Primary central nervous system lymphoma (PCNSL) is a rare but often rapidly fatal form of non-Hodgkin B-cell lymphoma that arises within the central nervous system (CNS) and has a low propensity to metastasize. We performed immunohistochemistry on formalin-fixed, paraffin-embedded brain biopsy specimens from 24 patients with PCNSL to investigate the expression of B cell-attracting chemokine 1 (BCA-1, CXCL13), a lymphoid chemokine involved in B-cell compartmental homing within secondary lymphoid organs and recently implicated in the pathogenesis of inflammatory and malignant lymphocyte-mediated diseases. Whereas BCA-1 was not detected in normal human brain, all 24 brain biopsy specimens containing PCNSL were positive for BCA-1. Double immunostaining on selected specimens localized BCA-1 to malignant B lymphocytes and vascular endothelium. In contrast, 2 chemokines implicated particularly in T-cell movement, secondary lymphoid tissue chemokine (SLC, CCL21) and Epstein-Barr virus-induced molecule 1 ligand chemokine (ELC, CCL19), were expressed only by occasional stromal cells in 2 and 4 of the 24 specimens, respectively. Tumor cells stained positively for CXCR5, the primary receptor for BCA-1. In situ hybridization verified the expression of BCA-1 mRNA by malignant B cells, but not vascular endothelium, within the tumor mass, suggesting that vascular endothelial BCA-1 expression may be consequent to transcytosis. In PCNSL, expression of BCA-1 by malignant lymphocytes and vascular endothelium may influence tumor development and localization to CNS.

BACKGROUND

B lymphocyte chemoattractant (BLC/CXCL13), a CXC chemokine, is involved in B1 and B2 cell trafficking for the activation of autoreactive T helper (Th) cells and autoantibody production in target organs during the development of lupus. CXCL13 can induce the trafficking of CXCR5+ T lymphocyte subset designated as follicular helper T lymphocytes (T(FH)) which are specifically involved in autoantibody production.

METHODS

We herein measured the plasma concentrations of CXCL13, B-cell-activating factor of the TNF family (BAFF), and T(FH)-cells-related cytokine IL-21 and cell surface expression of T(FH)-related receptor CXCR5 and IL-21R on CD4+Th and CD19+B cells in 35 systemic lupus erythematosus (SLE) patients and 23 sex- and age-matched control subjects (NC) using enzyme-linked immunosorbent assay and flow cytometry, respectively.

CONCLUSIONS

Plasma CXCL13, BAFF, and IL-21 concentrations were significantly higher in SLE patients than NC group (all p < 0.0001). Increase in CXCL13 concentration correlated positively and significantly with SLEDAI score in SLE patients (r = 0.399, p = 0.032). Cell surface expression of CXCR5 on Th and B cells and IL-21R on B cells was however significantly lower in SLE patients than controls (both p < 0.01). It may indicate that most differentiated T(FH) cells migrate out from circulation into lymphoid organ upon activation during the disease development of SLE.

CONCLUSIONS

The above results suggest that the elevated production of CXCL13, BAFF, and IL-21 may be associated with the function of T(FH) for the immunopathogenesis in SLE, and CXCL13 may serve as a potential disease marker of SLE.

The roles of microglia and macrophages during neuroinflammation and neurodegenerative diseases remain controversial. To date, at least 2 activations states have been suggested, consisting of a classical response (M1) and the alternative response (M2). Identifying selective biomarkers of microglia that representative their functional activation states may help elucidate disease course and enable a better understanding of repair mechanisms. Two cocktails containing either tumor necrosis factor (TNF)-α, interleukin (IL)-12, and IL-1β (referred to as CKT-1) or IL-13 and IL-4 (referred to CKT-2) were injections into the hippocampus of mice aged 6, 12, or 24 months. Microarray analysis was performed on hippocampal tissue 3 days postinjection. Gene transcripts were compared between CKT-1 versus CKT-2 stimulator cocktails. Several selective transcripts expressed for the CKT-1 included CXCL13, haptoglobin, MARCO, and calgranulin B, whereas a smaller subset of genes was selectively induced by the CKT-2 and consisted of FIZZ1, IGF-1, and EAR 11. Importantly, selective transcripts were induced at all ages by CKT-1, whereas selective gene transcripts induced by CKT-2 decreased with age suggesting an age-related reduction in the IL-4/ IL-13 signaling pathway.

