Tumor development and progression are multifactorial processes, regulated by a large variety of intrinsic and microenvironmental factors. A key role in cancer is played by members of the chemokine superfamily. Chemokines and their receptors are expressed by tumor cells and by host cells, in primary tumors and in specific metastatic loci. The effects of chemokines on tumorigenesis are diverse: While some members of the superfamily significantly support this process, others inhibit fundamental events required for tumor establishment and metastasis. The current review describes the multifaceted roles of chemokines in malignancy, addressing four major aspects of their activities: (1) inducing leukocyte infiltration to tumors and regulating immune functions, with emphasis on tumor-associated macrophages (and the chemokines CCL2, CCL5), T cells (and the chemokines CXCL9, CXCL10) and dendritic cells (and the chemokines CCL19, CCL20, CCL21); (2) directing the homing of tumor cells to specific metastatic sites (the CXCL12-CXCR4 axis); (3) regulating angiogenic processes (mainly the ELR(+)-CXC and non-ELR-CXC chemokines); (4) acting directly on the tumor cells to control their malignancy-related functions. Together, these different chemokine functions establish a net of interactions between the tumor cells and their microenvironment, and partly dictate the fate of the malignancy cascade.

De novo formation of tertiary lymphoid structures (TLS) has been described in lung cancers. Intratumoral TLS seem to be functional and are associated with a long-term survival for lung cancer patients, suggesting that they represent an activation site for tumor-specific T cells. Here, we characterized T-cell recruitment to TLS in human lung cancer to identify the adhesion molecules and chemoattractants orchestrating this migration. We found that most TLS T cells were CD62L+ and mainly of CD4+ memory phenotype, but naive T cells were highly enriched in these structures as compared with the rest of the tumor. A specific gene expression signature associated with T cell presence was identified in TLS, which included chemokines (CCL19, CCL21, CXCL13, CCL17, CCL22, and IL16), adhesion molecules (ICAM-2, ICAM-3, VCAM-1, and MAdCAM-1) and integrins (alphaL, alpha4, and alphaD). The presence of the corresponding receptors on TLS T cells was confirmed. Intratumoral PNAd+ high endothelial venules also were exclusively associated with TLS and colocalized with CD62L+ lymphocytes. Together, these data bring new insights into the T-cell recruitment to intratumoral TLS and suggest that blood T cell enter into TLS via high endothelial venules, which represent a new gateway for T cells to the tumor. Findings identify the molecules that mediate migration of tumor-specific T cells into TLS where T cell priming occurs, suggesting new strategies to enhance the efficacy of cancer immunotherapies.

The interactions of chemokine receptor CCR7 and its ligands are essential for migration of lymphocytes and dendritic cells to lymph nodes. In this study, we found that 4 of 6 (67%) gastric carcinoma cell lines tested expressed functional CCR7 for the chemokine CCL21/6Ckine, as demonstrated by calcium mobilization and actin polymerization assays. Moreover, we also showed that signaling through CCR7 induced chemotactic and invasive responses in CCR7-positive gastric carcinoma cells. In clinical samples, immunohistochemical assay showed that CCR7-positive carcinoma cells were detected in 42 of 64 (66%) cases and a significant difference in both lymph node metastasis (P < 0.001) and lymphatic invasion (P < 0.001) between CCR7-positive and -negative cases. Patients with CCR7-positive tumors had a significantly poorer prognosis than those with CCR7-negative tumors (P < 0.05). Stepwise regression analysis revealed that the most important factor related to lymph node metastasis was the expression of CCR7. These results indicated that CCR7 and its ligands interaction is associated with preferential lymph node metastasis of gastric carcinoma.

