The co-presence of histoincompatible fetal and maternal cells is a characteristic of human placental inflammation. Villitis of unknown etiology (VUE), a destructive inflammatory lesion of villous placenta, is characterized by participation of Hofbauer cells (placental macrophages) and maternal T cells. In contrast to acute chorioamnionitis of infection-related origin, the fundamental immunopathology of VUE is unknown. This study was performed to investigate the placental transcriptome of VUE and to determine whether VUE is associated with systemic maternal and/or fetal inflammatory response(s). Comparison of the transcriptome between term placentas without and with VUE revealed differential expression of 206 genes associated with pathways related to immune response. The mRNA expression of a subset of chemokines and their receptors (CXCL9, CXCL10, CXCL11, CXCL13, CCL4, CCL5, CXCR3, CCR5) was higher in VUE placentas than in normal placentas (p < 0.05). Analysis of blood cell mRNA showed a higher expression of CXCL9 and CXCL13 in the mother, and CXCL11 and CXCL13 in the fetus of VUE cases (p < 0.05). The median concentrations of CXCL9, CXCL10, and CXCL11 in maternal and fetal plasma were higher in VUE (p < 0.05). Comparison of preterm cases without and with acute chorioamnionitis revealed elevated CXCL9, CXCL10, CXCL11, and CXCL13 concentrations in fetal plasma (p < 0.05), but not in maternal plasma with chorioamnionitis. We report for the first time the placental transcriptome of VUE. A systemic derangement of CXC chemokines in maternal and fetal circulation distinguishes VUE from acute chorioamnionitis. We propose that VUE be a unique state combining maternal allograft rejection and maternal antifetal graft-vs-host disease mechanisms.

Ibrutinib (PCI-32765) is a highly potent oral Bruton tyrosine kinase (BTK) inhibitor in clinical development for treating B-cell lymphoproliferative diseases. Patients with chronic lymphocytic leukemia (CLL) often show marked, transient increases of circulating CLL cells following ibrutinib treatments, as seen with other inhibitors of the B-cell receptor (BCR) pathway. In a phase 1 study of ibrutinib, we noted similar effects in patients with mantle cell lymphoma (MCL). Here, we characterize the patterns and phenotypes of cells mobilized among patients with MCL and further investigate the mechanism of this effect. Peripheral blood CD19(+)CD5(+) cells from MCL patients were found to have significant reduction in the expression of CXCR4, CD38, and Ki67 after 7 days of treatment. In addition, plasma chemokines such as CCL22, CCL4, and CXCL13 were reduced 40% to 60% after treatment. Mechanistically, ibrutinib inhibited BCR- and chemokine-mediated adhesion and chemotaxis of MCL cell lines and dose-dependently inhibited BCR, stromal cell, and CXCL12/CXCL13 stimulations of pBTK, pPLCγ2, pERK, or pAKT. Importantly, ibrutinib inhibited migration of MCL cells beneath stromal cells in coculture. We propose that BTK is essential for the homing of MCL cells into lymphoid tissues, and its inhibition results in an egress of malignant cells into peripheral blood. This trial was registered at www.clinicaltrials.gov as #NCT00114738.

Human Vgamma9/Vdelta2 T cells comprise a small population of peripheral blood T cells that in many infectious diseases respond to the microbial metabolite, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), expanding to up to 50% of CD3(+) cells. This "transitional response," occurring temporally between the rapid innate and slower adaptive response, is widely viewed as proinflammatory and/or cytolytic. However, increasing evidence that different cytokines drive widely different effector functions in alphabeta T cells provoked us to apply cDNA microarrays to explore the potential pleiotropy of HMB-PP-activated Vgamma9/Vdelta2 T cells. The data and accompanying validations show that the related cytokines, IL-2, IL-4, or IL-21, each drive proliferation and comparable CD69 up-regulation but induce distinct effector responses that differ from prototypic alphabeta T cell responses. For example, the Th1-like response to IL-2 also includes expression of IL-5 and IL-13 that conversely are not induced by IL-4. The data identify specific molecules that may mediate gammadelta T cell effects. Thus, IL-21 induces a lymphoid-homing phenotype and high, unexpected expression of the follicular B cell-attracting chemokine CXCL13/BCA-1, suggesting a novel follicular B-helper-like T cell that may play a hitherto underappreciated role in humoral immunity early in infection. Such broad plasticity emphasizes the capacity of gammadelta T cells to influence the nature of the immune response to different challenges and has implications for the ongoing clinical application of cytokines together with Vgamma9/Vdelta2 TCR agonists.