OBJECTIVE

In mice, the B lymphocyte chemoattractant (BLC) CXC chemokine ligand 13 (CXCL13) is sufficient to induce a series of events leading to the formation of organised lymphoid tissue. Its receptor, CXCR5, is required for normal development of secondary lymphoid tissue. However, the human counterpart, B cell attracting chemokine 1 (BCA-1) has only been detected in the stomach and appendix and not in other parts of normal or diseased gut. Hence to elucidate the potential role of this chemokine and its receptor in human gut associated lymphoid tissue (GALT), we analysed their expression in normal intestine and ulcerative colitis (UC).

METHODS

Frozen sections of surgical specimens were studied by multicolour immunofluorescence staining, in situ mRNA hybridisation, and reverse transcription-polymerase chain reaction.

RESULTS

BCA-1 mRNA was detected in all normal colonic and UC specimens. BCA-1 was produced and accumulated in relation to peripheral dendritic elements of lymphoid follicles in Peyer's patches and normal colon, as well as in irregular lymphoid aggregates in UC lesions. BCA-1 was partially associated with the traditional follicular dendritic cell phenotype but also with extracellular fibrils in GALT structures. CXCR5 protein was expressed by mantle zone B cells and appeared at a high level on scattered germinal centre T cells.

CONCLUSIONS

BCA-1 and CXCR5 are expressed in normal GALT structures as well as in irregular lymphoid aggregates in UC. This strongly suggests that BCA-1 plays an important role not only in the formation of normal GALT but also in the generation of aberrant lymphoid tissue in inflammatory bowel disease.

B-cell-activating factor of the TNF family, (BAFF), and a proliferation-inducing ligand (APRIL) regulate B-lymphocyte survival and activation. We report that BAFF, but not APRIL, increased the chemotactic response of primary human B cells to CCL21, CXCL12, and CXCL13. The BAFF-induced increase in B-cell chemotaxis was totally abolished by blockade of BAFF-R and was strongly dependent on the activation of PI3K/AKT, NF-kappaB, and p38MAPK pathways. BAFF had similar effects on the chemotaxis of naive and memory B cells in response to CCL21 but increased more strongly that of memory B cells to CXCL13 than that of naive B cells. Our findings indicate a previously unreported role for the BAFF/BAFF-R pair in mature B-cell chemotaxis. The synergy between CXCL13 and BAFF produced by stromal cells and follicular dendritic cells may have important implications for B-cell homeostasis, the development of normal B-cell areas, and for the formation of germinal center-like follicles that may be observed in various autoimmune diseases.

Ectopic lymphoid tissue, such as bronchus-associated lymphoid tissue (BALT) in the lung, develops spontaneously at sites of chronic inflammation or during infection. The molecular mechanisms underlying the neogenesis of such tertiary lymphoid tissue are still poorly understood. We show that the type of inflammation-inducing pathogen determines which key factors are required for the formation and maturation of BALT. Thus, a single intranasal administration of the poxvirus modified vaccinia virus Ankara (MVA) is sufficient to induce highly organized BALT with densely packed B cell follicles containing a network of CXCL13-expressing follicular DCs (FDCs), as well as CXCL12-producing follicular stromal cells. In contrast, mice treated with P. aeruginosa (P.a.) develop BALT but B cell follicles lack FDCs while still harboring CXCL12-positive follicular stromal cells. Furthermore, in IL-17-deficient mice, P.a.-induced BALT largely lacks B cells as well as CXCL12-expressing stromal cells, and only loose infiltrates of T cells are present. We show that Toll-like receptor pathways are required for BALT induction by P.a., but not MVA, and provide evidence that IL-17 drives the differentiation of lung stroma toward podoplanin-positive CXCL12-expressing cells that allow follicle formation even in the absence of FDCs. Taken together, our results identify distinct pathogen-dependent induction and maturation pathways for BALT formation.

