Complement C3a promotes CXCL12-induced migration and engraftment of human and murine hemopoietic progenitor cells, suggesting a cross-influence between anaphylatoxin and chemokine axes. Here we have explored the underlying mechanism(s) of complement anaphylatoxin and chemokine cooperation. In addition to C3a, C3a-desArg and C4a but not C5a, are potent enhancers of CXCL12-induced chemotaxis of human and murine bone marrow (BM) stem/progenitor cells and B lineage cells. C3a enhancement of chemotaxis is chemokine specific because it is also observed for chemotaxis to CCL19 but not to CXCL13. The potentiating effect of C3a on CXCL12 is independent of the classical C3a receptor (C3aR). First, human BM CD34(+) and B lineage cells do not express C3aR by flow cytometry. Second, the competitive C3aR inhibitor SB290157 does not affect C3a-mediated enhancement of CXCL12-induced chemotaxis. Third, enhancement of chemotaxis of hemopoietic cells is also mediated by C3a-desArg, which does not bind to C3aR. Finally, C3a enhances CXCL12-induced chemotaxis of BM cells from C3aR knockout mice similar to BM cells from wild-type mice. Subsequent studies revealed that C3a increased the binding affinity of CXCL12 to human CXCR4(+)/C3aR(-), REH pro-B cells, which is compatible with a direct interaction between C3a and CXCL12. BM stromal cells were able to generate C3a, C3a-desArg, C4a, as well as CXCL12, suggesting that this pathway could function in vivo. Taken together, we demonstrate a C3a-CXCL12 interaction independent of the C3aR, which may provide a mechanism to modulate the function of CXCL12 in the BM microenvironment.

Chronic inflammation is characterized by the accumulation of leucocytes within tissues. In rheumatoid arthritis the inflammatory infiltrate shares many architectural features with lymphoid tissue. For example, CD4 T cells and B cells accumulate in perivascular lymphoid structures within synovial tissue. CD8 T cells and neutrophils are found predominantly within synovial fluid. What drives these distinctive lymphoid microstructures and the relative contribution of lymphocytes and stromal cells such as fibroblasts to this process is the subject of this review. Cellular interactions between leucocytes and stromal cells such as macrophages and fibroblasts are important in generating tumour necrosis factor-alpha within the inflamed synovium. Therefore understanding how leucocytes accumulate within the inflamed synovium is likely to provide new therapeutic approaches to modify the inflammatory process. We have found that fibroblasts play a dominant role in defining the disordered synovial microenvironment in rheumatoid arthritis. Through their production of a variety of cytokines (interferon-beta, transforming growth factor-beta) and constitutive chemokines (stromal cell-derived factor-1, CXCL12) they directly alter the behaviour of lymphocytes that accumulate within chronically inflamed joints leading to their inappropriate survival and retention. We have extended these observations to another chronic persistent rheumatic disease, Sjögren's syndrome, and found that ectopic production of the constitutive B cell-attracting chemokine BCA-1 (CXCL13) is associated with lymphocyte accumulation and lymphoid tissue formation. These findings suggest that stromal cells such as fibroblasts play an important role in the switch from acute resolving to chronic persistent arthritis by allowing lymphocytes to accumulate in the wrong place at the wrong time.

Oral squamous cell carcinomas (OSCC) are malignant tumors with a potent activity of local bone invasion/osteolysis. The chemokine ligand, CXCL13, has been identified as a prognostic marker for OSCC development and progression. Here in, we show that recombinant hCXCL13 treatment of OSCC cells stimulates (5-fold) RANK ligand (RANKL), a critical bone resorbing osteoclastogenic factor expression. Anti-CXCR5 chemokine receptor antibody abrogates CXCL13-induced RANKL expression in these cells. Also, CXCL13 stimulated (3.0-fold) hRANKL gene promoter activity in SCC14a cells. SuperArray screening for transcription factors by real-time RT-PCR identified significant increase in the levels of c-Jun and NFATc3 mRNA expression in CXCL13-stimulated OSCC cells. CXCL13 treatment significantly increased (3.5-fold) phospho-c-Jun levels in these cells and a c-Jun-NH(2)-kinase inhibitor abolished CXCL13-stimulated RANKL expression. Furthermore, we show that CXCL13 stimulation induced nuclear translocation of NFATc3 in OSCC cells. Chromatin-immune precipitation assay confirmed NFATc3 binding to the RANKL promoter region. We also show that overexpression of NFATc3 stimulates RANKL expression/promoter activity and that siRNA suppression of NFATc3 abolished CXCL13-stimulated RANKL expression. Thus, our results suggest that NFATc3 is a downstream target of the CXCL13/CXCR5 axis to stimulate RANKL expression in OSCC cells and implicates CXCL13 as a potential therapeutic target to prevent OSCC bone invasion/osteolysis.

