To explore the biological significance of the lymphatics in the autoimmune process, the thymus from non-obese diabetic (NOD) mice was evaluated by histochemistry and western blot analysis. Thymic lymphatic endothelial cells showed suggestive expression patterns of the functional molecules lymphatic vascular endothelial hyaluronan receptor (LYVE)-1, CCL21, CD31 and podoplanin. With increasing age, the expression of CCL21 was reduced in the medullary epithelial cells and lymphatics. Of note, LYVE-1-expressing lymphatics, filled with a cluster of thymocytes, increased in number and size and extended from the corticomedullary boundary into the medulla as the insulitis progressed. The development of lymphatic compartments was occasionally accompanied by a regional disappearance between the cortex and medulla. The CD4- and CD8-positive T cells frequently penetrated through the slender lymphatic walls. The epithelial reticular cell layer lining the perivascular spaces was extensively stained with cytokeratin, but the expression of cytokeratin showed an age-dependent decrease. These findings indicate that the occurrence of LYVE-1-expressing lymphatic compartments and the alteration of CCL21 expression in the lymphatics may be involved in defective thymocyte differentiation and migration, and play a significant role in insulitic and diabetic processes.

Type I interferons (IFN) have an essential role in antiviral defense, and they are produced upon viral infection in a variety of cells. IFN-alpha/beta treatment of immature dendritic cells (DC) is known to induce their phenotypic and functional maturation, but it remains unclear whether stimulation by this cytokine family influences the functions and maturation of Langerhans cells (LC). We used highly enriched (>95%) LC directly isolated from BALB/c mouse skin and addressed this issue, comparing LC with splenic CD11c(+) DC. Type I IFN-treated LC exhibited impaired ability to produce IL-12 and inflammatory cytokines, IL-6 and TNF-alpha, whereas IL-10 production was not augmented. In splenic DC, the production of inflammatory cytokines was rather enhanced by type I IFN treatment. With regard to chemokines, in both LC and splenic DC, type I IFN upregulated the production of inflammatory chemokines, such as CXCL10, CXCL11, CCL3, CCL4, and CCL5. Strikingly, IFN-beta treatment reduced the expression of CD40, CD54, CD80, and CD86 on LC, whereas IFN-beta-treated splenic DC showed enhanced expression of these molecules. Furthermore, IFN-beta-treated LC had impaired costimulatory activity for anti-CD3-induced proliferation of T cells. Finally, treatment with IFN-alpha/beta reduced the migratory capacity of LC to CCL21. These results indicate that type I IFN inhibit maturation and activation of LC in a direct manner. Our observations may provide a novel explanation for the reported inability of LC to act as potent antigen-presenting cells in cutaneous and mucosal viral infection.

Suppressors of cytokine signaling (SOCS) proteins control many aspects of lymphocyte function through regulation of STAT pathways. SOCS1-deficient mice develop severe skin and eye diseases that result from massive infiltration of inflammatory cells into these tissues. In this study, we have used SOCS1-, STAT1-, or STAT6-deficient mice, as well as, T cells with stable overexpression or deletion of SOCS1, to examine whether SOCS1 is involved in regulating lymphocyte trafficking to peripheral tissues. We show that SOCS1-deficient mice have increased numbers of T cells with characteristics of effector memory cells and expression of CCR7, a protein that promotes retention of T cells in lymphoid tissues, is markedly reduced in these cells. The decrease in CCR7 expression correlates with hyperactivation of STAT6, suggesting that aberrant recruitment of T cells into SOCS1-deficient mouse skin or eye results from abrogation of negative feedback regulation of STAT6 activation and CCR7 expression. Consistent with in vivo regulation of CCR7 expression and lymphocyte migration by SOCS1, forced overexpression of SOCS1 in T cells up-regulates CCR7 expression and enhances chemotaxis toward CCL19 or CCL21. CCR6 and CXCR3 are also up-regulated on SOCS1-deficient T cells and in situ analysis of the cornea or retina further reveal that these cells may mediate the chronic skin and eye inflammation through recruitment of Th1 and Th17 cells into these tissues. Collectively, these results suggest that SOCS1 regulates steady-state levels of chemokine receptors through its inhibitory effects on STAT pathways and this may underscore its role in regulating recruitment and retention of effector cells into nonlymphoid tissues.