The presence of tumor-infiltrating lymphocytes (TILs) is a strong prognostic parameter for local dissemination and overall survival in melanoma. Lymphocyte migration from blood into peripheral tissues is mainly regulated by vascular endothelium. However, the blood vessels and mechanisms governing the recruitment of TILs in melanoma tumors remain poorly understood. Here, we show that high endothelial venules (HEVs), specialized blood vessels for lymphocyte extravasation into lymphoid tissues, are frequently found in melanoma tumors and are associated with high levels of lymphocyte infiltration. The analysis of 225 primary melanomas revealed that lymphocytes specifically infiltrated HEV-rich areas of melanoma tumors and that the density of MECA-79+ HEVs was variable among patients and strongly correlated with CD3+, CD8+ and CD20+ TIL densities. Inflammatory (CCL5, CXCL9, CXCL10 and CXCL11) and lymphoid (CCL21, CCL19 and CXCL13) chemokines as well as TH1 and naïve T-cell genes were overexpressed in melanoma samples with high densities of tumor HEVs. Mature dendritic cells (mDCs) were frequently found around tumor HEVs and densities of HEVs and DC-LAMP+ mDCs within tumor stroma were strongly correlated. DCs which maintain HEVs in lymph nodes, may thus also contribute to the regulation of HEVs in melanomas. Finally, we found significantly higher densities of tumor HEVs in melanomas with tumor regression, low Clark level of invasion and thin Breslow thickness (all p < 0.001). The strong association between tumor HEVs, TILs, mDCs and clinical parameters of melanoma, supports a critical role for HEVs in limiting malignant melanoma development through both naïve and effector T-lymphocyte recruitment and activation.

Mycobacterium tuberculosis infection (Mtb) results in the generation of protective cellular immunity and formation of granulomatous structures in the lung. CXCL13, CCL21, and CCL19 are constitutively expressed in the secondary lymphoid organs and play a dominant role in the homing of lymphocytes and dendritic cells. Although it is known that dendritic cell transport of Mtb from the lung to the draining lymph node is dependent on CCL19/CCL21, we show in this study that CCL19/CCL21 is also important for the accumulation of Ag-specific IFN-gamma-producing T cells in the lung, development of the granuloma, and control of mycobacteria. Importantly, we also show that CXCL13 is not required for generation of IFN-gamma responses, but is essential for the spatial arrangement of lymphocytes within granulomas, optimal activation of phagocytes, and subsequent control of mycobacterial growth. Furthermore, we show that these chemokines are also induced in the lung during the early immune responses following pulmonary Mtb infection. These results demonstrate that homeostatic chemokines perform distinct functions that cooperate to mediate effective expression of immunity against Mtb infection.

Lymphoid neogenesis is associated with antibody-mediated autoimmune diseases such as Sjogren's syndrome and rheumatoid arthritis. Although systemic lupus erythematosus is the prototypical B-cell-mediated autoimmune disease, the role of lymphoid neogenesis in its pathogenesis is unknown. Intraperitoneal injection of 2,6,10,14-tetramethyl-pentadecane (TMPD, pristane) or mineral oil causes lipogranuloma formation in mice, but only TMPD-treated mice develop lupus. We report that lipogranulomas are a form of lymphoid neogenesis. Immunoperoxidase staining of lipogranulomas revealed B cells, CD4(+) T cells, and dendritic cells and in some cases organization into T- and B-cell zones. Lipogranulomas also expressed the lymphoid chemokines CCL21, CCL19, CXCL13, CXCL12, and CCL22. Expression of the type I interferon (IFN-I)-inducible genes Mx1, IRF7, IP-10, and ISG-15 was greatly increased in TMPD- versus mineral oil-induced lipogranulomas. Dendritic cells from TMPD lipogranulomas underwent activation/maturation with high CD86 and interleukin-12 expression. Magnetic bead depletion of dendritic cells markedly diminished IFN-inducible gene (Mx1) expression. We conclude that TMPD-induced lupus is associated with the formation of ectopic lymphoid tissue containing activated dendritic cells producing IFN-I and interleukin-12. In view of the increased IFN-I production in systemic lupus erythematosus, these studies suggest that IFN-I from ectopic lymphoid tissue could play a role in the pathogenesis of experimental lupus in mice.