Recent studies have implicated chemokines in microglial activation and pathogenesis of neuropathic pain. C-X-C motif chemokine 13 (CXCL13) is a B lymphocyte chemoattractant that activates CXCR5. Using the spinal nerve ligation (SNL) model of neuropathic pain, we found that CXCL13 was persistently upregulated in spinal cord neurons after SNL, resulting in spinal astrocyte activation via CXCR5 in mice. shRNA-mediated inhibition of CXCL13 in the spinal cord persistently attenuated SNL-induced neuropathic pain. Interestingly, CXCL13 expression was suppressed by miR-186-5p, a microRNA that colocalized with CXCL13 and was downregulated after SNL. Spinal overexpression of miR-186-5p decreased CXCL13 expression, alleviating neuropathic pain. Furthermore, SNL induced CXCR5 expression in spinal astrocytes, and neuropathic pain was abrogated in Cxcr5-/- mice. CXCR5 expression induced by SNL was required for the SNL-induced activation of spinal astrocytes and microglia. Intrathecal injection of CXCL13 was sufficient to induce pain hypersensitivity and astrocyte activation via CXCR5 and ERK. Finally, intrathecal injection of CXCL13-activated astrocytes induced mechanical allodynia in naive mice. Collectively, our findings reveal a neuronal/astrocytic interaction in the spinal cord by which neuronally produced CXCL13 activates astrocytes via CXCR5 to facilitate neuropathic pain. Thus, miR-186-5p and CXCL13/CXCR5-mediated astrocyte signaling may be suitable therapeutic targets for neuropathic pain.

We observed here that the expression of B lymphocyte chemokine (BLC/CXCL13) was markedly enhanced in the thymus and kidney in aged (NZB x NZW)F1 (BWF1) mice developing lupus nephritis, but not in similarly aged NZB and NZW mice. BLC-positive cells were present in the cellular infiltrates in the target organs with a reticular pattern of staining. CD11b+CD11c+ dendritic cells were increased in the thymus and spleen in aged BWF1 mice and identified as the major cell source for BLC. CD4+ T cells as well as B cells were dramatically increased in the thymus in aged BWF1 mice, whereas no increase was observed in aged NZB and NZW mice. B1/B2 ratio in the thymus was significantly higher than those in the spleen and peripheral blood in aged BWF1 mice. Interestingly, BLC showed preferential chemotactic activity for B1 cells derived from several mouse strains, including nonautoimmune mice. Cell surface CXCR5 expression on B1 cells was significantly higher than that on B2 cells. Thus, aberrant high expression of BLC by myeloid dendritic cells in the target organs in aged BWF1 mice may play a pivotal role in breaking immune tolerance in the thymus and in recruiting autoantibody-producing B cells in the development of murine lupus.

Although much has been learned recently of the mechanisms by which the differentiation of osteoclasts is induced, less is known of the factors that regulate their migration and localization, and their interactions with other bone cells. In related cell types, chemokines play a major role in these processes. We therefore systematically tested the expression of RNA for chemokines and their receptors by osteoclasts. Because bone is the natural substrate for osteoclasts and may influence osteoclast behavior, we also tested expression on bone slices. Quantitative RT-PCR using real-time analysis with SYBR Green was therefore performed on RNA isolated from bone marrow cells after incubation with macrophage-colony stimulating factor (M-CSF) with/without receptor-activator of NFkappaB ligand (RANKL), on plastic or bone. We found that RANKL induced expression of CCL9/MIP-1gamma to levels comparable to that of tartrate-resistant acid phosphatase (TRAP), a major specialized product of osteoclasts. CCL22/MDC, CXCL13/BLC/BCA-1, and CCL25/TECK were also induced. The dominant chemokine receptor expressed by osteoclasts was CCR1, followed by CCR3 and CX3CR1. Several receptors expressed on macrophages and associated with inflammatory responses, including CCR2 and CCR5, were down-regulated by RANKL. CCL9, which acts through CCR1, stimulated cytoplasmic motility and polarization in osteoclasts, identical to that previously observed in response to CCL3/MIP-1alpha, which also acts through CCR1 and is chemotactic for osteoclasts. These results identify CCL9 and its receptor CCR1 as the major chemokine and receptor species expressed by osteoclasts, and suggest a crucial role for CCL9 in the regulation of bone resorption.