Lymphocyte homing to secondary lymphoid tissue is defined by a multistep sequence of interactions between lymphocytes and endothelial cells in high endothelial venules (HEVs). After initial selectin-mediated tethering and rolling, firm adhesion of lymphocytes requires rapid upregulation of lymphocyte integrin adhesiveness. This step is mediated in part by the HEV-derived chemokine SLC (secondary lymphoid-tissue chemokine, or CCL21) that binds to the CC chemokine receptor (CCR)7 on lymphocytes. However, the CC chemokine ELC (Epstein-Barr virus-induced molecule 1 ligand chemokine, or CCL19) shares the same receptor, and ELC transcripts have been observed in the T cell areas of lymphoid organs. Here, we show that perivascular ELC is transcytosed to the luminal surfaces of HEVs and enables efficient T cell homing to lymph nodes. In situ hybridization on sections of human tonsil showed no ELC mRNA in HEVs, but immunostaining revealed ELC protein in cytoplasmic vesicles of HEV cells. Furthermore, ELC injected into the footpads of mice entered the draining lymph nodes and was presented by HEVs. Finally, intracutaneous injections of ELC in mice lacking functionally relevant ELC and SLC (plt/plt mice) restored T cell trafficking to draining lymph nodes as efficiently as SLC. We conclude that perivascular ELC is transcytosed to the luminal surfaces of HEVs and participates in CCR7-mediated triggering of lymphocyte arrest.

The efficacy of immune response to control human cancer remains controversial. It is particularly debated whether and to what extent the capacity of tumor-infiltrating dendritic cells (DC) to drive immunization can be turned off by transformed cells, leading to tumor-specific tolerance rather than immunization. To address this issue, we have characterized the DC isolated from human non-small cell lung cancer (NSCLC). These biopsy specimens contained CD11c(high) myeloid DC (mDC), but also CD11c(-) plasmacytoid DC (pDC) and a third DC subset expressing intermediate level of CD11c. Compared with peripheral blood, CD11c(high) tumor-infiltrating DC (TIDC) displayed a "semi-mature" phenotype, and TLR4 or TLR8 stimulation drove them to mature partially and to secrete limited amounts of cytokines. In contrast, most tumor-infiltrating pDC were immature but underwent partial maturation after TLR7 activation, whereas TLR9 ligation triggered low secretion of IFN-alpha. CD11c(int) mDC represented approximately 25% of total DC in tumoral and peritumoral tissues and expressed low levels of costimulatory molecules contrasting with high levels of the immunoinhibitory molecule B7-H1. Finally, the poor APC function of total TIDC even after TLR stimulation and the migratory response of both tumor-infiltrating mDC and pDC toward CCL21 and SDF-1 in vitro suggested their ability to compromise the tumor-specific immune response in draining lymph nodes in vivo. Further studies will be required to establish the specific role of the three TIDC subsets in tumor immunity and to draw conclusions for the design of therapeutic strategies.

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C(12)U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C(12)U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC -- but not CpG-C given at the same dose -- also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell-activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell-attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.

Lymph node (LN) function depends on T and B cell compartmentalization, antigen presenting cells, and high endothelial venules (HEVs) expressing mucosal addressin cell adhesion molecule (MAdCAM-1) and peripheral node addressin (PNAd), ligands for naive cell entrance into LNs. Luminal PNAd expression requires a HEV-restricted sulfotransferase (HEC-6ST). To investigate LT alpha beta's activities in lymphoid organogenesis, mice simultaneously expressing LT alpha and LT beta under rat insulin promoter II (RIP) control were compared with RIPLT alpha mice in a model of lymphoid neogenesis and with LT beta-/- mice. RIPLT alpha beta pancreata exhibited massive intra-islet mononuclear infiltrates that differed from the more sparse peri-islet cell accumulations in RIPLT alpha pancreata: separation into T and B cell areas was more distinct with prominent FDC networks, expression of lymphoid chemokines (CCL21, CCL19, and CXCL13) was more intense, and L-selectin+ cells were more frequent. In contrast to the predominant abluminal PNAd pattern of HEV in LT beta-/- MLN and RIPLT alpha pancreatic infiltrates, PNAd was expressed at the luminal and abluminal aspects of HEV in wild-type LN and in RIPLT alpha beta pancreata, coincident with HEC-6ST. These data highlight distinct roles of LT alpha and LT alpha beta in lymphoid organogenesis supporting the notion that HEC-6ST-dependent luminal PNAd is under regulation by LT alpha beta.

BACKGROUND

Lymphatic transport of peripheral interstitial fluid and dendritic cells (DCs) is important for both adaptive immunity and maintenance of tolerance to self-antigens. Lymphatic drainage can change rapidly and dramatically on tissue injury or inflammation, and therefore increased fluid flow may serve as an important early cue for inflammation; however, the effects of transmural flow on lymphatic function are unknown.