Glycosaminoglycans (GAGs), such as heparin or heparan sulfate, are required for the in vivo function of chemokines. Chemokines play a crucial role in the recruitment of leukocyte subsets to sites of inflammation and lymphocytes trafficking. GAG-chemokine interactions mediate cell migration and determine which leukocyte subsets enter tissues. Identifying the exact GAC sequences that bind to particular chemokines is key to understand chemokine function at the molecular level and develop strategies to interfere with chemokine-mediated processes. Here, we characterize the heparin binding profiles of eight chemokines (CCL21, IL-8, CXCL12, CXCL13, CCL19, CCL25, CCL28, and CXCL16) by employing heparin microarrays containing a small library of synthetic heparin oligosaccharides. The chemokines differ significantly in their interactions with heparin oligosaccharides: While some chemokines, (e.g., CCL21) strongly bind to a hexasaccharide containing the GlcNSO3(6-OSO3)-IdoA(2-OSO3) repeating unit, CCL19 does not bind and CXCL12 binds only weakly. The carbohydrate microarray binding results were validated by surface plasmon resonance experiments. In vitro chemotaxis assays revealed that dendrimers coated with the fully sulfated heparin hexasaccharide inhibit lymphocyte migration toward CCL21. Migration toward CXCL12 or CCL19 was not affected. These in vitro homing assays indicate that multivalent synthetic heparin dendrimers inhibit the migration of lymphocytes toward certain chemokine gradients by blocking the formation of a chemokine concentration gradient on GAG endothelial chains. These findings are in agreement with preliminary in vivo measurements of circulating lymphocytes. The results presented here contribute to the understanding of GAG-chemokine interactions, a first step toward the design of novel drugs that modulate chemokine activity.

Brain abscesses represent a significant medical problem despite recent advances made in detection and therapy. Using an established Staphylococcus aureus-induced brain abscess model, we have sought to define the functional importance of interleukin-1 (IL-1), tumor necrosis factor-alpha (TNF-alpha), and IL-6 in the host anti-bacterial immune response using cytokine gene knockout (KO) mice. Previous studies from our laboratory revealed that these cytokines are among the main proinflammatory mediators produced during the acute stage of brain abscess development. The results presented here demonstrate that although they share many redundant activities, IL-1 and TNF-alpha are important for containing bacterial infection in evolving brain abscesses as evident by increased mortality and bacterial burdens in IL-1 and TNF-alpha KO mice compared to wild type (WT) animals. In contrast, IL-6 was not found to be a major contributor to the host anti-bacterial immune response. Microarray analysis was used to evaluate the downstream consequences originating from the lack of IL-1 on subsequent proinflammatory mediator expression in brain abscesses from IL-1 KO and WT animals. Although numerous genes were significantly induced following S. aureus infection, only IL-1beta and 2 chemokines, CCL9 (macrophage inflammatory protein-1 gamma/MIP-1gamma) and CXCL13 (B lymphocyte chemoattractant/BLC), were differentially regulated in IL-1 KO versus WT animals. These results suggest that IL-1 and TNF-alpha play a pivotal role during the acute stage of brain abscess development through regulating the ensuing anti-bacterial inflammatory response.

OBJECTIVE

Chronic inflammation and B-cell hyperactivation are seen in HIV infection, contributing to an increased risk for the accrual of genetic errors that may result in B-cell lymphoma. The primary objective of this study was to determine the effect of highly active antiretroviral therapy (HAART) on serum levels of molecules that are associated with immune activation and/or inflammation, including several that are associated with B-cell activation, specifically IL-6, sCD30, sCD27, IgG, IgA, CXCL13 (B lymphocyte chemoattractant, BLC), a B-lymphocyte chemokine involved in B-cell trafficking, as well as C-reactive protein, an acute-phase protein.