Extranodal NK/T-cell lymphoma (ENKTL), nasal type, may be of NK or T-cell origin; however, the proportion of T-ENKTLs and whether they are of αβ or γδ type remains uncertain. To elucidate the cell of origin and detailed phenotype of ENKTL and assess any clinicopathologic associations, 67 cases of ENKTL from Thailand were investigated, together with 5 γδ enteropathy-associated T-cell lymphomas (EATLs) for comparison. In all, 70% of the ENKTL were T-cell receptor (TCR) β,γ and, in cases tested, δ negative (presumptive NK origin); 5% were TCR γδ, 3% were TCR αβ, 1% were TCR αβ/γδ, and 21% were indeterminate. Out of 17 presumptive NK-ENKTLs tested, 3 had clonal TCR rearrangements. All cases were EBV and TIA-1; >85% were positive for CD3, CD2, granzyme B, pSTAT3, and Lsk/MATK; and all were CD16. Presumptive NK-ENKTLs had significantly more frequent CD56 (83% vs. 33%) and CXCL13 (59% vs. 0%) but less frequent PD-1 (0% vs. 40%) compared with T-ENKTLs. Of the NK-ENKTLs, 38% were Oct-2 compared with 0% of T-ENKTLs, and 54% were IRF4/MUM1 compared with 20% of T-ENKTLs. Only αβ T-ENKTLs were CD5. Intestinal ENKTLs were EBV and had significantly more frequent CD30, pSTAT3, and IRF4/MUM1 expression but less frequent CD16 compared with γδ EATL. Significant adverse prognostic indicators included a primary non-upper aerodigestive tract site, high stage, bone marrow involvement, International Prognostic Index ≥2, lack of radiotherapy, Ki67 >40%, and CD25 expression. The upper aerodigestive tract ENKTLs of T-cell origin compared with those of presumptive NK origin showed a trend for better survival. Thus, at least 11% of evaluable ENKTLs are of T-cell origin. Although T-ENKTLs have phenotypic and some possible clinical differences, they share many similarities with ENKTLs that lack TCR expression and are distinct from intestinal γδ EATL.

Sjögren's syndrome is an autoimmune disease characterized by lymphocytic infiltrates resembling secondary lymphoid organs in salivary glands. In this study, we demonstrate the expression of the lymphoid tissue homing chemokine CXCL13 (BCA-1/BLC), which has attracting properties for B cells and subsets of activated T cells, in salivary glands of patients with Sjögren's syndrome using immunohistochemistry and in situ hybridization. CXCL13 expression was primarily observed in epithelial cells in acini and ducts of inflamed glands while its receptor, CXCR5 (BLR-1), was expressed on the infiltrating mononuclear cells. In addition, cells producing antibodies against one of the major autoantigens in Sjögren's syndrome, Ro 52, were identified at the periphery of the follicular infiltrates indicating that the ectopic lymphoid tissue is directly involved in the disease process. Identification of CXCL13 and CXCR5 in salivary glands suggests that the target organ plays an essential role in the inflammatory process by recruiting B and T cells. These results also provide a molecular mechanism by which lymphoid neogenesis and ectopic germinal centre formation might occur in the glands of these patients, which may be the key step in the development of the chronic inflammatory process in Sjögren's syndrome.

Myasthenia gravis (MG) is associated with ectopic germinal centers in the thymus. Thymectomy and glucocorticoids are the main treatments but they induce operative risks and side effects, respectively. The aim of this study was to propose new therapies more efficient for MG. We hypothesized that molecules dysregulated in MG thymus and normalized by glucocorticoids may play a key role in thymic pathogenesis. Using gene chip analysis, we identified 88 genes complying with these criteria, the most remarkable being the B-cell chemoattractant (CXCL13). Its expression was increased in thymus and sera of glucocorticoid-untreated patients and decreased in response to treatment in correlation with clinical improvement. Normal B cells were actively chemoattracted by thymic extracts from glucocorticoid-untreated patients, an effect inhibited by anti-CXCL13 antibodies. In the thymus, CXCL13 was preferentially produced by epithelial cells and overproduced by epithelial cells from MG patients. Altogether, our results suggest that a high CXCL13 production by epithelial cells could be responsible for germinal center formation in MG thymus. Furthermore, they show that this gene is a main target of corticotherapy. Thus, new therapies targeting CXCL13 could be of interest for MG and other autoimmune diseases characterized by ectopic germinal center formation.