OBJECTIVE

Here we tested the hypothesis that lymph drainage regulates the fluid and cell transport functions of lymphatic endothelium.

RESULTS

Using in vitro and in vivo models, we demonstrated that lymphatic endothelium is sensitive to low levels of transmural flow. Basal-to-luminal flow (0.1 and 1 mum/sec) increased lymphatic permeability, dextran transport, and aquaporin-2 expression, as well as DC transmigration into lymphatics. The latter was associated with increased lymphatic expression of the DC homing chemokine CCL21 and the adhesion molecules intercellular adhesion molecule-1 and E-selectin. In addition, transmural flow induced delocalization and downregulation of vascular endothelial cadherin and PECAM-1 (platelet/endothelial cell adhesion molecule-1). Flow-enhanced DC transmigration could be reversed by blocking CCR7, intercellular adhesion molecule-1, or E-selectin. In an experimental model of lymphedema, where lymphatic drainage is greatly reduced or absent, lymphatic endothelial expression of CCL21 was nearly absent.

CONCLUSIONS

These findings introduce transmural flow as an important regulator of lymphatic endothelial function and suggest that flow might serve as an early inflammatory signal for lymphatics, causing them to regulate transport functions to facilitate the delivery of soluble antigens and DCs to lymph nodes.

Spinal cord injury (SCI) results in the generation and amplification of pain caused in part by injury-induced changes in neuronal excitability at multiple levels along the sensory neuraxis. We have previously shown that activated microglia, through an ERK (extracellular signal-regulated kinase)-regulated PGE(2) (prostaglandin E(2)) signaling mechanism, maintain neuronal hyperexcitability in the lumbar dorsal horn. Here, we examined whether microglial cells in the thalamus contribute to the modulation of chronic pain after SCI, and whether microglial activation is governed by spinally mediated increases in the microglial activator cysteine-cysteine chemokine ligand 21 (CCL21). We report that CCL21 is upregulated in dorsal horn neurons, that tissue levels are increased in the dorsal horn and ventral posterolateral (VPL) nucleus of the thalamus 4 weeks after SCI, and that the increase can be differentially reduced by spinal blockade at T1 or L1. In intact animals, electrical stimulation of the spinothalamic tract induces increases in thalamic CCL21 levels. Recombinant CCL21 injected into the VPL of intact animals transiently activates microglia and induces pain-related behaviors, effects that could be blocked with minocycline. After SCI, intra-VPL antibody-mediated neutralization of CCL21 decreases microglial activation and evoked hyperexcitability of VPL neurons, and restores nociceptive thresholds to near-normal levels. These data identify a novel pathway by which SCI triggers upregulation of the neuroimmune modulator CCL21 in the thalamus, which induces microglial activation in association with pain phenomena.

Following lymphocyte depletion, homeostatic mechanisms drive the reconstitution of lymphocytes. We prospectively studied this process in 16 patients for 1 year after a single pulse of treatment with Campath-1H, a humanised anti-CD52 monoclonal antibody. We observed two phases of lymphocyte reconstitution. In the first 6 months after treatment the precursor frequency and proliferation index of the patients' autologous mixed lymphocyte reaction increased; the depleted T cell pool was dominated by memory T cells, especially (CD4+)CD25high T cells, a putative regulatory phenotype; and there was a non-significant rise in peripheral mononuclear cell FoxP3 mRNA expression and fall in constitutive cytokine mRNA expression. In the later phase, from 6-to-12 months after Campath-1H, these changes reversed and there was a rise in ROG mRNA expression. However, total CD4+ numbers remained below 50% of pre-treatment levels at 12 months, perhaps reflecting a failure in homeostasis. This was not due to an impaired IL-7 response, as in rheumatoid arthritis, nor to a lack of IL-7 receptors, which are found on fewer human (CD4+)CD25high than naive cells. We speculate that CCL21 and IL-15 responses to lymphopaenia may be suboptimal in multiple sclerosis.

OBJECTIVE

To better understand the contribution of age to the development of osteoarthritis (OA).

METHODS

Surgical destabilization of the medial meniscus (DMM) was used to model OA in 12-week-old and 12-month-old male C57BL/6 mice. OA severity was evaluated histologically. RNA used for microarray and real-time polymerase chain reaction analysis was isolated from joint tissue collected from the medial side of the joint, including cartilage, meniscus, subchondral bone, and the joint capsule with synovium. Computational analysis was used to identify patterns of gene expression, and immunohistochemistry was used to evaluate tissue distribution of selected proteins.