METHODS

We used a retrospective cohort study design, measuring serum levels of these markers at each of four 1-year intervals, 2 years before and 2 years after HAART initiation, in a subgroup of 290 HIV-infected men enrolled in the Multicenter AIDS Cohort Study (MACS).

METHODS

Serum levels of immune activation-associated molecules were measured by ELISA and multiplexed immunometric assays. Reference values were determined by the 5th to 95th percentiles from a sample of 109 HIV-uninfected MACS men.

RESULTS

HAART use was associated with a reduction, but not normalization, of most biomarkers tested. Serum levels of IL-6 and C-reactive protein appeared to be unaffected by HAART.

CONCLUSIONS

These results suggest a partial normalization of serum cytokine levels post HAART. However, a chronic state of B-cell hyperactivation continues 2-3 years after HAART initiation. These findings may explain, in part, the excess incidence of lymphoma still occurring in HIV-infected persons in the post-HAART era.

B-1 and marginal zone (MZ) B cells produce natural Abs, make Ab responses to microbial pathogens, and contribute to autoimmunity. Although the delta isoform of the PI3K p110 catalytic subunit is essential for development of these innate-like B cells, its role in the localization, activation, and function of normal B-1 and MZ B cells is not known. Using IC87114, a highly selective inhibitor of p110delta enzymatic activity, we show that p110delta is important for murine B-1 and MZ B cells to respond to BCR clustering, the TLR ligands LPS and CpG DNA, and the chemoattractants CXCL13 and sphingosine 1-phosphate. In these innate-like B cells, p110delta activity mediates BCR-, TLR- and chemoattractant-induced activation of the Akt prosurvival kinase, chemoattractant-induced migration, and TLR-induced proliferation. Moreover, we found that TLR-stimulated Ab responses by B-1 and MZ B cells, as well as the localization of MZ B cells in the spleen, depend on p110delta activity. Finally, we show that the in vivo production of natural Abs requires p110delta and that p110delta inhibitors can reduce in vivo autoantibody responses. Thus, targeting p110delta may be a novel approach for regulating innate-like B cells and for treating Ab-mediated autoimmune diseases.

Ectopic or tertiary lymphoid tissues (TLTs) are often induced at sites of chronic inflammation. They typically contain various hematopoietic cell types, high endothelial venules, and follicular dendritic cells; and are organized in lymph node-like structures. Although fibroblastic stromal cells may play a role in TLT induction and persistence, they have remained poorly defined. Herein, we report that TLTs arising during inflammation in mice and humans in a variety of tissues (eg, pancreas, kidney, liver, and salivary gland) contain stromal cell networks consisting of podoplanin(+) T-zone fibroblastic reticular cells (TRCs), distinct from follicular dendritic cells. Similar to lymph nodes, TRCs were present throughout T-cell-rich areas and had dendritic cells associated with them. They expressed lymphotoxin (LT) β receptor (LTβR), produced CCL21, and formed a functional conduit system. In rat insulin promoter-CXCL13-transgenic pancreas, the maintenance of TRC networks and conduits was partially dependent on LTβR and on lymphoid tissue inducer cells expressing LTβR ligands. In conclusion, TRCs and conduits are hallmarks of secondary lymphoid organs and of well-developed TLTs, in both mice and humans, and are likely to act as important scaffold and organizer cells of the T-cell-rich zone.

Inducible bronchus-associated lymphoid tissue (iBALT) is an organized tertiary lymphoid structure that is not pre-programmed but develops in response to infection or under chronic inflammatory conditions. Emerging research has shown that iBALT provides a niche for T-cell priming and B-cell education to assist in the clearance of infectious agents, highlighting the prospect that iBALT may be engineered and harnessed to enhance protective immunity against respiratory pathogens. Although iBALT formation is associated with several canonical factors of secondary lymphoid organogenesis such as lymphotoxin-α and the homeostatic chemokines, CXCL13, CCL19, and CCL21, these cytokines are not mandatory for its formation, even though they influence its organization and function. Similarly, lymphoid tissue-inducer cells are not a requisite of iBALT formation. In contrast, dendritic cells are emerging as pivotal players required to form and sustain the presence of iBALT. Regulatory T cells appear to be able to attenuate the development of iBALT, although the underlying mechanisms remain ill-defined. In this review, we discuss facets unique to iBALT induction, the cellular subsets, and molecular cues that govern this process, and the contribution of this ectopic structure toward the generation of immune responses in the pulmonary compartment.