Gene expression profiling identified genes uniquely expressed by human germinal-center T-helper (GCTh) cells, including programmed death-1 (PD-1) and CXCL13. Recently, we demonstrated that PD-1 is an immunophenotypic marker of GCTh cells and angioimmunoblastic T-cell lymphoma (AITL). The goal of this study was to investigate the expression pattern of CXCL13 in comparison with PD-1. We studied 63 cases of T-cell lymphoproliferative disorders, including 22 cases of AITL. In cases of AITL, PD-1+ and CXCL13+ neoplastic cells were seen at foci of expanded CD21+ follicular dendritic cell networks. CXCL13 expression was limited in other peripheral T-cell lymphomas. PD-1 and CXCL13 identified germinal-center T-helper cells, showed a similar pattern of expression in AITL, and should serve as useful new markers for AITL. The similar pattern of expression of CXCL13 and PD-1 in AITL provides further evidence that AITL is a neoplasm derived from germinal-center T-helper cells.

BACKGROUND

Most prostate cancer (PCa)-related deaths are due to metastasis, which is mediated in part by chemokine receptor and corresponding ligand interaction. We have previously shown that PCa tissue and cell lines express high levels of the chemokine receptor CXCR5, than compared to their normal counterparts, and interaction of CXCR5 with its specific ligand (CXCL13) promoted PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. This study dissects some of the molecular mechanisms following CXCL13-CXCR5 interaction that mediate PCa cell migration and invasion.

RESULTS

Using Western blot analysis, kinase-specific cell-based ELISAs, and migration and invasion assays, we show that PCa cell lines differentially express phosphoinositide-3 kinase (PI3K) catalytic subunit isoforms and dedicator of cytokinesis 2 (DOCK2). Specifically, we show that PC3 and normal prostatic epithelial (RWPE-1), but not LNCaP cell lines expressed DOCK2, while RWPE, PC3, and LNCaP cell lines expressed PI3K-p110alpha and -p110beta. Moreover, PC3 selectively expressed PI3K-p110gamma, but LNCaP and RWPE cell lines expressed PI3Kp110delta. CXCL13 caused CXCR5-dependent activation of the PI3Kp85alpha in LNCaP cells, and p85alpha as well as -p101 in PC3 cells. CXCL13-CXCR5 interaction regulated LNCaP and PC3 cell migration and invasion through extracellular signal-regulated kinase 1/2 (ERK1/2) activation that was primarily dependent on the PI3Kp110 isoform(s), Src, and focal adhesion kinase (FAK), but not DOCK2.

CONCLUSIONS

While additional studies will be needed to determine the PI3K-independent (i.e., DOCK2-mediated) and -dependent events that dictate PCa cell responsiveness to CXCL13, these data provide evidence of the existence of cell type- and stimulus-specific signaling events that support migration and invasion of PCa cells.

Dendritic cells (DC) attract both T and B lymphocytes to induce an efficient antigen-specific immune response. Recently, it was shown that naïve T cells are attracted to DC by dendritic cell chemokine 1 (DC-CK1, CCL18). The potent B lymphocyte chemoattractant BLC (CXCL13) was previously shown to be essential for homing of lymphocytes into secondary lymphoid organs and for the development of B cell follicles. As the cells that produce BLC are largely unknown and BLC could be a candidate chemokine for the recruitment of B cells to DC, we analyzed different DC subsets for expression of BLC. Here we demonstrate that monocyte-derived DC as well as activated blood DC indeed express and secrete BLC. Interestingly, ligation of the CD40 molecule down-regulated BLC expression in monocyte-derived DC. Staining of tonsilar sections indicated that BLC is expressed by follicular dendritic cells and germinal center dendritic cells (GCDC) in vivo. Real-time quantitative PCR confirmed the expression of BLC in isolated GCDC. Since both B cells and activated T cells express the receptor for BLC, our findings implicate an important role for BLC in establishing the interaction of DC with T cells and B cells. Furthermore, CD40/CD40 ligand interactions could modulate this process by down-regulating the expression of BLC.

BACKGROUND

Despite the discovery of CSF and serum diagnostic autoantibodies in autoimmune encephalitis, there are still very limited CSF biomarkers for diagnostic and monitoring purposes in children with inflammatory or autoimmune brain disease. The cause of encephalitis is unknown in up to a third of encephalitis cohorts, and it is important to differentiate infective from autoimmune encephalitis given the therapeutic implications.

OBJECTIVE

To study CSF cytokines and chemokines as diagnostic biomarkers of active neuroinflammation, and assess their role in differentiating demyelinating, autoimmune, and viral encephalitis.

METHODS

We measured and compared 32 cytokine/chemokines using multiplex immunoassay and APRIL and BAFF using ELISA in CSF collected prior to commencing treatment from paediatric patients with confirmed acute disseminated encephalomyelitis (ADEM, n = 16), anti-NMDAR encephalitis (anti-NMDAR E, n = 11), and enteroviral encephalitis (EVE, n = 16). We generated normative data using CSF from 20 non-inflammatory neurological controls. The sensitivity of CSF cytokine/chemokines to diagnose encephalitis cases was calculated using 95th centile of control values as cut off. We correlated CSF cytokine/chemokines with disease severity and follow up outcome based on modified Rankin scale. One-way hierarchical correlational cluster analysis of molecules was performed in different encephalitis and outcome groups.