The CC chemokine receptor 7 (CCR7) and its two ligands, CCL21 and CCL19, play an important role in migration of immune cells to lymphoid tissue. To analyze the function of CCR7 in T cell immunity to infectious agents in vivo, transgenic (tg) mice expressing CCL21 in an ubiquitous fashion were generated. These mice contained high amounts of CCL21 in the serum ( approximately 0.3 microg/ml that resulted in CCR7 down-regulation and in a strongly impaired migration of T cells toward CCL21 in vitro. Lymph nodes in CCL21-tg mice were reduced in size but with intact microanatomy and normal distribution of T and B cells. CCL21-tg mice showed a significantly decreased CD8 T cell response to lymphocytic choriomeningitis virus after footpad infection, whereas the response after systemic infection was not altered. Likewise, the CD4 T cell response to footpad infection with Leishmania major was considerably lowered and CCL21-tg mice failed to clear parasites from infected skin. Taken together, these data demonstrate the importance of CCR7 in mediating T cell immunity to viral and parasitic pathogens after local infection.

In T lymphocytes, Ca(2+) release-activated Ca(2+) (CRAC) channels composed of Orai1 subunits trigger Ag-induced gene expression and cell proliferation through the NFAT pathway. We evaluated the requirement of CRAC channel function for lymphocyte homing using expression of a dominant-negative Orai1-E106A mutant to suppress Ca(2+) signaling. To investigate homing and motility of human lymphocytes in immunocompromised mouse hosts, we transferred human lymphocytes either acutely or after stable engraftment after a second transfer from the same blood donor. Human and mouse lymphocyte homing was assessed, and cells were tracked within lymph nodes (LNs) by two-photon microscopy. Our results demonstrate that human T and B lymphocytes home into and migrate within the LNs of immunocompromised NOD.SCID mice similar to murine lymphocytes. Human T and B cells colocalized in atrophied or reconstituted mouse LNs, where T cells migrated in a random walk at velocities of 9-13 μm/min and B cells at 6 μm/min. Expression of Orai1-E106A inhibited CRAC channel function in human and mouse T cells, and prevented homing from high endothelial venules into murine LNs. Ca(2+) signals induced by CCL21 were also inhibited in T cells expressing Orai1-E106A. With CRAC channels inhibited, the high-affinity form of LFA-1 failed to become active, and T cells failed to migrate across endothelial cells in a transwell model. These results establish a requirement for CRAC channel-mediated Ca(2+) influx for T cell homing to LNs mediated by high-affinity integrin activation and chemokine-induced transendothelial migration.

The pharyngeal (Ph) and palatine (Pa) tonsils, although located in different regions of the upper aero-digestive tract (UADT), are thought to protect the respiratory tract similarly against infections by inducing and disseminating T and surface IgA(+) (sIgA(+)) B cells. We investigated the factors controlling the migratory properties of T and sIgA(+) B lymphocytes in the UADT of pigs by comparing the expression of vascular addressins, homing receptors and chemokine transcripts in Ph/Pa tonsils, Peyer's patches (PP) and their draining lymph nodes (LN). The vascular addressin PNAd was detected on high endothelial venules in both tonsils, whereas mucosal addressin cell adhesion molecule-1, otherwise present in PP and mesenteric LN, was not detected. More importantly, the vascular cell adhesion molecule-1 (VCAM-1) addressin was present in Ph tonsil and LN but neither in Pa tonsil nor in PP vascular cells, whereas both T and sIgA(+) B lymphocytes displayed similar levels of alpha4beta1(high) integrin, the ligand of VCAM-1. Analysis of transcript levels for several lymphoid (CCL19, CXCL12 and CCL21) and epithelial chemokines also demonstrated opposite chemokine mRNA ratios for Ph tonsil (CCL28>> CCL25) and PP, with Pa tonsil expressing very low levels of CCL28. Collectively, these data indicate that the differential compartmentalization of sIgA(+) lymphocytes between Pa and Ph tonsils may partly result from the differential expression of VCAM-1 and CCL28. They also suggest that tonsillar addressins and epithelial chemokines, rather than the cells intravasating it, control the regionalization of sIgA(+) lymphocytes in the UADT.