RESULTS

OA was more severe in older mice than in young mice. Only 55 genes showed a similar expression with DMM-induced OA in the 2 age groups, while 493 genes showed differential expression, the majority having increased expression in older mice. Functional categories for similarly expressed genes included extracellular matrix- and cell adhesion-related genes; differentially expressed genes included those related to muscle structure and development and immune response genes. Comparison of expression in sham-operated control joints revealed an age-related decrease in matrix gene expression and an increase in immune and defense response gene expression. Interleukin-33 was present in multiple joint tissue cells, while CCL21 was more localized to chondrocytes and meniscal cells. Periostin was found in the extracellular matrix of cartilage and meniscus.

CONCLUSIONS

Age affects both the basal pattern of gene expression in joint tissues and the response to surgically induced OA. Examining tissue from the joint beyond only cartilage revealed novel genes and proteins that would be important to consider in OA.

Chemokines and their receptors have emerged as attractive targets regulating the migration of tumor cells in vivo, a process known as cancer metastasis. The control of metastasis is critical to the control of cancer progression. Two chemokine receptors and their ligands stand out as likely targets for therapeutics: CCR7/CCL21 for lymph node metastases, and CXCR4/CXCL12 for lung, liver, bone marrow, and brain metastases. The most widely expressed chemokine receptor among cancers is likely to be CXCR4.

CXCL13 and CCL21 have been functionally implicated in lymphoid tissue organization both in the upstream phases of lymphoid tissue embryogenesis and in ectopic lymphoid neogenesis in transgenic mice. Here, we analyzed the relationship between CXCL13 and CCL21 production and lymphoid tissue organization in rheumatoid synovitis as a model of a naturally occurring ectopic lymphoneogenesis. Through systematic analysis of mRNA and protein expression, we defined the microanatomical relationship between CXCL13 and CCL21 in progressive aggregational and structural phases of synovial inflammatory infiltrate. We provide the first direct in situ evidence that production of CXCL13 and CCL21 (rather than simply protein binding) is associated with inflammatory lymphoid tissue formation and development with the demonstration, in organized aggregates, of a secondary lymphoid organ-like compartmentalization and vascular association. Notably, the presence of CXCL13 and CCL21 (protein and mRNA) was also demonstrated in non-organized clusters and minor aggregational stages, providing evidence that their induction can take place independently and possibly upstream of T-B compartmentalization, CD21(+) follicular dendritic cell network differentiation and germinal center formation. Our data support the concept that, under inflammatory conditions, CXCL13 and CCL21 participate in lymphoid tissue microanatomical organization, attempting to recapitulate, in an aberrant lymphoid neogenetic process, their homeostatic and morphogenetic physiologic functions.

Embryo implantation induces formation of the decidua, a stromal cell-derived structure that encases the fetus and placenta. Using the mouse as a model organism, we have found that this tissue reaction prevents DCs stationed at the maternal/fetal interface from migrating to the lymphatic vessels of the uterus and thus reaching the draining lymph nodes. Strikingly, decidual DCs remained immobile even after being stimulated with LPS and exhibiting responsiveness to CCL21, the chemokine that drives DC entry into lymphatic vessels. An analysis of maternal T cell reactivity toward a surrogate fetal/placental antigen furthermore revealed that regional T cell responses toward the fetus and placenta were driven by passive antigen transport and thus the tolerogenic mode of antigen presentation that predominates when there is negligible input from tissue-resident DCs. Indeed, the lack of involvement of tissue-resident DCs in the T cell response to the fetal allograft starkly contrasts with their prominent role in organ transplant rejection. Our results suggest that DC entrapment within the decidua minimizes immunogenic T cell exposure to fetal/placental antigens and raise the possibility that impaired development or function of the human decidua, which unlike that of the mouse contains lymphatic vessels, might lead to pathological T cell activation during pregnancy.

