organized lymphoid structure in idiopathic pulmonary fibrosis

Ectopic formation of secondary lymphoid tissue is initiated by the local attraction of naive T and B cells. In this study, we describe a novel type of organized lymphoid structure in the lung of human idiopathic pulmonary fibrosis, with key features of lymphoid neogenesis, including: 1) recently activated CD40 ligand (CD40L)+ T cells; 2) variable numbers of activated CD40+/CD40L+ B cells, sometimes organized in follicles; 3) fully mature dendritic cells (DC) expressing CD40, CD83, CD86, and DC-lysosome-associated membrane protein; 4) the expression of the chemokine CCL21; 5) the presence of vessels with characteristics of high endothelial venules; and 6) a dense network of follicular DC. Surprisingly, these structures are devoid of CCR7+ naive T cells, proliferating lymphocytes, and germinal centers, suggesting that newly recruited activated DC and Ag-experienced lymphocytes can drive lymphoid neogenesis and that factors present within the lymphoid aggregates, such as CD40L, are essential to induce DC maturation.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

A chemokine-dependent stromal induction mechanism for aberrant lymphocyte accumulation and compromised lymphatic return in rheumatoid arthritis

According to the current model for tissue-specific homing, specificity is conferred by the selective recruitment of lymphocyte populations from peripheral blood, based on their expression of chemokine and adhesion receptors (endothelial selection). In this study, we provide evidence for an alternative stromal induction mechanism that operates in chronic inflammation. We show that the human rheumatoid synovial microenvironment directly induces functional inflammatory (CCR5 and CXCR3) and constitutive (CCR7 and CXCR4) chemokine receptors on infiltrating CD4(+) T cells. Expression of the corresponding inflammatory chemokine ligands (CCL5 and CXCL11) was confined to stromal areas in the synovium. However, expression of the constitutive ligands (CCL19 and CXCL12) was inappropriately high on both vascular and lymphatic endothelium, suggesting that the vascular to lymphatic chemokine gradient involved in lymphatic recirculation becomes subverted in the rheumatoid synovium. These results challenge the view that leukocyte trafficking is regulated solely by selective recruitment of pre-existing chemokine receptor-positive cells from peripheral blood, by providing an alternative explanation based on aberrant lymphocyte retention and compromised lymphatic return.     

IFN-alpha beta released by Mycobacterium tuberculosis-infected human dendritic cells induces the expression of CXCL10: selective recruitment of NK and activated T cells

We recently reported that dendritic cells (DC) infected with Mycobacterium tuberculosis (Mtb) produce Th1/IFN-gamma-inducing cytokines, IFN-alpha beta and IL-12. In the present article, we show that maturing Mtb-infected DC express high levels of CCR7 and they become responsive to its ligand CCL21. Conversely, CCR5 expression was rapidly lost from the cell surface following Mtb infection. High levels of CCL3 and CCL4 were produced within 8 h after infection, which is likely to account for the observed CCR5 down-modulation on Mtb-infected DC. In addition, Mtb infection stimulated the secretion of CXCL9 and CXCL10. Interestingly, the synthesis of CXCL10 was mainly dependent on the Mtb-induced production of IFN-alpha beta. Indeed, IFN-alpha beta neutralization down-regulated CXCL10 expression, whereas the expression of CXCL9 appeared to be unaffected. The chemotactic activity of the Mtb-infected DC supernatants was evaluated by migration assays using activated NK, CD4(+), and CD8(+) cells that expressed both CCR5 and CXCR3. Mtb-induced expression of CCL3, CCL4, CXCL9, and CXCL10 was involved in the stimulation of NK and T cell migration. In accordance with the data on the IFN-alpha beta-induced expression of CXCL10, neutralization of IFN-alpha beta significantly reduced the chemotactic activity of the supernatant from Mtb-infected DC. This indicates that IFN-alpha beta may modulate the immune response through the expression of CXCL10, which along with CXCL9, CCL3, and CCL4 participates in the recruitment and selective homing of activated/effector cells, which are known to accumulate at the site of Mtb infection and take part in the formation of the granulomas.     

CCR7 expression in CD19 chimeric antigen receptor-engineered natural killer cells improves migration toward CCL19-expressing lymphoma cells and increases tumor control in mice with human lymphoma