The expression of distinct chemokines within the asthmatic lung suggests that specific regulatory mechanisms may mediate various stages of asthmatic disease. Global transcript expression profiling was used to define the spectrum and kinetics of chemokine involvement in an experimental murine model of asthma. Seventeen chemokines were induced in the lungs of allergen-inoculated mice, as compared with saline-treated mice. Two (CXCL13 and CCL9) of the 17 identified chemokines have not previously been associated with allergic airway disease. Seven (7 of 17; CCL2, CCL7, CCL9, CCL11, CXCL1, CXCL5, CXCL10) of the allergen-induced chemokines were induced early after allergen challenge and remained induced throughout the experimental period. Three chemokines (CXCL2, CCL3, and CCL17) were induced only during the early phase of the inflammatory response after the initial allergen challenge, while seven chemokines (CCL6, CCL8, CCL12, CCL22, CXCL9, CXCL12, and CXCL13) were increased only after a second allergen exposure. Unexpectedly, expression of only three chemokines, CCL11, CCL17, and CCL22, was STAT6 dependent, and many of the identified chemokines were overexpressed in STAT6-deficient mice, providing an explanation for the enhanced neutrophilic inflammation seen in these mice. Notably, IFN-gamma and STAT1 were shown to contribute to the induction of two STAT6-independent chemokines, CXCL9 and CXCL10. Taken together, these results show that only a select panel of chemokines (those targeting Th2 cells and eosinophils) is positively regulated by STAT6; instead, many of the allergen-induced chemokines are negatively regulated by STAT6. Collectively, we demonstrate that allergen-induced inflammation involves coordinate regulation by STAT1, STAT6, and IFN-gamma.

Naive and central memory CD8 T cells use CCR7 to recirculate through T cell zones of secondary lymphoid organs where they can encounter antigen. Here we describe a subset of human CD8 T cells expressing CXCR5 which enables homing in response to CXCL13 produced within B cell follicles. CXCR5+ CD8 T cells were found in tonsil B cell follicles, and isolated cells migrated towards CXCL13 in vitro. They expressed CD27, CD28, CD45RO, CD69, and were CD7low, and produced IFN-gamma and granzyme A but lacked perforin, a functional profile suggesting that these cells are early effector memory cells in the context of contemporary T cell differentiation models. Receptors important in the interaction with B cells, including CD70, OX40 and ICOS, were induced upon activation, and CXCR5+ CD8 T cells could to some extent support survival and IgG production in tonsil B cells. Furthermore, CXCR5+ CD8 T cells expressed CCR5 but no CCR7, suggesting a migration pattern distinct from that of follicular CD4 T cells. The finding that a subset of early effector memory CD8 T cells use CXCR5 to locate to B cell follicles indicates that MHC class I-restricted CD8 T cells are part of the follicular T cell population.

Prostate cancer (PC) is a slowly progressing malignancy that often responds to androgen ablation or chemotherapy by becoming more aggressive, acquiring a neuroendocrine phenotype, and undergoing metastatic spread. We found that B lymphocytes recruited into regressing androgen-deprived tumors by C-X-C motif chemokine 13 (CXCL13), a chemokine whose expression correlates with clinical severity, play an important role in malignant progression and metastatic dissemination of PC. We now describe how androgen ablation induces CXCL13 expression. In both allografted and spontaneous mouse PC, CXCL13 is expressed by tumor-associated myofibroblasts that are activated on androgen ablation through a hypoxia-dependent mechanism. The same cells produce CXCL13 after chemotherapy. Myofibroblast activation and CXCL13 expression also occur in the normal prostate after androgen deprivation, and CXCL13 is expressed by myofibroblasts in human PC. Hypoxia activates hypoxia-inducible factor 1 (HIF-1) and induces autocrine TGF-β signaling that promotes myofibroblast activation and CXCL13 induction. In addition to TGF-β receptor kinase inhibitors, myofibroblast activation and CXCL13 induction are blocked by phosphodiesterase 5 (PDE5) inhibitors. Both inhibitor types and myofibroblast immunodepletion block the emergence of castration-resistant PC in the transgenic adenocarcinoma of the mouse prostate (TRAMP) model of spontaneous metastatic PC with neuroendocrine differentiation.