The development of secondary lymphoid organs is a complex process dependent on a coordinated interaction of cells of hematopoietic and non-hematopoietic origin. In this context, chemokines and cytokines belonging to the tumor necrosis factor (TNF)/lymphotoxin (LT) family are critical signaling molecules during the initial steps of lymph node and Peyer's patch organogenesis. Homeostatic chemokines, such as CXCL13, CCL21, and CCL19, as well as their corresponding receptors, CXCR5 and CCR7, have now been shown to closely cooperate in the development of lymphoid organs and the maintenance of lymphoid tissue microarchitecture. We summarize recent data on the function of CXCR5 and CCR7 and their intricate connection to the TNF/LT system in order to refine the current model of lymphoid organ development.

Chemokines and adhesion molecules up-regulated in lymphatic endothelial cells (LECs) during tissue inflammation are thought to enhance dendritic cell (DC) migration to draining lymph nodes, but the in vivo control of this process is not well understood. We performed a transcriptional profiling analysis of LECs isolated from murine skin and found that inflammation induced by a contact hypersensitivity (CHS) response up-regulated the adhesion molecules ICAM-1 and VCAM-1 and inflammatory chemokines. Importantly, the lymphatic markers Prox-1, VEGFR3, and LYVE-1 were significantly down-regulated during CHS. By contrast, skin inflammation induced by complete Freund adjuvant induced a different pattern of chemokine and lymphatic marker gene expression and almost no ICAM-1 up-regulation in LECs. Fluorescein isothiocyanate painting experiments revealed that DC migration to draining lymph nodes was more strongly increased in complete Freund adjuvant-induced than in CHS-induced inflammation. Surprisingly, DC migration did not correlate with the induction of CCL21 and ICAM-1 protein in LECs. Although the requirement for CCR7 signaling became further pronounced during inflammation, CCR7-independent signals had an additional, albeit moderate, impact on enhancing DC migration. Collectively, these findings indicate that DC migration in response to inflammation is stimulus-specific, mainly CCR7-dependent, and overall only moderately enhanced by LEC-induced genes other than CCL21.

CD1d-restricted Valpha24-Jalpha18-invariant natural killer T cells (iNKTs) are potentially important in tumor immunity. However, little is known about their localization to tumors. We analyzed 98 untreated primary neuroblastomas from patients with metastatic disease (stage 4) for tumor-infiltrating iNKTs using TaqMan((R)) reverse transcription polymerase chain reaction and immunofluorescent microscopy. 52 tumors (53%) contained iNKTs, and oligonucleotide microarray analysis of the iNKT(+) and iNKT(-) tumors revealed that the former expressed higher levels of CCL2/MCP-1, CXCL12/SDF-1, CCL5/RANTES, and <em>CCL21</em>/SLC. Eight tested neuroblastoma cell lines secreted a range of CCL2 (0-21.6 ng/ml), little CXCL12 (</=0.1 ng/ml), and no detectable CCL5 or <em>CCL21</em>. CCR2, the receptor for CCL2, was more frequently expressed by iNKT compared with natural killer and T cells from blood (P < 0.001). Supernatants of neuroblastoma cell lines that produced CCL2 induced in vitro migration of iNKTs from blood of patients and normal adults; this was abrogated by an anti-CCL2 monoclonal antibody. CCL2 expression by tumors was found to inversely correlate with MYCN proto-oncogene amplification and expression (r = 0.5, P < 0.001), and MYCN-high/CCL2-low expression accurately predicted the absence of iNKTs (P < 0.001). In summary, iNKTs migrate toward neuroblastoma cells in a CCL2-dependent manner, preferentially infiltrating MYCN nonamplified tumors that express CCL2.

Dendritic cells (DCs) are key instigators of adaptive immune responses. Using an alphaviral expression cloning technology, we have identified the chemokine CCL19 as a potent inducer of T cell proliferation in a DC-T cell coculture system. Subsequent studies showed that CCL19 enhanced T cell proliferation by inducing maturation of DCs, resulting in upregulation of costimulatory molecules and the production of proinflammatory cytokines. Moreover, CCL19 programmed DCs for the induction of T helper type (Th) 1 rather than Th2 responses. Importantly, only activated DCs that migrated from the periphery to draining lymph nodes, but not resting steady-state DCs residing within lymph nodes, expressed high levels of CCR7 in vivo and responded to CCL19 with the production of proinflammatory cytokines. Migrating DCs isolated from mice genetically deficient in CCL19 and CCL21 (plt/plt) presented an only partially mature phenotype, highlighting the importance of these chemokines for full DC maturation in vivo. Our findings indicate that CCL19 and CCL21 are potent natural adjuvants for terminal activation of DCs and suggest that chemokines not only orchestrate DC migration but also regulate their immunogenic potential for the induction of T cell responses.