Chemokines are a large family of cytokines that direct normal leukocyte migration. They also have been implicated in leukocyte development and in the pathogenesis of many diseases. The CC chemokine CCL21, also known as Exodus-2, SLC, 6Ckine, and TCA4 induces both the adhesion and migration of human T cells. CCL21 is hypothesized to regulate the trafficking of T cells through secondary lymphoid tissues. To test this hypothesis, a transgenic mouse model was generated that placed the expression of mouse CCL21 (mCCL21) under the control of the T cell-specific lck promoter to abrogate the concentration gradient to which T cells normally respond. Overexpression of mCCL21 in T cells resulted in defects in CCL21- and CCL19-induced T-cell chemotaxis, node T-cell subpopulations, and lymph node architecture. The regulation of T-cell trafficking in secondary lymphoid tissues by CCL21 is therefore a tightly regulated system that can be altered by changes in the level of environmental CCL21 protein.

The T cell costimulatory molecule ICOS regulates Th2 effector function in allergic airway disease. Recently, several studies with ICOS(-/-) mice have also demonstrated a role for ICOS in Th2 differentiation. To determine the effects of ICOS on the early immune response, we investigated augmenting ICOS costimulation in a Th2-mediated immune response to Schistosoma mansoni Ags. We found that augmenting ICOS costimulation with B7RP-1-Fc increased the accumulation of T and B cells in the draining lymph nodes postimmunization. Interestingly, the increased numbers were due in part to increased migration of undivided Ag-specific TCR transgenic T cells and surprisingly B cells, as well as non-TCR transgenic T cells. B7RP-1-Fc also increased the levels of the chemokines CCL21 and CXCL13 in the draining lymph node, suggesting ICOS costimulation contributes to migration by direct or indirect effects on dendritic cells, stromal cells and high endothelial venules. Further, the effects of B7RP-1-Fc were not dependent on immunization. Our data support a model in which ICOS costimulation augments the pool of lymphocytes in the draining lymph nodes, leading to an increase in the frequency of potentially reactive T and B cells.

Inflammatory responses and associated products have been implicated in cancer metastasis. However, the relationship between these two processes is uncertain due to the lack of a suitable model. Taking advantage of localized and controllable inflammatory responses induced by biomaterial implantation and the capability of tissue scaffolds to release a wide variety of chemokines, we report a novel system for studying the molecular mechanisms of inflammation-mediated cancer metastasis. The animal model is comprised of an initial subcutaneous implantation of biomaterial microspheres which prompt localized inflammatory responses, followed by the transplantation of metastatic cancer cells into the peritoneal cavity or blood circulation. Histological results demonstrated that substantial numbers of B16F10 cells were recruited to the site nearby biomaterial implants. There was a strong correlation between the degree of biomaterial-mediated inflammatory responses and number of recruited cancer cells. Inflammation-mediated cancer cell migration was inhibited by small molecule inhibitors of CXCR4 but not by neutralizing antibody against CCL21. Using chemokine-releasing scaffolds, further studies were carried out to explore the possibility of enhancing cancer cell recruitment. Interestingly, erythropoietin (EPO) releasing scaffolds, but not stromal cell-derived factor-1α-releasing scaffolds, were found to accumulate substantially more melanoma cells than controls. Rather unexpectedly, perhaps by indirectly reducing circulating cancer cells, mice implanted with EPO-releasing scaffolds had ~30% longer life span than other groups. These results suggest that chemokine-releasing scaffolds may potentially function as implantable cancer traps and serve as powerful tools for studying cancer distraction and even selective annihilation of circulating metastatic cancer cells.