BACKGROUND

The chemokine CXCL13 is known to dictate homing and motility of B cells in lymphoid tissue and has been implicated in the formation of ectopic lymphoid tissue in chronic inflammation. Whether it influences B cell trafficking during acute infection, is largely unclear. In previous studies, we showed that (I) CXCL13 levels are markedly increased in the B cell-rich cerebrospinal fluid (CSF) of patients with acute Lyme neuroborreliosis (LNB), and (II) CXCL13 is released by monocytes upon recognition of borrelial outer surface proteins by Toll-like receptor 2. Here, we assessed the role of CXCL13--in comparison to other chemokines--in the recruitment of B cells to the CSF of patients with acute LNB.

METHODS

Measurement of chemokines was done by ELISA. B cells were isolated from whole blood using magnetic cell separation (MACS). For migration experiments, a modified Boyden chamber assay was used and the migrated B cells were further analysed by FACS. The migration was inhibited either by preincubation of the CSF samples with neutralizing antibodies, heating to 60 degrees C, removal of proteins >3 kDa, or by pre-treatment of the B cells with pertussis toxin. The principal statistical tests used were one-way analysis of variance and Bonferroni test (chemokine measurements) as well as paired Student's t-test (migration experiments).

RESULTS

Measurements of chemokine levels revealed an increase in three of the four known major B cell chemoattractants CXCL13, CCL19 and CXCL12 in LNB CSF. The CXCL13 CSF:serum ratio, as a measure of the chemotactic gradient, was substantially higher than that of CCL19 and CXCL12. Moreover, the chemotactic activity of LNB CSF was reduced up to 56% after preincubation with a neutralizing CXCL13 antibody, while combined preincubation with antibodies against CXCL13, CCL19, and CXCL12 did not lead to further reduction. Since treatment with pertussis toxin, heating to 60 degrees C, and removal of proteins >3 kDa abrogated the chemotactic activity, further not yet identified chemokines seem to be involved in B cell recruitment to LNB CSF.

CONCLUSIONS

Combined, our study suggests a key role of CXCL13 in B cell migration to sites of infection as shown here for the CSF of LNB patients.

Experimental autoimmune encephalomyelitis (EAE) is a Tcell-mediated autoimmune disease of the CNS that is widely used as an animal model of multiple sclerosis. In this study, we investigate the role of CXCL13, a chemokine involved in the development and organization of secondary lymphoid tissues, in the pathogenesis of EAE. We detected CXCL13 mRNA and protein in spinal cords of mice with EAE. CXCL13-deficient mice exhibited a mild, self-limited form of disease. CXCL13 appeared to be important for the establishment of chronic white matter lesions. Furthermore, adoptive transfer experiments with CXCL13-deficient hosts indicate that the chemokine plays a distinct role during the effector phase. Our findings raise the possibility that reagents that antagonize or inhibit CXCL13 might be useful for the treatment of neuroinflammatory diseases such as multiple sclerosis.

OBJECTIVE

Synovial B cells play a critical role in rheumatoid arthritis (RA), being involved in autoantibody synthesis, T cell activation, and cytokine production. CXCL13 is a B cell chemoattractant that is instrumental in synovial B cell organization; the regulatory determinants of CXCL13 in inflammation are poorly characterized. This study was undertaken to investigate the functional involvement of synovial T cells in the ectopic expression of CXCL13 in RA.

METHODS

CXCL13 production and regulation were addressed using immunohistochemistry, in situ hybridization, quantitative polymerase chain reaction, multicolor flow cytometry, and enzyme-linked immunosorbent assay, by in situ-ex vivo analysis and in vitro functional assays with rheumatoid synovial tissue and primary cells.

RESULTS

CXCL13 messenger RNA and protein expression and spontaneous CXCL13 secretion were detected in RA synovial fluid T cells but were not detected (or were detected only occasionally) in peripheral blood T cells. Analysis of tissue expression confirmed cytoplasm localization of CXCL13 in T lymphocytes infiltrating B cell follicles and small perivascular aggregates. Multicolor characterizations in synovial fluid demonstrated CXCL13 expression in antigen-experienced T helper cells, frequently characterized by terminal differentiation and the lack of the follicular helper T cell markers CXCR5 and BCL6 protein. In vitro functional assays revealed the enhancing effect of T cell receptor-CD28 engagement on CXCL13 production and secretion in primary cells.