RESULTS

In descending order, CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 had the best sensitivity (>79.1%) when all encephalitis patients were included. The combination of IL-6 and IFN-α was most predictive of inflammation on multiple logistic regression with area under the ROC curve 0.99 (CI 0.97-1.00). There were no differences in CSF cytokine concentrations between EVE and anti-NMDAR E, whereas ADEM showed more pronounced elevation of Th17 related (IL-17, IL-21) and Th2 (IL-4, CCL17) related cytokine/chemokines. Unlike EVE, heat map analysis showed similar clustering of cytokine/chemokine molecules in immune mediated encephalitis (ADEM and anti-NMDAR E). Th1 and B cell (CXCL13 and CXCL10) molecules clustered together in patients with severe encephalopathy at admission and worse disability at follow up in all encephalitis. There was no correlation between CSF neopterin and IFN-γ or IFN-α.

CONCLUSIONS

A combination panel of cytokine/chemokines consisting of CSF TNF-α, IL-10, IFN-α, IL-6, CXCL13 and CXCL10 measured using multiplex immunoassay may be used to diagnose and monitor intrathecal inflammation in the brain. Given their association with worse outcome, certain key chemokines (CXCL13, CXCL10) could represent potential therapeutic targets in encephalitis.

BACKGROUND

A key phenomenon in rheumatoid arthritis is the formation of lymphoid follicles in the inflamed synovial membrane. C-X-C motif chemokine 13 (CXCL13) is central in this process as it attracts C-X-C chemokine receptor type 5 (CXCR5)-expressing B cells and T follicular helper cells to the follicle. We here examine the role of CXCL13 and its association with disease in patients with treatment-naïve early rheumatoid arthritis.

METHODS

Plasma samples from patients in the OPERA trial were examined for CXCL13 at treatment initiation and after 6 months of treatment with either methotrexate plus placebo (DMARD) (n = 37) or methotrexate plus adalimumab (DMARD + ADA) (n = 39). Treatment outcome was evaluated after 1 and 2 years. CXCL13 plasma levels in healthy volunteers (n = 38) were also examined.

RESULTS

Baseline CXCL13 plasma levels were increased in early rheumatoid arthritis patients in comparison with healthy volunteers. Also, plasma CXCL13 correlated positively with disease activity parameters; swollen joint count 28 (rho = 0.34) and 40 (rho = 0.39), visual analog score (rho = 0.38) and simplified disease activity index (rho = 0.25) (all P <0.05). CXCL13 levels decreased a significantly twofold more in the DMARD + ADA group than in the DMARD group. Baseline CXCL13 plasma levels in the DMARD group correlated inversely with disease activity parameters; disease activity score in 28 joints, four variables, C-reactive protein based (DAS28CRP) (rho = 0.58, P < 0.05) at 12 months. High baseline CXCL13 was associated with remission (DAS28CRP less than 2.6) after 2 years.

CONCLUSIONS

In treatment-naïve early rheumatoid arthritis patients, plasma CXCL13 levels were associated with joint inflammation. Furthermore, patients with high baseline plasma CXCL13 levels had an improved chance of remission after 2 years. We propose that high CXCL13 concentrations indicate recent onset of inflammation that may respond better to early aggressive treatment. Thus, high levels of CXCL13 could reflect the 'the window of opportunity' for optimal treatment effect.

BACKGROUND

Clinicaltrial.gov NCT00660647. Registered 10 April 2008.

BACKGROUND

The aim of our study was to evaluate the prognostic role of immunological microenvironnement in stage II-III CRC patients.

METHODS

We constructed a tissue microarray from 196 consecutive patients with stage II-III CRC and compared CD3, CD4, CD8, CD57, CD68, CXCL9/MIG, CXCL13, and PPARγ immunoreactivity in tumour samples and their matched non-tumour tissue. We assessed their association with relapse-free survival (RFS; primary endpoint) and overall survival (OS) in multivariate Cox models.

RESULTS

Low densities of CD57+ and CD68+ tumour-infiltrating cells (TIC) independently predicted worse outcomes. A prognostic score combining CD57 (+,>> vs -, ≤2 cells per spot) and CD68 (+, >0 vs -, =0 cells per spot) TIC density discriminated CRC patients at low (CD68+/CD57+), intermediate (CD68+/CD57-), or high (CD68-/CD57-) risk, with hazard ratios for the intermediate-risk and high-risk groups of 2.7 (95% confidence interval (CI): 1.3-5.8) and 9.0 (3.2-25.4) for RFS, and 2.5 (1.2-5.1) and 10.6 (3.8-29.2) for OS, respectively, as compared with the low-risk group. Corresponding 5-year survival rates (95% CI) in the low-, moderate- and high-risk groups were 84% (71-91), 65% (54-74), and 12% (2-47), respectively, for RFS, and 91% (80-96), 76% (66-84), and 25% (7-59), respectively, for OS.

