Epstein-Barr virus (EBV) causes a persistent infection in human B cells by establishing specific transcription programs to control B cell activation and differentiation. Transcriptional reprogramming of EBV infected B cells is predominantly driven by the action of EBV nuclear antigens, among them the transcriptional repressor EBNA3A. By comparing gene expression profiles of wt and EBNA3A negative EBV infected B cells, we have previously identified a broad array of cellular genes controlled by EBNA3A. We now find that genes repressed by EBNA3A in these cells are significantly enriched for the repressive histone mark H3K27me3, which is installed by Polycomb group (PcG) proteins. This PcG-controlled subset of genes also carries H3K27me3 marks in a variety of other tissues, suggesting that the commitment to PcG silencing is an intrinsic feature of these gene loci that can be used by EBNA3A. In addition, EBNA3A targets frequently reside in co-regulated gene clusters. To study the mechanism of gene repression by EBNA3A and to evaluate the relative contribution of PcG proteins during this process, we have selected the genomic neighbors CXCL10 and CXCL9 as a model for co-repressed and PcG-controlled genes. We show that EBNA3A binds to CBF1 occupied intergenic enhancers located between CXCL10 and CXCL9 and displaces the transactivator EBNA2. This impairs enhancer activity, resulting in a rapid transcriptional shut-down of both genes in a CBF1-dependent manner and initiation of a delayed gain of H3K27me3 marks covering an extended chromatin domain. H3K27me3 marks increase gradually and are maintained by EBNA3A. Our study provides direct evidence that repression by EBNA3A requires CBF1 and that EBNA3A and EBNA2 compete for access to CBF1 at identical genomic sites. Most importantly, our results demonstrate that transcriptional silencing by EBNA3A precedes the appearance of repressive PcG marks and indicate that both events are triggered by loss of enhancer activity.

Individual cytokines and groups of cytokines that might represent networks in chronic lymphocytic leukemia (CLL) were analyzed and their prognostic values determined. Serum levels of 23 cytokines were measured in 84 patients and 49 age-matched controls; 17 levels were significantly elevated in patients. Unsupervised hierarchical bicluster analysis identified 3 clusters (CLs) of highly correlated but differentially expressed cytokines: CL1 (CXCL9, CXCL10, CXCL11, CCL3, CCL4, CCL19, IL-5, IL-12, and IFNγ), CL2 (TNFα, IL-6, IL-8, and GM-CSF), and CL3 (IL-1β, IL-2, IL-4, IL-15, IL-17, and IFNα). Combination scores integrating expression of CL1/CL2 or CL1/CL3 strongly correlated (P < .005) with time-to-first-treatment and overall survival (OS), respectively. Patients with the worst course had high CL1 and low CL2 or CL3 levels. Multivariate analysis revealed that CL1/CL2 combination score and immunoglobulin heavy chain variable region mutation status were independent prognostic indicators for time-to-first-treatment, whereas CL1/CL3 combination score and immunoglobulin heavy chain variable region mutation status were independent markers for OS. Thus, we identified groups of cytokines differentially expressed in CLL that are independent prognostic indicators of aggressive disease and OS. These findings indicate the value of multicytokine analyses for prognosis and suggest therapeutic strategies in CLL aimed at reducing CL1 and increasing CL2/CL3 cytokines.