Background aimsChimeric antigen receptor (CAR) T-cell therapy can be associated with significant toxicities. CAR-engineered natural killer (NK) cells provide a safer alternative while maintaining anti-tumor effects. Activated NK (aNK) cells are a clinical-grade cellular product obtained from the NK-92 cell line that have demonstrated both safety and potent cytotoxicity toward a wide range of cancers in phase 1 trials. Genetically engineered variants of aNK cells expressing a high-affinity Fc receptor (haNK) or co-expressing a CAR (t-haNK) are currently in phase 1/2 clinical trials. A key factor in the efficacy of cellular immunotherapies is biodistribution and tumor infiltration, which affect the local effector:target ratio. The chemokines CCL19 and CCL21 can drive recruitment of CCR7 receptor-expressing immune cells to secondary lymphoid organs.MethodsSince NK-92 cells do not spontaneously express CCR7, clinical-grade aNK cells were transfected with a non-viral vector containing the CCR7 receptor, an anti-CD19 CAR and a high-affinity CD16 Fc receptor.ResultsCCR7-engineered CD19 t-haNK showed significant migration in vitro toward K562 cells engineered to secrete CCL19. This observation was confirmed in a NOD.Cg-Prkdc^scid Il2rg^tm1Wjl/SzJ (NSG) mouse model in which subcutaneous tumors of CCL19-expressing K562 cells displayed a higher number of infiltrating CCR7_CD19 t-haNK cells than CCR7-negative CD19 t-haNK cells. In NSG mice inoculated either intravenously or subcutaneously with CCL19-secreting Raji cells, treatment with CCR7_CD19 t-haNK improved survival and tumor control compared with CD19 t-haNK or vehicle.ConclusionsExpression of CCR7 receptor by off-the-shelf t-haNK cells improves their homing toward lymph node chemokines both in vitro and in vivo, resulting in superior tumor control.     

Toxoplasma gondii: comparison of human CD34+ and monocyte-derived dendritic cells after parasite infection

Human dendritic cells (DC) obtained in vitro from CD34(+) progenitors (CD34-DC) or blood monocytes (mo-DC) are different DC which may be used in a model of T. gondii infection. We compared the survival, infection rate and cell surface receptor expression of both DC types after living T. gondii tachyzoite infection. CD34-DC appeared less resistant to the parasite than mo-DC. At 48h post-infection, chemokine receptors responsible for DC homing and migration were absent in mo-DC, while down regulation of CCR6 and up regulation of CCR7 was observed in CD34-DC. This result, suggesting migration ability of CD34-DC, was confirmed by in vitro migration experiments against different chemokines. Tachyzoite supernatant, used as chemokine, attracted immature CD34-DC as observed by MIP3alpha, while MIP3beta, as expected, attracted mature CD34-DC. Under similar conditions, no significant difference was noticed between mature or immature mo-DC. These data indicated that CD34-DC represent an alternative model that allows migration assay of infected DC by T. gondii.     

Opposite fate of endocytosed CCR7 and its ligands: recycling versus degradation

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 play a crucial role for the homing of lymphocytes and dendritic cells to secondary lymphoid tissues. Nevertheless, how CCR7 senses the gradient of chemokines and how migration is terminated are poorly understood. In this study, we demonstrate that CCR7(-GFP) is endocytosed into early endosomes containing transferrin receptor upon CCL19 binding, but less upon CCL21 triggering. Internalization of CCR7 was independent of lipid rafts but relied on dynamin and Eps15 and was inhibited by hypertonic sucrose, suggesting clathrin-dependent endocytosis. After chemokine removal, internalized CCR7 recycled back to the plasma membrane and was able to mediate migration again. In contrast, internalized CCL19 was sorted to lysosomes for degradation, showing opposite fate for endocytosed CCR7 and its ligand.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

Semimature stage: a checkpoint in a dendritic cell maturation program that allows for functional reversion after signal-regulatory protein-alpha ligation and maturation signals

CD47 on live cells actively engages signal-regulatory protein-alpha (SIRP-alpha) on phagocytes and delivers a negative signal that prevents their elimination. We evaluated the biological consequences of SIRP-alpha ligation on the dendritic cell (DC) response to maturation signals and the potential interplay with the IL-10/IL-10R inhibitory pathway. At first, CD47/SIRP-alpha allowed the generation of mature migratory DCs not producing IL-12, IFN-gamma-inducible protein-10, and CCL19. Rather, they secreted neutrophils attracting chemokine CXCL5 and IL-1beta, reflecting a partial block in functional DC maturation. Afterward, semimature DCs functionally regressed in an IL-10-independent fashion toward cells that retrieved the cardinal features of immature DCs: re-expression of CCR5, loss of DC-lysosome-associated membrane protein, high endocytosis, and impaired allostimulatory functions. The global gene expression profile of IL-10 and SIRP-alpha-ligated DC demonstrated two distinct molecular pathways. IL-10R and SIRP-alpha expression were reciprocally down-regulated by CD47 and IL-10, respectively. These results emphasize that the SIRP-alpha pathway might be part of the molecular machinery used by the DC to dampen or resolve an inflammatory response in an IL-10-independent manner.     

CCR7 ligand-enhanced phagocytosis of various antigens in mature dendritic cells-time course and antigen distribution different from phagocytosis in immature dendritic cells

CC chemokine receptor 7 (CCR7) is selectively expressed on mature dendritic cells (DC). The CCR7 ligands, CC chemokine ligand (CCL) 19 and CCL21, facilitate migration of mature DC from the peripheral tissues to regional lymph nodes. We previously demonstrated that CCR7 ligands induced rapid receptor-mediated endocytosis of dextran in mature DC. In the present study, we further examined the effects of CCR7 ligands on endocytosis of other kinds of antigen, mannosilated bovine serum albumin (Mann-BSA), Escherichia coli(E. coli), or ovalbumin-containing immune complex (OVA-IC), by mature DC. We found that CCR7 ligands enhanced the endocytosis of Mann BSA, E. coli, and OVA-IC in mature DC but not in immature DC. The endocytosis of BSA was not enhanced by CCR7 ligands. Furthermore, the phagocytosis of OVA-IC was significantly inhibited by anti-Fcgamma receptor III/II antibody. These results demonstrate that CCR7 ligands enhance only receptor-mediated endocytosis by mature DC. When rapidly phagocytosed E. coli were traced in CCL19-treated mature DC, most of the phagocytosed E. coli did not colocalize with the lysosomal marker: lysosome-associated membrane protein-1 (Lamp-1), whereas most of E. coli taken up relatively slowly by immature DC colocalized with Lamp-1. These results suggest that phagocytosis of antigens by immature and mature DC plays different functional roles.     