CONCLUSIONS

Tumour CD57+ and CD68+ TIC density assessment independently predicts survival in patients with stage II-III CRC. If validated, our score based on a quick, inexpensive, and well-established method such as point counting on diagnostic tissue sections could be used routinely as a prognostic tool in CRC patients.

BACKGROUND

The functional relevance of synovial ectopic lymphoid neogenesis (ELN) in rheumatoid arthritis (RA) remains unknown. As ELN correlates with the degree of tissue inflammation, we investigated whether ELN was associated with specific cytokine profiles.

METHODS

Synovial ELN was determined by immunohistology and long CD21 isoform (CD21L) expression. Cytokine expression was determined by multiplex enzyme-linked immunosorbent assay (ELISA) and quantitative polymerase chain reaction (PCR) as well as immunohistology in synovial fluid (SF) (n = 44) and tissue (ST) (n = 108), respectively. Production of ELN-associated chemokines by fibroblast-like synoviocytes (FLS) was studied in vitro.

RESULTS

Screening analysis of SF by multiplex ELISA showed higher protein levels of interleukin (IL)-23 (p = 0.018) and IL-17F (p = 0.028) in ELN+ versus ELN- samples. Other cytokines, including IL-17A, IL-6, and tumor necrosis factor (TNF)-α, were not different. The association between IL-23 and ELN was not biased by disease activity or other clinical features and was confirmed by higher IL-23 mRNA expression in ELN+ versus ELN- ST samples (p = 0.030), a correlation between IL-23 and CD21L expression in the same samples (r = 0.70 p < 0.0001), and a similar correlation in two independent ST sample sets (r = 0.778 p < 0.0001 and r = 0.817 p = 0.011). IL-23 p19 staining was neither restricted nor enhanced in close proximity of ectopic lymphoid follicles, and neither IL-23 nor IL-17A stimulation induced expression of the ELN-associated CC chemokine ligand, CCL21 and CXC chemokine ligand CXCL13, by FLS. Downstream of IL-23, CD21L expression was significantly associated with IL-17F, IL-21, and IL-22, but not IL-17A in two independent ST sample sets.

CONCLUSIONS

Synovial ELN in RA is strongly associated with activation of the IL-23 pathway but not with IL-17A.

To assess the effect of abatacept (CTLA-4Ig), which limits T cell activation, on homeostasis of CD4+ T cell subsets and T cell-dependent B cell hyperactivity in patients with primary Sjögren's syndrome (SS).

Fifteen patients with primary SS treated with abatacept were included. Circulating CD4+ T cell and B cell subsets were analyzed by flow cytometry at baseline, during the treatment course, and after treatment was completed. CD4+ effector T cell subsets and Treg cells were identified based on expression of CD45RA, CXCR3, CCR6, CCR4, CXCR5, programmed death 1, inducible costimulator (ICOS), and FoxP3. Serum levels of anti-SSA/anti-SSB and several T cell-related cytokines were measured. Expression of ICOS and interleukin-21 (IL-21) protein was examined in parotid gland tissue at baseline and after treatment. Changes in laboratory parameters and associations with systemic disease activity (EULAR Sjögren's Syndrome Disease Activity Index [ESSDAI]) over time were analyzed using generalized estimating equations.

Abatacept selectively reduced percentages and numbers of circulating follicular helper T (Tfh) cells and Treg cells. Other CD4+ effector T cell subsets were unaffected. Furthermore, expression of the activation marker ICOS by circulating CD4+ T cells and expression of ICOS protein in parotid gland tissue declined. Reduced ICOS expression on circulating Tfh cells correlated significantly with lower ESSDAI scores during treatment. Serum levels of IL-21, CXCL13, anti-SSA, and anti-SSB decreased. Among circulating B cells, plasmablasts were decreased by treatment. After cessation of treatment, all parameters gradually returned to baseline.

Abatacept treatment in patients with primary SS reduces circulating Tfh cell numbers and expression of the activation marker ICOS on T cells. Lower numbers of activated circulating Tfh cells contribute to attenuated Tfh cell-dependent B cell hyperactivity and may underlie the efficacy of abatacept.

Trigeminal nerve damage-induced neuropathic pain is a severely debilitating chronic orofacial pain syndrome. Spinal chemokine CXCL13 and its receptor CXCR5 were recently demonstrated to play a pivotal role in the pathogenesis of spinal nerve ligation-induced neuropathic pain. Whether and how CXCL13/CXCR5 in the trigeminal ganglion (TG) mediates orofacial pain are unknown.