Synovial inflammation in rheumatoid arthritis is closely related to the formation of ectopic lymphoid microstructures. In synovial tissue from some patients, one finds seemingly diffuse infiltrates; in others, T cells and B cells cluster in aggregates with interdigitating dendritic cells (DCs) but no follicular DCs (FDCs). In a third group, T cell/B cell follicles with germinal center (GC) reactions are generated. Within a given patient, aggregates and GCs are mutually exclusive and stable over time. Because antigen storage capacity, lymphoid density, and three-dimensional topography of GCs optimize immune responses, synovial GCs should play a crucial role in the breakdown of self-tolerance. We have identified factors critical for ectopic GCs, thereby transforming the synovial inflammatory process. Tissues with GCs produced 10- to 20-fold higher amounts of the chemokines CXCL13 and CCL21. CXCL13 derived from three sources, endothelial cells, synovial fibroblasts, and FDC networks. The level of CXCL13 transcripts strongly predicted GCs; however, some tissues had high levels of CXCL13 but lacked GCs. Tissue expression of LT-beta emerged as a second key factor. LT-beta protein was detected on follicular center and mantle zone B cells. Multivariate regression analysis identified CXCL13 and LT-beta as the only cytokines predicting GCs. Remarkably, LT-alpha did not contribute independently. The contribution of B cells to ectopic lymphoid organogenesis was not limited to LT-beta production. Rather, synovial tissue B cells were critical in regulating T cell activation. In adoptive transfer experiments in human synovium-SCID mouse chimeras, activation of synovium-derived CD4 T cells was strictly dependent on T cell/B cell follicles. Depletion of synovial tissue B cells abrogated T cell function, and non-B cell antigen-presenting cells could not maintain T cell stimulation. Unexpectedly, GC function in the rheumatoid lesion was also dependent on CD8 T cells. The majority of T cell receptors derived from CD8 T cells were shared between distinct GCs. Depletion of CD8 T cells disrupted synovial GCs, FDC networks disappeared, and transcription of LT-beta, IgG, and Igkappa declined. Follicle-sustaining CD8 T cells were located at the edge of or within the mantle zone. Cell-cell communication in the mantle zone, including CD8 T cells, appears to be critical for ectopic GC formation in rheumatoid synovitis.

Lymphotoxins (LT alpha and LT beta), LIGHT [homologous to LT, inducible expression, competes with herpes simplex virus (HSV) glycoprotein D for HSV entry mediator (HVEM), a receptor expressed on T lymphocytes], tumor necrosis factor (TNF), and their specific receptors LT beta R, HVEM, and TNF receptor 1 (TNFR1) and TNFR2, form the immediate family of the larger TNF superfamily. These cytokines establish a critical communication system required for the development of secondary lymphoid tissues; however, knowledge of the target genes activated by these signaling pathways is limited. Target genes regulated by the LT alpha beta-LT beta R pathway include the tissue-organizing chemokines, CXCL13, CCL19, and CCL21, which establish cytokine circuits that regulate LT expression on lymphocytes, leading to organized lymphoid tissue. Infectious disease models have revealed that LT alpha beta pathways are also important for innate and adaptive immune responses involved in host defense. Here, regulation of interferon-beta by LT beta R and TNFR signaling may play a crucial role in certain viral infections. Regulation of autoimmune regulator in the thymus via LT beta R implicates LT/LIGHT involvement in central tolerance. Dysregulated expression of LIGHT overrides peripheral tolerance leading to T-cell-driven autoimmune disease. Blockade of TNF/LT/LIGHT pathways as an intervention in controlling autoimmune diseases is attractive, but such therapy may have risks. Thus, identifying and understanding the target genes may offer an opportunity to fine-tune inhibitory interventions.