There is accumulating evidence that points to a role of serotonin (5-hydroxytryptamine [5-HT]) in the pathophysiology of asthma. Therefore, we analyzed the expression of serotoninergic receptors (5-HTR), its linkage to intracellular calcium homeostasis, and its influence on the production and secretion of IL-6, prostaglandin E(2), the CCL-Chemokine CCL5/Rantes, and the CXC-chemokines CXCL8/IL-8, CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC in primary alveolar epithelial cells type II and the human lung cell lines A549 and BEAS-2B. Employing a PCR approach we were able to demonstrate mRNA expression of several 5-HTR, such as the heptahelical receptors 5-HTR1A, 5-HTR1B, 5-HTR1E, 5-HTR1F, 5-HTR2A, 5-HTR4, 5-HTR6, and 5-HTR7, as well as the ligand-gated ion channel 5-HTR3 in alveolar epithelial cells type II (AEC-II), A549, and BEAS-2B cells. To verify functional expression of 5-HTR subtypes, Ca(2+)-transients were analyzed. This enabled us to show that 5-HT induced an increase in intracellular calcium. Further experiments with isotype-selective receptor agonists allowed us to demonstrate that 5-HT induced calcium transients via activation of 5-HTR1, 5-HTR2, and 5-HTR3 in A549 and BEAS-2B cells. Moreover, we revealed that stimulation of 5-HTR1 and 5-HTR2 induced Ca(2+) mobilization from intracellular stores, whereas activation of 5-HTR3 induced Ca(2+) influx from the extracellular space. Functional studies indicated that activation of 5-HTR1B, 5-HTR1E/F, 5-HTR2, 5-HTR3, 5-HTR4, and 5-HTR7 regulated the release of the cytokine IL-6 and the CXC-chemokine CXCL8/IL-8. Our study shows that 5-HT stimulates different signaling pathways and regulates cytokine release in airway epithelial cells. In summary, our data implicate a pathophysiologic role of 5-HT in the asthmatic inflammatory responses in human airway epithelial cells.

BACKGROUND

CD8+ T cells may participate in cigarette smoke (CS) induced-lung inflammation in mice. CXCL10/IP-10 (IFNgamma-inducible protein 10) and CXCL9/Mig (monokine induced by IFN-gamma) are up-regulated in CS-induced lung injury and may attract T-cell recruitment to the lung. These chemokines together with CXCL11/ITAC (IFN-inducible T-cell alpha chemoattractant) are ligands for the chemokine receptor CXCR3 which is preferentially expressed chiefly in activated CD8+ T cells. The purpose of this investigation was to study the contribution of CXCR3 to acute lung inflammation induced by CS using CXCR3 knockout (KO) mice.

METHODS

Mice (n = 8 per group) were placed in a closed plastic box connected to a smoke generator and were exposed whole body to the tobacco smoke of five cigarettes four times a day for three days. Lung pathological changes, expression of inflammatory mediators in bronchoalveolar lavage (BAL) fluid and lungs at mRNA and protein levels, and lung infiltration of CD8+ T cells were compared between CXCR3-/- mice and wild type (WT) mice.

RESULTS

Compared with the WT littermates, CXCR3 KO mice showed less CS-induced lung inflammation as evidenced by less infiltration of inflammatory cells in airways and lung tissue, particularly fewer CD8+ T cells, lower levels of IFNgamma and CXCR3 ligands (particularly CXCL10).

CONCLUSIONS

Our findings show that CXCR3 is important in promoting CD8+ T cell recruitment and in initiating IFNgamma and CXCL10 release following CS exposure. CXCR3 may represent a promising therapeutic target for acute lung inflammation induced by CS.

Ambient air ultrafine particles (UFPs) have gained enormous attention to many researchers with recent evidence showing them to have more hazardous effects on human health than larger ambient particles. Studies focusing the possibility of effects on brain are quite limited. To examine the effect of ultrafine carbon black (ufCB) on mice brain, we instilled 125 microg of 14 nm or 95 nm CB into the nostrils of 8-week-old male BALB/c mice, once a week for 4 weeks. Four hours after the last instillation, we collected olfactory bulb and hippocampus and detected the expression of cytokine and chemokine mRNA by quantitative real-time PCR method. In this study, we found the induction of proinflammatory cytokines (interleukin-1 beta and tumor necrosis factor-alpha and chemokines (monocyte chemoattractant protein-1/CCL2, macrophage inflammatory protein-1 alpha/CCL3), and monokine induced interferon-gamma/CXC chemokine ligand (CXCL9) mRNA in brain olfactory bulb, not in the hippocampus of mice instilled with 14 nm ufCB intranasally. We suggest that the intranasal instillation of ufCB may influence the brain immune function depending on their size. To our knowledge, this is the first study to demonstrate region-specific brain cytokine and chemokine mRNA-induction in mice triggered by intranasal instillation of specific-sized ufCB, in a physiologically relevant condition.