The partial infraorbital nerve ligation (pIONL) was used to induce trigeminal neuropathic pain in mice. The expression of ATF3, CXCL13, CXCR5, and phosphorylated extracellular signal-regulated kinase (pERK) in the TG was detected by immunofluorescence staining and western blot. The effect of shRNA targeting on CXCL13 or CXCR5 on pain hypersensitivity was checked by behavioral testing.

pIONL induced persistent mechanical allodynia and increased the expression of ATF3, CXCL13, and CXCR5 in the TG. Inhibition of CXCL13 or CXCR5 by shRNA lentivirus attenuated pIONL-induced mechanical allodynia. Additionally, pIONL-induced neuropathic pain and the activation of ERK in the TG were reduced in Cxcr5 (-/-) mice. Furthermore, MEK inhibitor (PD98059) attenuated mechanical allodynia and reduced TNF-α and IL-1β upregulation induced by pIONL. TNF-α inhibitor (Etanercept) and IL-1β inhibitor (Diacerein) attenuated pIONL-induced orofacial pain. Finally, intra-TG injection of CXCL13 induced mechanical allodynia, increased the activation of ERK and the production of TNF-α and IL-1β in the TG of WT mice, but not in Cxcr5 (-/-) mice. Pretreatment with PD98059, Etanercept, or Diacerein partially blocked CXCL13-induced mechanical allodynia, and PD98059 also reduced CXCL13-induced TNF-α and IL-1β upregulation.

CXCL13 and CXCR5 contribute to orofacial pain via ERK-mediated proinflammatory cytokines production. Targeting CXCL13/CXCR5/ERK/TNF-α and IL-1β pathway in the trigeminal ganglion may offer effective treatment for orofacial neuropathic pain.

Multiple sclerosis (MS) is an autoimmune disease of unknown cause, in which chronic inflammation drives multifocal demyelination of axons in both white and gray matter in the CNS. The pathological course of the disease is heterogeneous and involves an early, predominantly inflammatory demyelinating disease phase of relapsing-remitting MS (RRMS), which, over a variable period of time, evolves into a progressively degenerative stage associated with axonal loss and scar formation, causing physical and cognitive disability. For patients with RRMS, there is a growing arsenal of disease-modifying agents (DMAs), with varying degrees of efficacy, as defined by reduced relapse rates, improved magnetic resonance imaging outcomes, and preservation of neurological function. Establishment of personalized treatment plans remains one of the biggest challenges in therapeutic decision-making in MS because the disease prognosis and individual therapeutic outcomes are extremely difficult to predict. Current research is aimed at discovery and validation of biomarkers that reliably measure disease progression and effective therapeutic intervention. Individual biomarker candidates with evident clinical utility are highlighted in this review and include neutralizing autoantibodies against DMAs, fetuin-A, osteopontin, isoprostanes, chemokine (C-X-C motif) ligand 13 (CXCL13), neurofilament light and heavy, and chitinase 3-like protein. In addition, application of more advanced screening technologies has opened up new categories of biomarkers that move beyond detection of individual soluble proteins, including gene expression and autoantibody arrays, microRNAs, and circulating microvesicles/exosomes. Development of clinically useful biomarkers in MS will not only shape the practice of personalized medicine but will also serve as surrogate markers to enable investigation of innovative treatments within clinical trials that are less costly, are of shorter duration, and have more certainty of outcomes.

OBJECTIVE

Several histological scoring systems, including the focus score, performed in minor salivary glands (MSGs) by hematoxylin-eosin (H&E) staining, have been employed in clinical practice to assess the inflammatory infiltrate and provide the diagnosis of primary Sjo¨gren׳s syndrome (pSS). Aims of this study were to integrate different scoring systems and identify potential differences in the molecular profile of lymphoid cytokines related to germinal center (GC) formation and clinical subsets in pSS.

METHODS

Overall, 104 pSS patients and 40 subjects with sicca non-pSS were retrospectively evaluated. MSG biopsies were evaluated by H&E and immunofluorescence to assess histological pattern, Chisholm and Mason grading system, Tarpley score, a grading for the severity of inflammatory infiltrate, T-/B-cell segregation, and the presence of GC. MSGs from 50 pSS patients and 30 sicca non-pSS patients were processed by real-time PCR to assess LTα, LTβ, BAFF, CXCR4, CXCL12, CXCR5, CXCL13, CCR7, CCL19, and CCL21.

RESULTS

GCs presence was associated with anti-Ro/SSA and anti-La/SSB antibodies, hypergammaglobulinemia, salivary gland swelling, higher Tarpley score and focus score, and extraglandular involvement but, at multivariate analysis, only extraglandular involvement was independently associated to GC. pSS patients displayed higher level of all cytokines compared to those with sicca symptoms. GC(+) pSS patients displayed higher level of all cytokines compared to those GC(-).