We assessed the effect of voriconazole (VRC) on the expression and release of selected cytokines and chemokines in the THP-1 human monocytic cell line in response to Aspergillus fumigatus hyphal fragments (HF) by cDNA microarray analysis, reverse transcriptase (RT) PCR, and enzyme-linked immunosorbent assay. Stimulation of THP-1 cells by HF alone caused a significant up-regulation of CCL4 (MIP1B) and CCL16, while CCL2 (MCP1) was down-regulated. By comparison, in the presence of VRC, a large number of genes such as CCL3 (MIP1A), CCL4 (MIP1B), CCL5 (RANTES), CCL7 (MCP3), CCL11 (EOTAXIN), CCL15 (MIP1Delta), CXCL6, and CXCL13 were strongly up-regulated in THP-1 cells challenged by HF, whereas CCL20 (MIP3A) and CCL21 (MIP2) were down-regulated. Among five genes differentially expressed in THP-1 cells, IL12A, IL12B, and IL-16 were down-regulated whereas IL-11 and TGFB1 were significantly up-regulated in the presence of VRC. The inflammation-related genes IFNgamma, IL1R1, and TNFA were also up-regulated in THP-1 cells exposed to HF only in the presence of VRC. RT-PCR of four selected genes validated the results of microarrays. The release of interleukin 1beta (IL-1beta) and IL-12 was significantly increased from monocytes stimulated either by HF alone (P < 0.05) or in the presence of VRC (P < 0.01 and P < 0.05, respectively). In contrast, tumor necrosis factor alpha release from monocytes was enhanced only in the presence of VRC (P < 0.01). The chemokines monocyte chemoattractant protein 1 and macrophage inflammatory protein 1beta were decreased under both conditions (P < 0.01). These results demonstrate that in the presence of VRC, HF induces a more pronounced profile of gene expression in THP-1 cells than HF alone, potentially leading to more-efficient host resistance to A. fumigatus.

Over the last decade, the significance of the homeostatic CC chemokine receptor-7 and its ligands CC chemokine ligand-19 (CCL19) and CCL21, in various types of cancer, particularly mammary carcinoma, has been highlighted. The chemokine receptor CCX-CKR is a high-affinity receptor for these chemokine ligands but rather than inducing classical downstream signalling events promoting migration, it instead sequesters and targets its ligands for degradation, and appears to function as a regulator of the bioavailability of these chemokines in vivo. Therefore, in this study, we tested the hypothesis that local regulation of chemokine levels by CCX-CKR expressed on tumours alters tumour growth and metastasis in vivo. Expression of CCX-CKR on 4T1.2 mouse mammary carcinoma cells inhibited orthotopic tumour growth. However, this effect could not be correlated with chemokine scavenging in vivo and was not mediated by host adaptive immunity. Conversely, expression of CCX-CKR on 4T1.2 cells resulted in enhanced spontaneous metastasis and haematogenous metastasis in vivo. In vitro characterisation of the tumourigenicity of CCX-CKR-expressing 4T1.2 cells suggested accelerated epithelial-mesenchymal transition (EMT) revealed by their more invasive and motile character, lower adherence to the extracellular matrix and to each other, and greater resistance to anoikis. Further analysis of CCX-CKR-expressing 4T1.2 cells also revealed that transforming growth factor (TGF)-β1 expression was increased both at mRNA and protein levels leading to enhanced autocrine phosphorylation of Smad 2/3 in these cells. Together, our data show a novel function for the chemokine receptor CCX-CKR as a regulator of TGF-β1 expression and the EMT in breast cancer cells.

Development of type 1 diabetes (T1D) is preceded by invasive insulitis. Although CD4(+)CD25(+) regulatory T cells (nTregs) induce tolerance that inhibits insulitis and T1D, the in vivo cellular mechanisms underlying this process remain largely unclear. Using an adoptive transfer model and noninvasive imaging-guided longitudinal analyses, we found nTreg depletion did not affect systemic trafficking and tissue localization of diabetogenic CD4(+) BDC2.5 T (BDC) cells in recipient mice prior to development of T1D. In addition, neither the initial expansion/activation of BDC cells nor the number of CD11c(+) or NK cells in islets and pancreatic lymph nodes were altered. Unexpectedly, our results showed nTreg depletion led to accelerated invasive insulitis dominated by CD11c(+) dendritic cells (ISL-DCs), not BDC cells, which stayed in the islet periphery. Compared with control mice, the phenotype of ISL-DCs and their ability to stimulate BDC cells did not change during invasive insulitis development. However, ISL-DCs from nTreg-deficient recipient mice showed increased in vitro migration toward CCL19 and CCL21. These results demonstrated invasive insulitis dominated by DCs, not CD4(+) T cells, preceded T1D onset in the absence of nTregs, and suggested a novel in vivo function of nTregs in T1D prevention by regulating local invasiveness of DCs into islets, at least partly, through regulation of DC chemotaxis toward CCL19/CCL21 produced by the islets.