Gliomas are highly aggressive and accompanied by numerous microglia/macrophages (MG/MP) in and about the tumor. Little is known about what MG/MP do in this setting, or whether modulating MG/MP activation might affect glioma progression. Here, we used a glioma-microglia in culture system to establish the effects the tumor and microglia have on each other. We assessed glioma progression in vivo after MG/MP ablation or in the setting of exaggerated MG/MP activation. We show that glioma cells activate microglia but inhibit their phagocytic activities. Local ablation of MG/MP in vivo decreased tumor size and improved survival curves. Conversely, pharmacological activation of MG/MP increased glioma size through stimulating tumor proliferation and inhibiting apoptosis. In agreement with recent reports, expression of the chemokine CCL21 is enhanced after MG/MP activation and correlates with tumor growth. Taken together, our findings demonstrate that inhibition of MG/MP activation may constitute a new and effective contribution towards suppressing glioma proliferation.

Follicular dendritic cells (FDCs) retain and display opsonized antigens in primary follicles and germinal centers (GCs). However, their roles beyond antigen presentation have been incompletely defined. In this study, we tested the impact of selective FDC ablation on short-term follicle and GC function. Within 2 d of FDC ablation, primary follicles lost their homogeneity and became disorganized bands of cells around T zones. These B cell areas retained CXCL13-expressing stromal cells but often exhibited inappropriate ER-TR7 and CCL21 expression. Ablation of GC FDCs led to the disappearance of GCs. When B cell death was prevented using a Bcl2 transgene, FDC ablation led to splenic GC B cell dispersal. Mesenteric lymph node GCs were more resistant but became dispersed when sphingosine-1-phosphate receptor-2 was also removed. These experiments indicate that FDCs help maintain primary follicles as a B cell exclusive niche and define a critical role for FDCs in cell retention within GCs.

Thymus seeding by T-lymphoid progenitor cells is a prerequisite for T-cell development. However, molecules guiding thymus colonization and their roles before and after thymus vascularization are unclear. Here we show that mice doubly deficient for chemokine receptors CCR7 and CCR9 were defective specifically in fetal thymus colonization before, but not after, thymus vascularization. The defective prevascular fetal thymus colonization was followed by selective loss of the first wave of T-cell development generating epidermal Vgamma3(+) gammadelta T cells. Unexpectedly, CCL21, a CCR7 ligand, was expressed not by Foxn1-dependent thymic primordium but by Gcm2-dependent parathyroid primordium, whereas CCL25, a CCR9 ligand, was predominantly expressed by Foxn1-dependent thymic primordium, revealing the role of the adjacent parathyroid in guiding fetal thymus colonization. These results indicate coordination between Gcm2-dependent parathyroid and Foxn1-dependent thymic primordia in establishing CCL21/CCR7- and CCL25/CCR9-mediated chemokine guidance essential for prevascular fetal thymus colonization.

BACKGROUND

The chemokines and cytokines CXCL13, CXCL12, CCL19, CCL21, BAFF and APRIL are believed to play a role in the recruitment of B cells to the central nervous system (CNS) compartment during neuroinflammation. To determine which chemokines/cytokines show the strongest association with a humoral immune response in the cerebrospinal fluid (CSF), we measured their concentrations in the CSF and correlated them with immune cell subsets and antibody levels.

METHODS

Cytokine/chemokine concentrations were measured in CSF and serum by ELISA in patients with non-inflammatory neurological diseases (NIND, n = 20), clinically isolated syndrome (CIS, n = 30), multiple sclerosis (MS, n = 20), Lyme neuroborreliosis (LNB, n = 8) and patients with other inflammatory neurological diseases (OIND, n = 30). Albumin, IgG, IgA and IgM were measured by nephelometry. CSF immune cell subsets were determined by seven-color flow cytometry.

RESULTS

CXCL13 was significantly elevated in the CSF of all patient groups with inflammatory diseases. BAFF levels were significantly increased in patients with LNB and OIND. CXCL12 was significantly elevated in patients with LNB. B cells and plasmablasts were significantly elevated in the CSF of all patients with inflammatory diseases. CXCL13 showed the most consistent correlation with CSF B cells, plasmablasts and intrathecal Ig synthesis.

CONCLUSIONS

CXCL13 seems to be the major determinant for B cell recruitment to the CNS compartment in different neuroinflammatory diseases. Thus, elevated CSF CXCL13 levels rather reflect a strong humoral immune response in the CNS compartment than being specific for a particular disease entity.