CONCLUSIONS

Our study demonstrates that different histopathological patterns, including GC presence, reflect different cytokine expression and different clinical subsets. We believe that the combined immunofluorescence/molecular approach in MSGs would help to tailor diagnostic and therapeutic approach for different subsets of pSS patients.

Obesity is recognized as a low-grade chronic inflammatory state which involves a chemokine network contributing to a variety of diseases. As a first step toward understanding the roles of the obesity-driven chemokine network, we used a 3T3-L1 cell differentiation model to identify the chemokine profiles elicited during adipogenesis and how this profile is modified by epidermal growth factor (EGF) and tumor necrosis factor-α (TNF) as a growth and proinflammatory factor, respectively. The chemokine network was monitored using PCR arrays and qRT-PCR while main signaling pathways of EGF and TNF were measured using immunoblotting. The dominant chemokines in preadipocytes were CCL5, CCL8, CXCL1, and CXCL16, and in adipocytes CCL6 and CXCL13. The following chemokines were found in both preadipocytes and adipocytes: CCL2, CCL7, CCL25, CCL27, CXCL5, CXCL12, and CX3CL1. Among chemokine receptors, CXCR7 was specific for preadipocytes and CXCR2 for adipocytes. These findings indicate the development of a CXCL12-CXCR7 axis in preadipocytes and a CXCL5-CXCR2 axis in adipocytes. In addition to induction of CCL2 and CCL7 in both preadipocytes and adipocytes, EGF enhanced specifically CXCL1 and CXCL5 in adipocytes, indicating the potentiation of CXCR2-mediated pathway in adipocytes. TNF induced CCL2, CCL7, and CXCL1 in preadipocytes but had no response in adipocytes. EGFR downstream activation was dominant in adipocytes whereas NFκB activation was dominant in preadipocytes. Taken together, the adipocyte-driven chemokine network in the 3T3-L1 cell differentiation model involves CXCR2-mediated signaling which appears more potentiated to growth factors like EGF than proinflammatory factors like TNF.

Inflammatory mediators have crucial roles in leukocyte recruitment and subsequent central nervous system (CNS) neuroinflammation. The extent of neuronal injury and axonal loss are associated with the degree of CNS inflammation and determine physical disability in multiple sclerosis (MS). The aim of this study was to explore possible associations between a panel of selected cerebrospinal fluid biomarkers and robust clinical and demographic parameters in a large cohort of patients with MS and controls (n = 1066) using data-driven multivariate analysis. Levels of matrix metalloproteinase 9 (MMP9), chemokine (C-X-C motif) ligand 13 (CXCL13), osteopontin (OPN) and neurofilament-light chain (NFL) were measured by ELISA in 548 subjects comprising different MS subtypes (relapsing-remitting, secondary progressive and primary progressive), clinically isolated syndrome and persons with other neurological diseases with or without signs of inflammation/infection. Principal component analyses and orthogonal partial least squares methods were used for unsupervised and supervised interrogation of the data. Models were validated using data from a further 518 subjects in which one or more of the four selected markers were measured. There was a significant association between increased patient age and lower levels of CXCL13, MMP9 and NFL. CXCL13 levels correlated well with MMP9 in the younger age groups, but less so in older patients, and after approximately 54 years of age the levels of CXCL13 and MMP9 were consistently low. CXCL13 and MMP9 levels also correlated well with both NFL and OPN in younger patients. We demonstrate a strong effect of age on both inflammatory and neurodegenerative biomarkers in a large cohort of MS patients. The findings support an early use of adequate immunomodulatory disease modifying drugs, especially in younger patients, and may provide a biological explanation for the relative inefficacy of such treatments in older patients at later disease stages.

OBJECTIVE

Better biomarkers are needed for guiding management of patients with vasculitis. Large cohorts and technological advances had led to an increase in preclinical studies of potential biomarkers.

RESULTS

The most interesting markers described recently include a gene expression signature in CD8+ T cells that predicts tendency to relapse or remain relapse-free in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, and a pair of urinary proteins that are elevated in Kawasaki disease but not other febrile illnesses. Both of these studies used 'omics' technologies to generate and then test hypotheses. More conventional hypothesis-based studies have indicated that the following circulating proteins have potential to improve upon clinically available tests: pentraxin-3 in giant cell arteritis and Takayasu's arteritis; von Willebrand factor antigen in childhood central nervous system vasculitis; eotaxin-3 and other markers related to eosinophils or Th2 immune responses in eosinophilic granulomatosis with polyangiitis (Churg-Strauss syndrome); and matrix metalloproteinase-3, tissue inhibitor of metalloproteinase-1, and CXCL13 in ANCA-associated vasculitis.

CONCLUSIONS

New markers testable in blood and urine have the potential to assist with diagnosis, staging, assessment of current disease activity, and prognosis. However, the standards for clinical usefulness, in particular, the demonstration of either very high sensitivity or very high specificity have yet to be met for clinically relevant outcomes.