BACKGROUND

There is increasing recognition of the importance of B lymphocytes in the immunopathogenesis of multiple sclerosis (MS), encouraging the evaluation of B cell-associated biomarkers in the cerebrospinal fluid (CSF). We aimed to evaluate the relevance of the B cell chemoattractant CXCL13 as a prognostic marker in patients with clinically isolated syndrome (CIS) regarding conversion to MS, and to compare it to Barkhof criteria in magnetic resonance imaging (MRI), oligoclonal bands (OCB) and the polyspecific intrathecal B cell response against measles, rubella and varicella zoster virus (MRZR).

RESULTS

CXCL13 was determined in a prospective study over 2 years including 46 patients that remained CIS over follow-up (CIS-CIS), 45 patients that developed MS (CIS-RRMS), and 30 controls using ELISA. CSF CXCL13 was significantly elevated in CIS-RRMS as compared to CIS-CIS and controls (p<0.001). It was significantly elevated in CIS with OCB (p<0.001), positive MRZR (p=0.04), and gadolinium enhancement in MRI (p=0.02) and showed a significant correlation with CSF leukocyte count (p<0.001) and QIgG (p<0.001). CXCL13 showed the best positive predictive value (PPV) of all parameters investigated (70%, 95%-CI: 53-84%), which could be further increased by combination with Barkhof criteria in MRI (80%).

CONCLUSIONS

Our data indicate the relevance of CXCL13 in CIS to predict conversion to MS. It furthermore shows CXCL13 to be an important mediator in the inflammatory cascade associated with the polyspecific intrathecal B cell response that manifests itself in OCB and MRZR.

Establishing the diagnosis of focal brain lesions in patients with unexplained neurologic symptoms represents a challenge. The goal of this study is to provide evidence supporting functional roles for CXC chemokine ligand (CXCL)13 and interleukin (IL)-10 in central nervous system (CNS) lymphomas and to evaluate the utility of each as prognostic and diagnostic biomarkers. We demonstrate for the first time that elevated CXCL13 concentration in cerebrospinal fluid (CSF) is prognostic and that CXCL13 and CXCL12 mediate chemotaxis of lymphoma cells isolated from CNS lymphoma lesions. Expression of the activated form of Janus kinase 1 supported a role for IL-10 in prosurvival signaling. We determined the concentration of CXCL13 and IL-10 in CSF of CNS lymphoma patients and control cohorts including inflammatory and degenerative neurologic disease in a multicenter study involving 220 patients. Bivariate elevated CXCL13 plus IL-10 was 99.3% specific for primary and secondary CNS lymphoma, with sensitivity significantly greater than reference standard CSF tests. These results identify CXCL13 and IL-10 as potentially important biomarkers of CNS lymphoma that merit further evaluation and support incorporation of CXCL13 and IL-10 into diagnostic algorithms for the workup of focal brain lesions in which lymphoma is a consideration.

CD4+CD3- cells are the predominant hematopoietic cells found in mouse fetal intestine. We prove their role as Peyer's patch (PP)-inducing cells by transfer into neonatal PP-deficient mice. To test the requirement of chemokines and adhesion molecules in induction of PP, we studied mice deficient in CXCR5 and/or alpha4beta1 integrin-mediated adhesion. CXCR5-/- mice have CD4+CD3- cells, which are inefficient in inducing PP formation. We show here that CXCR5/CXCL13 signaling activates alpha4beta1 integrin on CD4+CD3- cells. Blocking of beta1 integrin or VCAM-1, the ligand of alpha4beta1 integrin, inhibits PP formation. This study demonstrates the link between chemokine receptors and adhesion molecules that regulates stromal/hematopoietic cell interaction leading to PP formation.