Experimental autoimmune prostatitis (EAP) is a murine model of chronic prostatitis/chronic pelvic pain syndrome (CPPS) in men, a syndrome characterized by chronic pelvic pain. We have demonstrated that chemokine ligands CCL2 and CCL3 are biomarkers that correlate with pelvic pain symptoms. We postulated that CCL2 and CCL3 play a functional role in CPPS and therefore examined their expression in EAP. Upon examination of the prostate 5 days after induction of EAP, CCL2 mRNA was elevated 2- to 3-fold, CCL8 by 15-fold, CCL12 by 12- to 13-fold, and CXCL9 by 2- to 4-fold compared with control mice. At 10 days the major chemokines were CXCL13 and CXCL2; at 20 days CCL2 (1- to 2-fold), CCL3 (2- to 3-fold) and CCL11 (2- to 3-fold); and at 30 days, CCL12 (20- to 35-fold) and smaller increases in CCL2, CCL3, and XCL1. Chemokine elevations were accompanied by increases in mast cells and B cells at 5 days, monocytes and neutrophils at day 10, CD4+ T cells at day 20, and CD4+ and CD8+ T cells at day 30. Anti-CCL2 and anti-CCL3 neutralizing antibodies administered at EAP onset attenuated pelvic pain development, but only anti-CCL2 antibodies were effective therapeutically. CCL2- and its cognate receptor CCR2-deficient mice were completely protected from development of pain symptoms but assumed susceptibility after reconstitution with wild-type bone marrow. CCL3-deficient mice showed resistance to the maintenance of pelvic pain while CCR5-deficient mice did not show any lessening of pelvic pain severity. These results suggest that the CCL2-CCR2 axis and CCL3 are important mediators of chronic pelvic pain in EAP.

Chemokines regulate the migration of hemopoietic cells and play an important role in the pathogenesis of many immune-mediated diseases. Intradermal recruitment of CD8(+) T cells by CXCL10 is a central feature of the pathogenesis of cutaneous acute GVHD (aGVHD), but very little is known about the pathogenesis of chronic GVHD (cGVHD). Serum concentrations of the 3 CXCR3-binding chemokines, CXCL9, CXCL10, and CXCL11, were found to be markedly increased in patients with active cGVHD of the skin (n = 8). An 80% decrease in CD4(+) cells expressing CXCR3 was seen in the blood of these patients (n = 5), whereas CD4(+) cells were increased in tissue biopsies and were clustered around the central arterioles of the dermis. The well-documented increase in expression of CXCL10 in aGVHD therefore diversifies in cGVHD to include additional members of the CXCR3-binding family and leads to preferential recruitment of CD4(+) T cells. These observations reveal a central role for chemokine-mediated recruitment of CXCR3(+) T cells in cGVHD.

BACKGROUND

Systemic inhibition of the inflammatory enzyme cyclooxygenase (COX) 2 decreases the risk of breast cancer and its recurrence. However, the biology of COX-2 in the multicellular tumor microenvironment is poorly defined.

METHODS

Mammary tumor onset and multiplicity were examined in ErbB2 transgenic mice that were deficient in mammary epithelial cell COX-2 (COX-2(MEC)KO) compared to wild type (WT) mice. Tumors were analyzed, by real time PCR, immune-staining and flow cytometry, for proliferation, apoptosis, angiogenesis and immune microenvironment. Lentiviral shRNA delivery was used to knock down (KD) COX-2 in ErbB2-transformed mouse breast cancer cells (COX-2KD), and growth as orthotopic tumors was examined in syngenic recipient mice, with or without depletion of CD8+ immune cells.