Follicular lymphomas (FLs) localize in lymphoid tissues and recapitulate the structure of normal secondary follicles. The chemokine/chemokine receptor pair CXCL13/CXCR5 is required for the architectural organization of B cells within lymphoid follicles. In this study, we showed that CXCL13 was secreted by FL cells. FL cells expressed CXCR5 and migrated in response to CXCL13. Furthermore, we observed a synergistic effect between CXCL13 and CXCL12 (SDF-1), a chemokine produced by stromal cells in lymphoid tissues. The production of CXCL13 by FL cells and CXCL12 by stromal cells probably directs and participates in the accumulation of FL cells within specific anatomic sites.

The investigation of trophoblast chemoattractive molecules in humans is of high interest for the reproductive field. Current evidence in ruminants demonstrates that CXCL10, formerly the interferon-gamma-inducible protein 10 (IP-10), is a potent chemotactic molecule implicated in the migration of trophoblast cells during early gestation. The aim of this work was to explore the existence of CXCL10/CXCR3 in the human model. Furthermore, chemotaxis assays were performed to demonstrate CXCL10 chemotactic activity in the human trophoblast cell lines JEG-3 and AC-1M88. Surprisingly, the conditioned media from epithelial endometrial cells (EEC) induced the highest trophoblast migration rate. Cytokine and chemokine membrane protein arrays were used to identify the secreted protein profile of EEC-conditioned media, and IL-6 was found to be the most abundant and CXCL13 the second most abundant molecule. Using a chemotaxis assay on AC-IM88, IL-6 antibody blocked the effect of EEC, indicating IL-6 to be an effective chemoattractive factor for trophoblast cells in the human model.

The mechanism of nasal-associated lymphoid tissue (NALT) development is incompletely understood with regard to the roles of cytokines, chemokines, and vascular addressins. Development of the wild-type NALT continued in the immediate postnatal period with gradual increases in cellularity, compartmentalization into T- and B-cell zones, and expression of lymphotoxin (LT)-alpha, LT-beta, and lymphoid chemokines (CCL21, CCL19, CXCL13). High endothelial venules (HEVs) developed that expressed GlyCAM-1, HEC-6ST [an enzyme crucial for expression of luminal peripheral node addressin (PNAd)], and PNAd itself. LT-beta(-/-) and LT-alpha(-/-) NALTs had fewer cells than those of wild-type mice, reduced (LT-beta(-/-)) or absent (LT-alpha(-/-)) lymphoid chemokines, and no T- and B-cell compartmentalization. LT-beta(-/-) HEVs expressed only abluminal PNAd and no HEC-6ST or GlyCAM-1. LT-alpha(-/-) HEVs had no PNAd, HEC-6ST, or GlyCAM-1. Because intranasal immunization gives rise to vaginal IgA, immunization of LT-beta(-/-) mice, which retain cervical lymph nodes, might generate such a response. Intranasal immunization with ovalbumin and cholera toxin revealed lower cytokine levels in the LT-alpha(-/-) and LT-beta(-/-) NALTs, and undetectable vaginal IgA. In contrast, splenic cytokines and serum IgG titers, although reduced, were detectable. These data indicate that LT-alpha(3) and LT-alpha(1)beta(2) cooperatively contribute to NALT development and function through regulation of lymphoid chemokines and adhesion molecules; they are the first to implicate LT-alpha(1)beta(2) in GlyCAM-1 regulation in NALT HEV development.

Trafficking of B-cell chronic lymphocytic leukemia (CLL) cells to the bone marrow and interaction with supporting stromal cells mediates important survival and proliferation signals. Previous studies have demonstrated that deletion of Rhoh led to a delayed disease onset in a murine model of CLL. Here we assessed the impact of RhoH on homing, migration, and cell-contact dependent interactions of CLL cells. Rhoh(-/-) CLL cells exhibited reduced marrow homing and subsequent engraftment. In vitro migration toward the chemokines CXCL12 and CXCL13 and cell-cell interactions between Rhoh(-/-) CLL cells and the supporting microenvironment was reduced. In the absence of RhoH the distribution of phosphorylated focal adhesion kinase, a protein known to coordinate activation of the Rho GTPases RhoA and Rac, appeared less polarized in chemokine-stimulated Rhoh(-/-) CLL cells, and activation and localization of RhoA and Rac was dysregulated leading to defective integrin function. These findings in the Rhoh(-/-) CLL cells were subsequently demonstrated to closely resemble changes in GTPase activation observed in human CLL samples after in vitro and in vivo treatment with lenalidomide, an agent with known influence on microenvironment protection, and suggest that RhoH plays a critical role in prosurvival CLL cell-cell and cell-microenvironment interactions with this agent.