BACKGROUND

There is microscopic spatial and temporal heterogeneity of pathological changes in idiopathic pulmonary fibrosis (IPF) lung tissue, which may relate to heterogeneity in pathophysiological mediators of disease and clinical progression. We assessed relationships between gene expression patterns, pathological features, and systemic biomarkers to identify biomarkers that reflect the aggregate disease burden in patients with IPF.

METHODS

Gene expression microarrays (N=40 IPF; 8 controls) and immunohistochemical analyses (N=22 IPF; 8 controls) of lung biopsies. Clinical characterisation and blood biomarker levels of MMP3 and CXCL13 in a separate cohort of patients with IPF (N=80).

RESULTS

2940 genes were significantly differentially expressed between IPF and control samples (|fold change| >1.5, p<0.05). Two clusters of co-regulated genes related to bronchiolar epithelium or lymphoid aggregates exhibited substantial heterogeneity within the IPF population. Gene expression in bronchiolar and lymphoid clusters corresponded to the extent of bronchiolisation and lymphoid aggregates determined by immunohistochemistry in adjacent tissue sections. Elevated serum levels of MMP3, encoded in the bronchiolar cluster, and CXCL13, encoded in the lymphoid cluster, corresponded to disease severity and shortened survival time (p<10(-7) for MMP3 and p<10(-5) for CXCL13; Cox proportional hazards model).

CONCLUSIONS

Microscopic pathological heterogeneity in IPF lung tissue corresponds to specific gene expression patterns related to bronchiolisation and lymphoid aggregates. MMP3 and CXCL13 are systemic biomarkers that reflect the aggregate burden of these pathological features across total lung tissue. These biomarkers may have clinical utility as prognostic and/or surrogate biomarkers of disease activity in interventional studies in IPF.

Lyme neuroborreliosis, caused by the spirochete Borrelia burgdorferi, often manifests by causing neurocognitive deficits. As a possible mechanism for Lyme neuroborreliosis, we hypothesized that B. burgdorferi induces the production of inflammatory mediators in the central nervous system with concomitant neuronal and/or glial apoptosis. To test our hypothesis, we constructed an ex vivo model that consisted of freshly collected slices from brain cortex of a rhesus macaque and allowed live B. burgdorferi to penetrate the tissue. Numerous transcripts of genes that regulate inflammation as well as oligodendrocyte and neuronal apoptosis were significantly altered as assessed by DNA microarray analysis. Transcription level increases of 7.43-fold (P = 0.005) for the cytokine tumor necrosis factor-alpha and 2.31-fold (P = 0.016) for the chemokine interleukin (IL)-8 were also detected by real-time-polymerase chain reaction array analysis. The immune mediators IL-6, IL-8, IL-1beta, COX-2, and CXCL13 were visualized in glial cells in situ by immunofluorescence staining and confocal microscopy. Concomitantly, significant proportions of both oligodendrocytes and neurons undergoing apoptosis were present in spirochete-stimulated tissues. IL-6 production by astrocytes in addition to oligodendrocyte apoptosis were also detected, albeit at lower levels, in rhesus macaques that had received in vivo intraparenchymal stereotaxic inoculations of live B. burgdorferi. These results provide proof of concept for our hypothesis that B. burgdorferi produces inflammatory mediators in the central nervous system, accompanied by glial and neuronal apoptosis.

Mouse bone marrow-derived macrophages (BMDM) grown in M-CSF (CSF-1) have been used widely in studies of macrophage biology and the response to TLR agonists. We investigated whether similar cells could be derived from the domestic pig using human rCSF-1 and whether porcine macrophages might represent a better model of human macrophage biology. Cultivation of pig bone marrow cells for 5-7 d in presence of human rCSF-1 generated a pure population of BMDM that expressed the usual macrophage markers (CD14, CD16, and CD172a), were potent phagocytic cells, and produced TNF in response to LPS. Pig BMDM could be generated from bone marrow cells that had been stored frozen and thawed so that multiple experiments can be performed on samples from a single animal. Gene expression in pig BMDM from outbred animals responding to LPS was profiled using Affymetrix microarrays. The temporal cascade of inducible and repressible genes more closely resembled the known responses of human than mouse macrophages, sharing with humans the regulation of genes involved in tryptophan metabolism (IDO, KYN), lymphoattractant chemokines (CCL20, CXCL9, CXCL11, CXCL13), and the vitamin D3-converting enzyme, Cyp27B1. Conversely, in common with published studies of human macrophages, pig BMDM did not strongly induce genes involved in arginine metabolism, nor did they produce NO. These results establish pig BMDM as an alternative tractable model for the study of macrophage transcriptional control.