CONCLUSIONS

Our findings define a new functional property of synovial T cells, demonstrating their active involvement in the local production of B cell chemoattractants, and support a direct contribution of the adaptive immune system and antigen-dependent signals in the mechanisms of B cell localization in RA.

Despite improved understanding of the pathogenesis of neuroinflammatory disorders of the brain and development of new diagnostic markers, our biomarker repertoire to demonstrate and monitor inflammation remains limited. Using PubMed database, we reviewed 83 studies on CSF cytokines and chemokines and describe the pattern of elevation and possible role of cytokines/chemokines as biomarkers in viral and autoimmune inflammatory neurological disorders of the CNS. Despite inconsistencies and overlap of cytokines and chemokines in different neuroinflammation syndromes, there are some trends regarding the pattern of cytokines/chemokine elevation. Namely B cell markers, such as CXCL13 and BAFF are predominantly investigated and found to be elevated in autoantibody-associated disorders, whereas interferon gamma (IFN-γ) is elevated mainly in viral encephalitis. Th2 and Th17 cytokines are frequently elevated in acute disseminated encephalomyelitis (ADEM) and neuromyelitis optica (NMO), whereas Th1 and Th17 cytokines are more commonly elevated in multiple sclerosis (MS). Cytokine/chemokine profiling might provide new insights into disease pathogenesis, and improve our ability to monitor inflammation and response to treatment.

This study investigated the role of several chemokines and their receptors on malignant B lymphocytes recovered from 13 patients with chronic lymphocytic leukemia (CLL), 9 with hairy cell leukemia (HCL), 5 with mantle cell lymphoma (MCL), 5 with marginal zone B-cell lymphoma (MZL), 6 with small lymphocytic lymphoma (SLL), and 5 with follicular cell lymphoma (FCL). Flow cytometry analysis demonstrated that CXCR4 and CXCR5 were expressed on all malignant and normal B cells. Considering CC receptors, CCR1 was expressed in 70% of patients with CLL and 40% of those with HCL but was lacking in patients with MCL, MZL, SLL, and normal B cells. CCR2 showed a heterogeneous pattern of expression. CCR3 was found in almost all patients with CLL and in the majority of those with HCL, whereas it was usually lacking in patients with MZL and SLL and in healthy subjects. CCR5 was expressed in patients with HCL and MCL. Migration assays showed that different chemokines, mainly CXCL12 and CXCL13, are able to trigger migration of malignant B lymphocytes. Some of these chemokines induce calcium mobilization. These data indicate that different patterns of chemokine receptor expression identify different malignant B-cell subsets and that these receptors are functional and might play a role in malignant B-cell circulation.

The FDC-specific molecular signals required in the formation of FDC networks, B cell follicles, and germinal centers (GCs) have remained poorly understood. We used FDC-specific gene targeting to investigate the function of p55TNFR and IKK2 in lymphoid organ structure and function. Here we show that FDC-specific expression of p55TNFR is necessary and sufficient to promote FDC network and B cell follicle formation, restore the expression of CXCL13 and VCAM-1/ICAM-1 in FDCs, and lead to productive GCs. Notably, FDC-specific disruption of IKK2 does not affect formation of FDC networks. Yet, after antigen engagement or immune complex (IC) deposition, FDCs lacking IKK2 fail to upregulate VCAM-1 and ICAM-1, and GCs remain sterile. These findings demonstrate that IKK2-independent function of p55TNFR on FDCs is sufficient to support the development of FDC networks and GCs, while FDC-specific IKK2 is indispensable for the generation of efficient humoral immune responses.