Mouse formylpeptide receptor 2 (Fpr2) is a homologue of the human G-protein coupled chemoattractant receptor FPR2, which interacts with pathogen and host-derived chemotactic agonists. Our previous studies revealed reduced allergic airway inflammation and immune responses in Fpr2-deficient (Fpr2(-/-)) mice in association with diminished dendritic cell (DC) recruitment into the airway and draining lymph nodes. These defects prompted us to investigate the potential changes in the differentiation and maturation of DCs caused by Fpr2 deficiency. Bone marrow monocytes from Fpr2(-/-) mouse mice incubated with GM-CSF and IL-4 in vitro showed normal expression of markers of immature DCs. However, upon stimulation with the TLR4 agonist LPS, Fpr2(-/-) mouse DCs failed to express normal levels of maturation markers with reduced production of IL-12 and diminished chemotaxis in response to the DC homing chemokine CCL21. Fpr2(-/-) DCs also failed to induce allogeneic T-cell proliferation in vitro, and their recruitment into the T-cell zones of the spleen was reduced after antigen immunization. The capacity of Fpr2 to sustain normal DC maturation was dependent on its interaction with an endogenous ligand CRAMP expressed by DCs, because neutralization of either Fpr2 or CRAMP inhibited DC maturation in response to LPS. We additionally observed that the presence of exogenous CRAMP in culture increased the sensitivity of WT mouse DCs to LPS stimulation. The importance of CRAMP for DC maturation was further demonstrated by the observations that DCs from CRAMP(-/-) mice expressed lower levels of costimulatory molecules and MHC II and exhibited poor chemotaxis in response to CCL21 after LPS stimulation. Our observations indicate a nonredundant role for Fpr2 and its agonist CRAMP in DC maturation in immune responses.

CCL21 is a homeostatic chemokine that is expressed constitutively in secondary lymph nodes and attracts immune cells via chemokine receptor CCR7. In the brain however, CCL21 is inducibly expressed in damaged neurons both in vitro and in vivo and has been shown to activate microglia in vitro, albeit not through CCR7 but through chemokine receptor CXCR3. Therefore, a role for CCL21 in CXCR3-mediated neuron-microglia signaling has been proposed. It is well established that human and mouse astrocytes, like microglia, express CXCR3. However, effects of CCL21 on astrocytes have not been investigated yet. In this study, we have examined the effects of CCL21 on calcium transients and proliferation in primary mouse astrocytes. We show that similar to CXCR3-ligand CXCL10, CCL21 (10(-9) M and 10(-8) M) induced calcium transients in astrocytes, which were mediated through CXCR3. However, in response to high concentrations of CCL21 (10(-7) M) calcium transients persisted in CXCR3-deficient astrocytes, whereas CXCL10 did not have any effect in these cells. Furthermore, prolonged exposure to CXCL10 or CCL21 promoted proliferation of wild type astrocytes. Although CXCL10-induced proliferation was absent in CXCR3-deficient astrocytes, CCL21-induced proliferation of these cells did not significantly differ from wild type conditions. It is therefore suggested that primary mouse astrocytes express an additional (chemokine-) receptor, which is activated at high CCL21 concentrations.

Previous studies have demonstrated that the cell surface receptor Slamf1 (CD150) is requisite for optimal NADPH-oxidase (Nox2) dependent reactive oxygen species (ROS) production by phagocytes in response to Gram- bacteria. By contrast, Slamf8 (CD353) is a negative regulator of ROS in response to Gram+ and Gram- bacteria. Employing in vivo migration after skin sensitization, induction of peritonitis, and repopulation of the small intestine demonstrates that in vivo migration of Slamf1-/- dendritic cells and macrophages is reduced, as compared to wt mice. By contrast, in vivo migration of Slamf8-/- dendritic cells, macrophages and neutrophils is accelerated. These opposing effects of Slamf1 and Slamf8 are cell-intrinsic as judged by in vitro migration in transwell chambers in response to CCL19, CCL21 or CSF-1. Importantly, inhibiting ROS production of Slamf8-/- macrophages by diphenyleneiodonium chloride blocks this in vitro migration. We conclude that Slamf1 and Slamf8 govern ROS-dependent innate immune responses of myeloid cells, thus modulating migration of these cells during inflammation in an opposing manner.

BACKGROUND

The genitourinary tract is regarded as part of the mucosal immune system. However, the structural and functional aspects of the human prostate-associated lymphoid tissue (PALT) have never been extensively explored.