Crohn's disease (CD) and ulcerative colitis (UC), two forms of inflammatory bowel disease (IBD), are chronic, relapsing, and tissue destructive lesions that are accompanied by the uncontrolled activation of effector immune cells in the mucosa. Recent estimates indicate that there are 1.3 million annual cases of IBD in the United States, 50% of which consists of CD and 50% of UC. Chemokines and cytokines play a pivotal role in the regulation of mucosal inflammation by promoting leukocyte migration to sites of inflammation ultimately leading to tissue damage and destruction. In recent years, experimental studies in rodents have led to a better understanding of the role played by these inflammatory mediators in the development and progression of colitis. However, the clinical literature on IBD remains limited. Therefore, the aim of this study was to evaluate systemic concentrations of key chemokines and cytokines in forty-two IBD patients with a range of disease activity compared to levels found in ten healthy donors. We found a significant increase in an array of chemokines including macrophage migration factor (MIF), CCL25, CCL23, CXCL5, CXCL13, CXCL10, CXCL11, MCP1, and CCL21 in IBD patients as compared to normal healthy donors (P<0.05). Further, we also report increases in the inflammatory cytokines IL-16, IFN-γ, IL-1β and TNF-α in IBD patients when compared to healthy donors (P<0.05). These data clearly indicate an increase in circulating levels of specific chemokines and cytokines that are known to modulate systemic level through immune cells results in affecting local intestinal inflammation and tissue damage in IBD patients. Blockade of these inflammatory mediators should be explored as a mechanism to alleviate or even reverse symptoms of IBD.

Homeostatic chemokines participate in the development of secondary lymphoid organs and later on in the functional organization of these tissues. The development of lymph nodes (LNs) and Peyer's patches depends on the recruitment of CD3- CD4+ interleukin (IL)-7R alpha hi cells to sites of future organ development. CD3- CD4+ IL-7R alpha hi cells express the chemokine receptor CXCR5 and might be attracted by its ligand CXCL13, which is secreted by mesenchymal cells. Mesenchymal cells also secrete CCL19, a ligand for CCR7, yet it is not clear whether CCR7 and CCL19 are important for secondary lymphoid organ development. Analyzing CXCR5-/- CCR7-/- double deficient mice we now show that these mice lack all examined peripheral LNs suggesting a profound role for both receptors in secondary lymphoid organ development. We demonstrate that CD3- CD4+ IL-7R alpha hi cells express CXCR5 as well as CCR7 indicating that both receptors cooperate during an early step of secondary lymphoid organ development. Furthermore, CXCR5-/- CCR7-/- mice display a severely disturbed architecture of mesenteric LN and spleen. Due to an impaired migration of B cells into the white pulp, CXCR5-/- CCR7-/- mice fail to develop B cell follicles but show small clusters of unorganized lymphocytes in the spleen. These data demonstrate a cooperative function of CXCR5 and CCR7 in lymphoid organ organogenesis and organization.

Normal lymphoid tissue development and function depend upon directed cell migration. Providing guideposts for cell movement and positioning within lymphoid tissues, chemokines signal through cell surface receptors that couple to heterotrimeric G proteins, which are in turn subject to regulation by regulator of G protein signaling (RGS) proteins. In this study, we report that germinal center B lymphocytes and thymic epithelial cells strongly express one of the RGS family members, RGS13. Located between Rgs1 and Rgs2, Rgs13 spans 42 kb on mouse chromosome 1. Rgs13 encodes a 157-aa protein that shares 82% amino acid identity with its 159-aa human counterpart. In situ hybridization with sense and antisense probes localized Rgs13 expression to the germinal center regions of mouse spleens and Peyer's patches and to the thymus medulla. Affinity-purified RGS13 Abs detected RGS13-expressing cells in the light zone of the germinal center. RGS13 interacted with both Gialpha and Gqalpha and strongly impaired signaling through G(i)-linked signaling pathways, including signaling through the chemokine receptors CXCR4 and CXCR5. Prolonged CD40 signaling up-regulated RGS13 expression in human tonsil B lymphocytes. These results plus previous studies of RGS1 indicate the germinal center B cells use two RGS proteins, RGS1 and RGS13, to regulate their responsiveness to chemokines.