The development of secondary lymphoid organs, such as lymph nodes (LNs), in the embryo results from the reciprocal action between lymphoid tissue inducer (LTi) cells and stromal cells. However, the initial events inducing LN anlagen formation before the LTi stromal cells cross-talk interactions take place are not fully elucidated. In this study, we show that the inguinal LN anlagen in mouse embryos developed from mesenchymal cells surrounding the lymph sacs, spherical structures of endothelial cells that bud from veins. Using inguinal and mesenteric LNs (mLNs), we provide evidence supporting a two-step maturation model for stromal cells: first, ICAM-1(-)VCAM-1(-) mesenchymal precursor cells become ICAM-1(int)VCAM-1(int) cells, in a process independent of LTi cells and lymphotoxin beta receptor (LTbetaR) signaling. The second step involves the maturation of ICAM-1(int)VCAM-1(int) cells to ICAM-1(high)VCAM-1(high) mucosal addressin cell adhesion molecule-1(+) organizer cells and depends on both LTi cells and LTbetaR. Addition of alphaLTbetaR agonist to LN organ cultures was sufficient to induce ICAM-1(int)VCAM-1(int) cells to mature. In LtbetaR(-/-) embryos, both inguinal and mLN stromal cells showed a block at the ICAM-1(int)VCAM-1(int) stage, and, contrary to inguinal LNs, mLNs persist longer and contained LTi cells, which correlated with the sustained gene expression of Il-7, Cxcl13, and, to a lesser degree, Ccl21. Taken together, these results highlight the importance of the signals and cellular interactions that induce the maturation of stromal cells and ultimately lead to the formation of lymphoid tissues.

OBJECTIVE

To determine whether the plasma levels of a range of inflammatory proteins have utility as biomarkers of disease activity in rheumatoid arthritis (RA) patients.

METHODS

Plasma proteins (n = 163) were profiled in 44 patients with RA diagnosed according to the American College of Rheumatology 1987 criteria (22 with active and 22 with quiescent disease) and in 16 age- and sex-matched healthy controls. The utility of a subset of differentially expressed proteins as predictors of RA disease activity was investigated using partial least-squares discriminant analysis, and their response to therapeutic intervention was evaluated in plasma from an additional cohort of 16 patients with active RA treated with anti-tumor necrosis factor alpha (anti-TNFalpha).

RESULTS

The protein profiling study identified 25 proteins that were differentially expressed in plasma samples from patients with active RA (P for the false discovery rate < or = 0.01) compared with those with quiescent RA, including the previously described interleukin-6 (IL-6), oncostatin M, and IL-2, and the 5 less-established markers macrophage colony-stimulating factor (M-CSF), tumor necrosis factor receptor superfamily member 9, CCL23, transforming growth factor alpha, and CXCL13. Systemic levels of these 5 markers correlated with the C-reactive protein level, erythrocyte sedimentation rate, rheumatoid factor level, tender joint count in 68 joints, and Disease Activity Score in 28 joints (DAS28), and their combined plasma levels were shown to be good predictors of disease activity (kappa = 0.64). In anti-TNFalpha-treated RA patients, plasma levels of CXCL13 were reduced after 1 and 7 days of therapy, and levels of CCL23, M-CSF, and CXCL13 showed a statistically significant positive correlation with the DAS28 score.

CONCLUSIONS

This exploratory study for biomarker discovery led to the identification of several proteins predictive of RA disease activity that may be useful in the definition of disease subphenotypes and in the measurement of response to therapy in clinical studies.

The chemokines (CKs) CXCL13, CCL21, and CXCL12 are known to play differential roles in the organization of the lymphoid tissues and the development of lymphoid malignancies. We investigated the expression of these CKs and their receptors in the salivary glands of Sjogren's syndrome patients with lymphoepithelial lesions (lymphoepithelial sialadenitis or LESA) and in MALT lymphoma to understand their involvement in salivary gland lymphomagenesis. We demonstrate that within salivary glands with LESA and MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are selectively associated with areas of reactive lymphoid proliferation, whereas no significant expression of these molecules was detected in the malignant lymphoid aggregate. Conversely, CXCL12 was observed predominantly in infiltrated ducts and malignant B cells. Accordingly, CXCL13 and CCL21 transcript levels were significantly increased in LESA samples while CXCL12 levels were increased in MALT lymphoma and isolated tumor cells. Low levels of CK receptors were detected on lymphoma-extracted lymphocytes, suggesting down-regulation in the abundance of ligands. Our findings suggest that in salivary gland MALT lymphoma the lymphoid CKs CXCL13 and CCL21 are directly implicated in the organization of ectopic reactive lymphoid tissue, whereas CXCL12 is associated with the infiltrated epithelium and malignant B cell component and is possibly involved in the regulation of malignant B cell survival.