METHODS

This article describes our investigation of this issue by means of immunohistological, confocal, and ultrastructural examination of the normal human prostate.

RESULTS

PALT consists of two main components: (1) intraepithelial leukocytes, namely CD3(+)T cells with prevalent CD8(+) and CD45RA(-)CD45RO(+) phenotype, sometimes CD69(+), followed by CD94(+)NK, CD11c(+)DCs, some expressing CD86, DC-SIGN(+)DCs and a few B lymphocytes; (2) lymphoid aggregates, frequently below the epithelia, arranged in B cell follicles, endowed with a central ICAM-1(+)VCAM-1(+)CD21(+)FDCs network expressing BLC/CXCL13, and parafollicular T cell areas crossed by PNAd(+)HEV-like vessels showing SLC/CCL21 expression. Parafollicular areas were formed of prevalent CD4(+)T lymphocytes, both CD45RA(-) and CD45RO(+), and intermingled with CD11c(+)DCs. Germinal-center-containing follicles are few and their parafollicular areas are scantily infiltrated by Foxp3(+)CD69(-) highly suppressive regulatory T cells. Most lymphoid follicles lack a distinct germinal center and their parafollicular area harbor numerous Foxp3(+)CD69(-) cells.

CONCLUSIONS

Comparison with the tonsils shows that PALT displays immunomorphological features required for the onset of cellular and humoral immune responses, while its T regulatory cells appear to function as suppressor-regulators of T and B cell responses.

BACKGROUND

Type 1 diabetes mellitus (T1D) is a chronic autoimmune disease caused by the selective destruction of pancreatic β cells, followed by hyperglycemia, oxidative stress and the subsequent extensive impairment of immune cell functions, a phenomenon responsible for the development of chronic diabetic complications. Propolis, a natural bee product that is extensively used in foods and beverages, significantly benefits human health. Specifically, propolis exerts antioxidant, anti-inflammatory and analgesic effects that may improve diabetic complications. To further elucidate the potential benefits of propolis, the present study investigated the effect of dietary supplementation with propolis on the plasma cytokine profiles, free radical levels, lipid profile and lymphocyte proliferation and chemotaxis in a streptozotocin (STZ)-induced type I diabetic mouse model.

METHODS

Thirty male mice were equally distributed into 3 experimental groups: group 1, non-diabetic control mice; group 2, diabetic mice; and group 3, diabetic mice supplemented daily with an ethanol-soluble derivative of propolis (100 mg/kg body weight) for 1 month.

RESULTS

First, the induction of diabetes in mice was associated with hyperglycemia and significant decreases in the insulin level and the lymphocyte count. In this context, diabetic mice exhibited severe diabetic complications, as demonstrated by a significant decrease in the levels of IL-2, IL-4 and IL-7, prolonged elevation of the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and reactive oxygen species (ROS) and altered lipid profiles compared with control non-diabetic mice. Moreover, antigen stimulation of B and T lymphocytes markedly reduced the proliferative capacity and chemotaxis of these cells towards CCL21 and CXCL12 in diabetic mice compared with control mice. Interestingly, compared with diabetes induction alone, treatment of diabetic mice with propolis significantly restored the plasma cytokine and ROS levels and the lipid profile to nearly normal levels. Most importantly, compared with untreated diabetic mice, diabetic mice treated with propolis exhibited significantly enhanced lymphocyte proliferation and chemotaxis towards CCL21 and CXCL12.

CONCLUSIONS

Our findings reveal the potential immuno-modulatory effects of propolis, which acts as a natural antioxidant to enhance the function of immune cells during diabetes.

OBJECTIVE

Toxoplasma gondii, the parasite responsible for ocular toxoplasmosis, accesses the retina from the bloodstream. We investigated the dendritic cell as a potential taxi for T. gondii tachyzoites moving across the human retinal endothelium, and examined the participation of adhesion molecules and chemokines in this process.

METHODS

CD14-positive monocytes were isolated from human peripheral blood by antibody-mediated cell enrichment, and cultured in granulocyte-macrophage colony-stimulating factor and interleukin-4 to generate dendritic cells. Transmigration assays were performed over 18 hours in transwells seeded with human retinal endothelial cells and using dendritic cells exposed to laboratory or natural strains of T. gondii tachyzoites. Parasites were tagged with yellow fluorescent protein to verify infection. In some experiments, endothelial monolayers were preincubated with antibody directed against adhesion molecules, or chemokine was added to lower chambers of transwells.