Plasma cells (PC) localize to discrete areas of secondary lymphoid tissue and bone marrow (BM). The positioning of PC in different sites is believed to be regulated by chemokines and adhesion molecules expressed by accessory cells in the lymphoid tissue microenvironment. However, the mechanisms responsible for the positioning of PC within the red pulp (RP) of human spleen have not been elucidated. Therefore, we examined the contribution of human splenic stromal cells to the migration and function of human PC. Splenic PC expressed the chemokine receptor CXCR4 and responded to its ligand CXCL12. In contrast, PC lacked CXCR5 and CCR7, and consequently exhibited minimal migration towards CXCL13 and CCL21. Splenic stromal cells proved to be a rich source of CXCL12, and could induce the migration of human B cells. Furthermore, they supported Ig production by splenic PC mainly by secreting IL-6. Lastly, a striking difference between splenic and BM PC was the constitutive expression of CD11a by only splenic PC. Notably, splenic stromal cells expressed high levels of CD54, the counter-structure of CD11a, and splenic PC were positioned adjacent to stromal cells in the RP. Thus, we propose that stromal cells attract PC to the RP and contribute to their retention and function through the combined expression of CXCL12, CD54 and IL-6.

Recruitment of leukocytes is essential for eventual control of virus infections. Macrophages represent a leukocyte population involved in the first line of defense against many infections, including herpes simplex virus (HSV) infection. Through presentation of antigens to T cells and production of cytokines and chemokines, macrophages also constitute an important link between the innate and adaptive immune systems. Here, we have investigated the chemokine expression profile of macrophages after HSV infection and the virus-cell interactions involved. By reverse transcription-PCR and cDNA arrays, we found that HSV type 1 (HSV-1) and HSV-2 induced expression of the CC chemokine RANTES/CCL5 in murine macrophage cell lines and peritoneal cells. The CXC chemokine BCA-1/CXCL13 was also induced in peritoneal cells. Twenty-six other chemokines tested were not affected. Accumulation of RANTES mRNA was detectable after 5 h of infection, was sensitive to UV irradiation of the virus, and was preceded by accumulation of viral immediate-early mRNA and proteins. The viral components responsible for initiation of RANTES expression were examined with virus mutants and RAW 264.7 macrophage-like cells expressing a dominant negative mutant of the double-stranded-RNA-activated protein kinase (PKR). The PKR mutant cell line displayed reduced constitutive and HSV-inducible RANTES expression compared to the control cell line. HSV-1 mutants deficient in genes encoding the immediate-early proteins ICP4, ICP22, and ICP27 remained fully capable of inducing RANTES expression in macrophages. By contrast, the ability of an ICP0-deficient HSV-1 mutant to induce RANTES expression was compromised. Thus, HSV selectively induces expression of RANTES in macrophages through a mechanism dependent on cellular PKR and viral ICP0.

BACKGROUND

Various inflammatory biomarkers show prognostic potential for multiple sclerosis (MS)-risk after clinically isolated syndromes. However, biomarkers are often examined singly and their interrelation and precise aspects of their associated pathological processes remain unclear. Clarification of these relationships could aid the appropriate implementation of prognostic biomarkers in clinical practice.

OBJECTIVE

To investigate the interrelation between biomarkers of inflammation, demyelination and neurodegeneration in acute optic neuritis and to assess their association to measures of MS risk.

METHODS

A prospective study at a tertiary referral centre from June 2011 to December 2012 of 56 patients with optic neuritis as a first demyelinating symptom and 27 healthy volunteers. Lumbar puncture was performed within 28 (median 16) days of onset. CSF levels of CXCL13, matrix metalloproteinase (MMP)-9, CXCL10, CCL-2, osteopontin and chitinase-3-like-1, myelin basic protein (MBP) and neurofilament light-chain (NF-L) were determined. MS-risk outcome measures were dissemination in space (DIS) of white matter lesions on cerebral MRI, CSF oligoclonal bands and elevated IgG-index.

RESULTS

IN THE INTERRELATION ANALYSIS THE BIOMARKERS SHOWED CLOSE CORRELATIONS WITHIN TWO DISTINCT GROUPS: Biomarkers of leukocyte infiltration (CXCL13, MMP-9 and CXCL10) were strongly associated (p<0.0001 for all). Osteopontin and chitinase-3-like-1 were also tightly associated (p<0.0001) and correlated strongly to tissue damage markers (NF-L and MBP). The biomarkers of leukocyte infiltration all associated strongly with MS-risk parameters, whereas CHI3L1 and MBP correlated with MRI DIS, but not with CSF MS-risk parameters and osteopontin and NF-L did not correlate with any MS-risk parameters.

CONCLUSIONS

OUR FINDINGS SUGGEST TWO DISTINCT INFLAMMATORY PROCESSES: one of leukocyte infiltration, represented by CXCL13, CXCL10 and MMP-9, strongly associated with and potentially predicting MS-risk; the other represented by osteopontin and CHI3L1, suggesting tissue damage-related inflammation, potentially predicting residual disabilities after attack and perhaps cumulative damage over time. These hypotheses should be further addressed in follow-up studies.