Primary Sjögren's syndrome (pSS) is an autoimmune disorder characterized by specific pathological features. A hallmark of pSS is B-cell hyperactivity as manifested by the production of autoantibodies, hypergammaglobulinemia, formation of ectopic lymphoid structures within the inflamed tissues, and enhanced risk of B-cell lymphoma. Changes in the distribution of peripheral B-cell subsets and differences in post-recombination processes of immunoglobulin variable region (IgV) gene usage are also characteristic features of pSS. Comparison of B cells from the peripheral blood and salivary glands of patients with pSS with regard to their expression of the chemokine receptors CXCR4 and CXCR5, and their migratory capacity towards the corresponding ligands, CXCL12 and CXCL13, provide a mechanism for the prominent accumulation of CXCR4+ CXCR5+ memory B cells in the inflamed glands. Glandular B cells expressing distinct features of IgV light and heavy chain rearrangements, (re)circulating B cells with increased mutations of cmu transcripts in both CD27- and CD27+ memory B-cell subsets, and enhanced frequencies of individual peripheral B cells containing IgV heavy chain transcripts of multiple isotypes indicate disordered selection and incomplete differentiation processes of B cells in the inflamed tissues in pSS. This may possibly be related to a lack of appropriate censoring mechanisms or different B-cell activation pathways within the ectopic lymphoid structures of the inflamed tissues. These findings add to our understanding of the pathogenesis of this autoimmune inflammatory disorder and may result in new therapeutic approaches.

Using protein expression profiling, the authors identified an upregulation of the chemokine B lymphocyte chemoattractant (BLC) in the CSF of patients with neuroborreliosis but not in patients with noninflammatory and various other inflammatory neurologic diseases. This upregulation was confirmed by ELISA, showing increased BLC levels in every neuroborreliosis patient while being undetectable in patients with noninflammatory neurologic diseases. These results point to BLC as a putative additional diagnostic marker for neuroborreliosis.

Circulation of mature lymphocytes between blood and secondary lymphoid tissues plays a central role in the immune system. Homing of lymphocytes from blood into secondary lymphoid tissues beyond high endothelial venules is highly dependent on the interaction between the chemokines CCL19, CCL21, CXCL12, and CXCL13, and their receptors CCR7, CXCR4 and CXCR5. However, the molecular mechanism(s) of lymphocyte egress from secondary lymphoid tissues to lymph remained unclear. We have found a new class of immunomodulator, FTY720 by chemical modification of vegetative wasp-derived natural product, ISP-I (myriocin). FTY720 has been shown to be highly effective in experimental allograft and autoimmune disease models. A striking feature of FTY720 is the induction of a marked decrease in peripheral blood lymphocytes at doses that show immunomodulating activity in these models. The reduction of circulating lymphocytes by FTY720 is caused by sequestration of lymphocytes into secondary lymphoid tissues and thymus. FTY720 is rapidly converted to (S)-enantiomer of FTY720-phosphate [(S)-FTY720-P] by sphingosine kinase 2 in vivo. (S)-FTY720-P acting as a potent agonist of S1P receptor type 1 (S1P1), induces long-term down-regulation of S1P1 on lymphocytes, and thereby inhibits the migration of lymphocytes toward S1P. Thus, it is presumed that FTY720-induced lymphocyte sequestration is due to the inhibition of S1P/S1P1-dependent lymphocyte egress from secondary lymphoid tissues and thymus by its active metabolite (S)-FTY720-P. Throughout the analysis of the mechanism of action of FTY720, it is clarified that S1P/S1P1 interaction plays an important role for lymphocyte egress from secondary lymphoid tissues and thymus.