CXC chemokine ligand 13 and CC chemokine ligand 19 cooperatively render resistance to apoptosis in B cell lineage acute and chronic lymphocytic leukemia CD23+CD5+ B cells

CXCL13/CXCR5 and CCL19/CCR7 play a quite important role in normal physiological conditions, but the functions of both chemokine/receptor pairs in pathophysiological events are not well-investigated. We have investigated expression and functions of CXCL13/CXCR5 and CCL19/CCR7 in CD23+CD5+ and CD23+CD5- B cells from cord blood (CB) and patients with B cell lineage acute or chronic lymphocytic leukemia (B-ALL or B-CLL). CXCR5 and CCR7 are selectively expressed on B-ALL, B-CLL, and CB CD23+CD5+ B cells at high frequency, but not on CD23+CD5- B cells. Although no significant chemotactic responsiveness was observed, CXCL13 and CCL19 cooperatively induce significant resistance to TNF-alpha-mediated apoptosis in B-ALL and B-CLL CD23+CD5+ B cells, but not in the cells from CB. B-ALL and B-CLL CD23+CD5+ B cells express elevated levels of paternally expressed gene 10 (PEG10). CXCL13 and CCL19 together significantly up-regulate PEG10 expression in the same cells. We have found that CXCL13 and CCL19 together by means of activation of CXCR5 and CCR7 up-regulate PEG10 expression and function, subsequently stabilize caspase-3 and caspase-8 in B-ALL and B-CLL CD23+CD5+ B cells, and further rescue the cells from TNF-alpha-mediated apoptosis. Therefore, we suggest that normal lymphocytes, especially naive B and T cells, use CXCL13/CXCR5 and CCL19/CCR7 for migration, homing, maturation, and cell homeostasis as well as secondary lymphoid tissues organogenesis. In addition, certain malignant cells take advantages of CXCL13/CXCR5 and CCL19/CCR7 for infiltration, resistance to apoptosis, and inappropriate proliferation.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

IL-27-producing CD14(+) cells infiltrate inflamed joints of rheumatoid arthritis and regulate inflammation and chemotactic migration

Interleukin (IL)-27, a heterodimeric cytokine, has been reported to be involved in the pathogenesis of autoimmune diseases through mediating differentiation of Th1 or Th17 cells and immune cell activity or survival. However, the origin and effects of IL-27 in joints of rheumatoid arthritis (RA) remain unclear. In this study, we investigated the distribution and anti-inflammatory roles of IL-27 in RA synovium. The IL-27 levels in plasma of RA patients, osteoarthritis (OA) patients, or healthy volunteers (n=15 per group) were equivalent and were at most 1 ng/ml, but the IL-27 level in synovial fluid of RA patients (n=15, mean 0.13 ng/ml; range 0.017-0.37 ng/ml) was significantly higher than that in synovial fluid of OA patients (n=15, mean 0.003 ng/ml; range 0-0.033 ng/ml) and potentially lower than in plasma. We analyzed the protein level of IL-27 produced by RA fibroblast-like synoviocytes (FLSs) or mononuclear cells (MNCs) from RA or OA synovial fluid or peripheral blood and showed that IL-27 in RA joints was derived from MNCs but not from FLSs. We also found by flow cytometry that IL-27-producing MNCs were CD14(+), and that these CD14(+)IL-27(+) cells were clearly detected in RA synovium but rarely in OA synovium by immunohistochemistry. Furthermore, we demonstrated that a relatively physiological concentration of IL-27 below 10 ng/ml suppressed the production of IL-6 and CCL20 from RA FLSs induced by proinflammatory cytokines through the IL-27/IL-27R axis. In the synovial fluid of RA, the IL-27 level interestingly had positive correlation with the IFN-γ level (r=0.56, p=0.03), but weak negative correlation with the IL-17A level (r=-0.30, p=0.27), implying that IL-27 in inflammatory joints of RA induces Th1 differentiation and suppresses the development or the migration of Th17 cells. These findings indicate that circulating IL-27-producing CD14(+) cells significantly infiltrate into inflamed regions such as RA synovium and have anti-inflammatory effects in several ways: both directly through the reduction of IL-6 production, and possibly through the induction of Th1 development and the suppression of Th17 development; and indirectly by regulation of recruitment of CCR6(+) cells, such as Th17 cells, through the suppression of CCL20 production. Our results suggest that such a serial negative feedback system could be applied to RA therapy.