RESULTS

Mammary tumor onset was delayed, and multiplicity halved, in COX-2(MEC)KO mice compared to WT. COX-2(MEC)KO tumors showed decreased expression of Ki67, a proliferation marker, as well as reduced VEGFA, its receptor VEGFR2, endothelial NOS and the vascular endothelial marker CD31, indicating reduced tumor vascularization. COX-2(MEC)KO tumors contained more CD4+ T helper (Th) cells and CD8+ cytotoxic immune cells (CTL) consistent with increased immune surveillance. The ratio of Th markers Tbet (Th1) to GATA3 (Th2) was higher, and levels of Retnla, a M2 macrophage marker, lower, in COX-2(MEC)KO tumor infiltrating leukocytes compared to WT, suggesting a prevalence of pro-immune Th1 over immune suppressive Th2 lymphocytes, and reduced macrophage polarization to the immune suppressive M2 phenotype. Enhanced immune surveillance in COX-2(MEC)KO tumors was coincident with increased intratumoral CXCL9, a T cell chemoattractant, and decreased expression of T lymphocyte co-inhibitory receptors CTLA4 and PD-1, as well as PD-L1, the ligand for PD-1. PD-L1 was also decreased in IFNγ-treated COX-2KD mouse mammary cancer cells in vitro and, compared to control cells, growth of COX-2KD cells as orthotopic tumors in immune competent mice was markedly suppressed. However, robust growth of COX-2KD tumor cells was evident when recipients were depleted of CD8+ cells.

CONCLUSIONS

The data strongly support that, in addition to its angiogenic function, tumor cell COX-2 suppresses intratumoral cytotoxic CD8+ immune cell function, possibly through upregulation of immune checkpoints, thereby contributing to tumor immune escape. COX-2 inhibition may be clinically useful to augment breast cancer immunotherapy.

Chemokines and chemokine receptors play a role in cell recruitment during granulomatous inflammatory reactions. Here, we evaluated the expression of chemokines and chemokine receptors and their regulation by IFN-gamma in the course of Paracoccidioides brasiliensis (Pb) infection in mice. We found an association between KC and MIP-1alpha (CCL3) production and neutrophil infiltration in the lungs of Pb-infected mice during the early acute phase of infection. High levels of RANTES/CCL5, MCP-1/CCL2, IP-10/CXCL10, and Mig/CXCL9 simultaneously with mononuclear cell infiltration in the lungs was found. In the absence of IFN-gamma (GKO mice) we observed increased production of KC and MIP-1alpha and chronic neutrophilia. Moreover, we found a change in the chemokine receptor profiles expressed by wild-type (WT) versus GKO animals. Increased expression of CXCR3 and CCR5, and low levels of CCR3 and CCR4 were observed in the lungs of Pb-infected WT mice, whereas the opposite effect was observed in the lungs of GKO mice. Consistent with these results, infected cells from WT mice preferentially migrated in response to IP-10 (CXCR3 ligand), while those from GKO mice migrated in response to eotaxin/CCL11 (CCR3 ligand). These results suggest that IFN-gamma modulates the expression of chemokines and chemokine receptors as well as the kind of cells that infiltrate the lungs of Pb-infected mice.

Sindbis virus (SINV) infection of neurons results in nonfatal viral encephalomyelitis and provides a model system for understanding recovery from virus infection of the central nervous system (CNS). Infection is followed by clearance of infectious virus, a gradual decrease in viral RNA, and then long-term maintenance of low levels of viral RNA. Antibody to the E2 glycoprotein is important for virus clearance, and B cells enter the CNS along with CD4(+) and CD8(+) T cells during the early clearance phase. Antibody-secreting cells (ASCs) are present in the CNS and become enriched for SINV-specific ASCs. We have evaluated the factors within the CNS that facilitate continued local antibody production after infection. Expression of CXCL9, CXCL10, CCL1, CCL2, and CCL5 chemokine mRNAs increased early, and infiltrating B cells expressed CXCR3, CXCR5, and CCR7. The mRNAs for IL-10 and IL-21, cytokines important for B cell proliferation and differentiation, rose rapidly and remained elevated long after clearance of infectious virus. Active proliferation of B cells, as indicated by Ki-67 expression, continued for months. Bromodeoxyuridine (BrdU) labeling of proliferating cells showed that ASCs produced in the draining cervical lymph nodes during the early germinal center response were preferentially retained in the CNS. Sustained increase in B-cell-activating factor (BAFF) mRNA in the CNS and BAFF receptor expression by B cells coincided with the long-term maintenance of SINV-specific ASCs in the brain. We conclude that multiple changes in the brain microenvironment facilitate B-cell entry and support proliferation and differentiation and long-term survival of antiviral ASCs during recovery from alphaviral encephalomyelitis.