RESULTS

Human monocyte-derived dendritic cell preparations infected with laboratory or natural strain T. gondii tachyzoites transmigrated in larger numbers across simulated human retinal endothelium than uninfected dendritic cells (P ≤ 0.0004 in 5 of 6 experiments). Antibody blockade of intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and activated leukocyte cell adhesion molecule (ALCAM) inhibited transmigration (P ≤ 0.007), and CCL21 or CXCL10 increased transmigration (P ≤ 0.031).

CONCLUSIONS

Transmigration of human dendritic cells across retinal endothelium is increased following infection with T. gondii. Movement may be impacted by locally produced chemokines and is mediated in part by ICAM-1, VCAM-1, and ALCAM. These findings have implications for development of novel therapeutics aimed at preventing retinal infection by T. gondii.

Chemokines are essential guidance cues orchestrating cell migration in health and disease. Cognate chemokine receptors sense chemokine gradients over short distances to coordinate directional cell locomotion. The chemokines CCL19 and CCL21 are essential for recruiting CCR7-expressing dendritic cells bearing pathogen-derived antigens and lymphocytes to lymph nodes, where the two cell types meet to launch an adaptive immune response against the invading pathogen. CCR7-expressing cancer cells are also recruited by CCL19 and CCL21 to metastasize in lymphoid organs. In contrast, atypical chemokine receptors (ACKRs) do not transmit signals required for cell locomotion but scavenge chemokines. ACKR4 is crucial for internalizing and degrading CCL19 and CCL21 to establish local gradients, which are sensed by CCR7-expressing cells. Here, we describe the production of fluorescently tagged chemokines by fusing CCL19 and CCL21 to monomeric red fluorescent protein (mRFP). We show that purified CCL19-mRFP and CCL21-mRFP are versatile and powerful tools to study CCR7 and ACKR4 functions, such as receptor trafficking and chemokine scavenging, in a spatiotemporal fashion. We demonstrate that fluorescently tagged CCL19 and CCL21 permit the visualization and quantification of chemokine gradients in real time, while CCR7-expressing leukocytes and cancer cells sense the guidance cues and migrate along the chemokine gradients.

Orf virus (ORFV) is a zoonotic parapoxvirus that induces acute pustular skin lesions in sheep and humans. ORFV can reinfect its host and the discovery of several secreted immune modulatory factors that include a chemokine-binding protein (CBP) may explain this phenomenon. Dendritic cells (DC) are professional antigen presenting cells that induce adaptive immunity and their recruitment to sites of infection in skin and migration to peripheral lymph nodes is critically dependent on inflammatory and constitutive chemokine gradients respectively. Here we examined whether ORFV-CBP could disable these gradients using mouse models. Previously we established that ORFV-CBP bound murine inflammatory chemokines with high affinity and here we show that this binding spectrum extends to constitutive chemokines CCL19 and CCL21. Using cell-based chemotaxis assays, ORFV-CBP inhibited the movement of both immature and mature DC in response to these inflammatory and constitutive chemokines respectively. Moreover in C57BL/6 mice, intradermally injected CBP potently inhibited the recruitment of blood-derived DC to lipopolysaccharide-induced sites of skin inflammation and inhibited the migration of ex vivo CpG-activated DC to inguinal lymph nodes. Finally we showed that ORFV-CBP completely inhibited T responsiveness in the inguinal lymph nodes using intradermally injected DC pulsed with ovalbumin peptide and transfused transgenic T cells.

Chemokines expressed in neurons are important mediators in neuron-neuron and neuron-glia signaling. One of these chemokines is CCL21 that activates microglia via the chemokine receptor CXCR3. As neurons also express CXCL10, a main ligand for CXCR3, we have thus investigated in detail the expression pattern of CXCL10 in neurons. We show that CXCL10 is constitutively expressed by neurons, is stored in large dense-core vesicles and is not regulated by neuronal injury or stress. Neuronal CXCL10 release occurred constitutively at low level. In vivo CXCL10 expression was found in the developing brain at various embryonic stages and its peak expression correlates with the presence of CD11b- and GFAP-positive cells expressing CXCR3. These results suggest a possible role of neuronal CXCL10 in recruitment and homing of glial cells during embryogenesis.

Chemokine and chemokine receptors expressed by normal and neoplastic lymphocytes play a key role in cell recruitment into skin and lymph nodes. The aim of this study was to get further insights into the role of chemokines in pathogenesis and progression of cutaneous T-cell lymphoma (CTCL) with particular regard to Sézary Syndrome (SS), a CTCL variant with blood involvement. Here, we show that functional CXCL13 homeostatic chemokine is strongly up-regulated in SS cells, well-detectable in skin lesions and lymph nodes, and measurable at high concentration in plasma of SS patients, at different levels during disease progression. Furthermore, we show that the addition of CXCL13 to CCL19 or to CCL21, the selective CCR7 agonists responsible for lymph node homing, strongly enhances the migration of CCR7+ SS cells. We also show that neutralization of the CCR7 receptor strongly impairs CCL19/21-induced chemotaxis of SS cells both in the absence or presence of CXCL13. Additional experiments performed to investigate the survival, adhesion, and metalloproteases secretion indicate that CXCL13 combined with CCL19 and CCL21 mainly affects the chemotaxis of SS cells. Our findings suggest that this newly described CXCL13 expression in SS represents a new pathogenetic mechanism of diagnostic significance.

In the inflammatory mucosal microenvironment of head and neck SCC (HNSCC), DC express CD16 and are usually in direct contact with tumor cells. Mucosal and inflammation-associated DC develop from monocytes, and monocyte-derived DC are used in HNSCC immunotherapy. However, beyond apoptotic tumor cell uptake and presentation of tumor antigens by DC, HNSCC cell interactions with DC are poorly understood. Using co-cultures of monocyte-derived DC and two established HNSCC cell lines that represent well- and poorly-differentiated SCC, respectively, we found that carcinoma cells induced significant increases in CD16 expression on DC while promoting a CD1a(+)CD86(dim) immature phenotype, similar to that observed in HNSCC specimens. Moreover, HNSCC cells affected steady-state and CCL21-induced migration of DC, and these effects were donor-dependent. The CCL21-induced migration directly correlated with HNSCC-mediated effects on CCR7 and CD38 expression on DC-SIGN-high DC. The dominant pattern seen in six out of nine donors was the increase in steady-state and CCL21-induced DC migration in co-cultures with HNSCC, while the reverse pattern, i.e., decreased DC migration in co-cultures with SCC, was identified in two donors. A split in migratory DC behavior, i.e. increase with one HNSCC cell line and a decrease with the second cell line, was observed in one donor. Remarkably, the numbers of live detached HNSCC cells were orders of magnitude higher in DC-HNSCC co-cultures than in parallel HNSCC cell cultures without DC. This study provides novel insights into the effects of DC-HNSCC interactions relevant to the tumor microenvironment.

The organization of secondary lymphoid tissues into distinct T and B cell compartments supports proper regulation of an immune response to foreign Ags. In the splenic white pulp, this compartmentalization is also thought to be important in the maintenance of B cell tolerance. Using lymphotoxin-alpha-(LT-alpha)-, TNF-alpha-, or TNFRp55-deficient mice, all with disrupted splenic architecture, we tested whether normal T/B segregation and/or intact follicular structure are necessary for the maintenance of anti-dsDNA B cell anergy. This study demonstrates that anti-dsDNA B cells remain tolerant in LT-alpha(-/-), TNF-alpha(-/-), and TNFRp55(-/-) mice; however, TNF-alpha or a TNF-alpha-dependent factor is required for their characteristic positioning to the T/B interface. Providing a TNF-alpha signal in TNF-alpha(-/-) mice by systemic administration of an agonist anti-TNFRp55 mAb induces the maturation of the anti-dsDNA B cells and their movement away from the T cell area toward the B cell area. Additionally, the agonist Ab induces changes in the follicular environment, including FDC clustering, up-regulation of the CXC chemokine ligand CXCL13, and down-regulation of the CC chemokine ligands CCL19 and CCL21. Therefore, this study suggests that a balance between B and T cell tropic chemokine signals may be an important mechanism for positioning anergic B cells at the T/B interface of the splenic white pulp.