BACKGROUND

In murine breast cancer models, the two interferon-gamma (IFN-γ) inducible chemokines and CXC-chemokine receptor 3 (CXCR3) receptor ligands, monokine induced by γ-interferon (CXCL9) and interferon-γ-inducible protein-10 (CXCL10) impair tumor growth and metastasis formation through recruitment of natural killer (NK) cells and tumor-suppressive T lymphocytes. In human breast cancer, CXCL9 mRNA overexpression correlates with the number of tumor infiltrating lymphocytes and predicts response to different chemotherapeutic regimens. Raising the intratumoral CXCR3 ligand concentration is therefore a possible way to enhance immune intervention in breast cancer. Little is known, however, about expression levels and regulation of these chemokines in human breast cancer. Since the inhibition of cyclooxygenases (COX) has been shown to reduce tumor growth and incidence of metastases in a lymphocytic and IFN-γ dependent manner, we argued that COX isoenzymes are a pharmacologic target to increase intratumoral CXCR3 ligand concentration in human breast cancer.

METHODS

CXCL9 was visualized in breast cancer specimens by immunohistochemistry, expression levels of CXCL9 and cyclooxygenases were determined by ELISA and western blotting, respectively. For regulation studies, Michigan Cancer Foundation-7 (MCF-7) and M.D. Anderson - Metastatic Breast 231 (MDA-MB 231) breast cancer cells were stimulated with IFN-γ with or without prostaglandin E2 (PGE2) or COX inhibitors (indomethacin, acetylsalicylic acid (ASA), celecoxib). CXCR3 ligand release from cells was measured by ELISA.

RESULTS

Within the tumor microenvironment, cancer cells are the major source of CXCL9. PGE2 impairs IFN-γ mediated CXCL9 and CXCL10 release from MCF-7 and MDA-MB 231 cells, and inhibition of endogenous cyclooxygenases by indomethacin or ASA correspondingly increases this secretion. Otherwise, high concentrations of the Cyclooxygenase-2 (COX-2) specific antagonist celecoxib have opposite effects and impair CXCL9 and CXCL10 release. In human breast cancer tissue specimens there is an inverse correlation between COX-2 overexpression and CXCL9 concentration, suggesting that the observed in vitro effects are of importance in vivo as well.

CONCLUSIONS

Suppressing endogenous PGE2 synthesis by cyclooxygenase inhibition increases CXCL9 and CXCL10 release from breast cancer cells and is therefore a pharmacologic candidate to enhance intratumoral immune infiltration. Yet, to this end the unselective COX inhibitors ASA and indomethacin seem preferable to celecoxib that at higher concentrations reduces CXCR3 ligand release most probably due to COX independent mechanisms.

Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with immune dysbiosis. In this study, we found that P. gingivalis did not induce the expression of the T-cell chemokine IP-10 (CXCL10) from neutrophils, peripheral blood mononuclear cells (PBMCs), or gingival epithelial cells. Furthermore, P. gingivalis suppressed gamma interferon (IFN-γ)-stimulated release of IP-10, ITAC (CXCL11), and Mig (CXCL9) from epithelial cells and inhibited IP-10 secretion in a mixed infection with the otherwise stimulatory Fusobacterium nucleatum. Inhibition of chemokine expression occurred at the level of gene transcription and was associated with downregulation of interferon regulatory factor 1 (IRF-1) and decreased levels of Stat1. Ectopic expression of IRF-1 in epithelial cells relieved P. gingivalis-induced inhibition of IP-10 release. Direct contact between P. gingivalis and epithelial cells was not required for IP-10 inhibition. These results highlight the immune-disruptive potential of P. gingivalis. Suppression of IP-10 and other Th1-biasing chemokines by P. gingivalis may perturb the balance of protective and destructive immunity in the periodontal tissues and facilitate the pathogenicity of oral microbial communities.

OBJECTIVE

To assess whether serum levels of CC and CXC chemokines correlate with disease activity in patients with rheumatoid arthritis (RA), and to determine whether these effects predict clinical response.

METHODS

Serum levels of the chemokines CC (CCL2, CCL5) and CXC (CXCL8, CXCL9, CXCL10) were quantified at baseline and after 12 weeks of treatment with disease-modifying antirheumatic drugs or biologic agents in 28 patients using flow cytometry. Serum from 40 healthy individuals was collected for comparison at baseline. Response to treatment was classified according to the European League Against Rheumatism (EULAR) response criteria. Remission of disease was defined as a Disease Activity Score < 2.6.

RESULTS

The baseline serum concentrations of CC and CXC chemokines were significantly elevated in patients with active RA compared to healthy controls (p < 0.05) except for CCL2. Significant improvement in all disease activity measurements was observed after 12 weeks of treatment. Seventeen (60.7%) patients achieved good to moderate response based on the EULAR response criteria, and 5 (17.9%) patients achieved remission. The improvement in clinical activity in patients with RA was accompanied by a significant reduction in the serum concentration of CXCL9 and CXCL10 (p < 0.001). A significant reduction in the serum level of CXCL10 was also observed in the group that achieved EULAR response. Serum concentration of CCL5 remained significantly elevated in patients with RA (n = 5) who achieved remission compared to the healthy controls (p < 0.05).

CONCLUSIONS

Serum concentration of CXCL9 and CXCL10 may serve as sensitive biomarkers for disease activity in patients with RA.

OBJECTIVE

We assayed mRNA expression of the TRP family of channels and ASIC1 in bladder tissue from patients with interstitial cystitis.

METHODS

Bladder biopsies of 1) nonclassic interstitial cystitis, 2) nonulcerative portions of classic interstitial cystitis, 3) ulcerative portions of classic interstitial cystitis and 4) noncancerous portions of bladder cancer as the control were placed immediately in ice-cold RNAlater® and subjected to real-time reverse transcriptase-polymerase chain reaction. We compared the mRNA expression of TRP channels, ASIC1, NGF, CXCL9 and UPK3A with that of controls, and correlated expression with symptom severity.

RESULTS

We analyzed specimens from 17 patients with nonclassic interstitial cystitis, 22 with classic interstitial cystitis and 11 controls. In nonclassic interstitial cystitis samples TRPV2 and NGF showed significantly increased expression. In classic interstitial cystitis samples nonulcerative portions demonstrated a significant increase in the expression of TRPA1, TRPM2 and 8, TRPV1 and 2, ASIC1, NGF and CXCL9, and a significant decrease in UPK3A and TRPV4. Ulcerative portions showed similar changes for TRPM2, TRPV1, 2 and 4, CXCL9 and UPK3A. Increased expression of TRPM2, first noted in interstitial cystitis tissue, was the most pronounced one of the TRP family. All symptom measures correlated with TRPM2 and TRPV2 expression, and partially with that of the other genes.

CONCLUSIONS

This study showed increased expression of the genes involved in pronociceptive inflammatory reactions in interstitial cystitis, including TRPV1, 2 and 4, ASIC1, NGF and CXCL9, and to our knowledge TRPM2 for the first time. The different expression patterns suggest distinct pathophysiologies for classic and nonclassic interstitial cystitis. The genes and their products are potential candidates for use as biomarkers or novel therapy targets.

BACKGROUND

Interleukin (IL)-27 is a novel member of the IL-6/IL-12 family cytokines that are produced early by antigen-presenting cells in T helper (Th)1-mediated inflammation. Elevated expression of IL-27 has been detected in the synovial membranes and fluid of rheumatoid arthritis (RA).

METHODS

We investigated the in vitro effects of IL-27, alone or in combination with inflammatory cytokine tumor necrosis factor (TNF)-α or IL-1 β on the pro-inflammatory activation of human primary fibroblast-like synoviocytes (FLS) from RA patients and normal control subjects, and the underlying intracellular signaling molecules were determined by intracellular staining using flow cytometry.

RESULTS

Significantly higher plasma concentration of IL-27 was found in RA patients (n = 112) than control subjects (n = 46). Both control and RA-FLS constitutively express functional IL-27 receptor heterodimer, gp130 and WSX-1, with more potent IL-27-mediated activation of signal transducers and activators of transcription (STAT)1 in RA-FLS. IL-27 was found to induce significantly higher cell surface expression of intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 and release of inflammatory chemokine IL-6, CCL2, CXCL9, CXCL10 and matrix metalloproteinase-1 of RA-FLS than that of control FLS (all P < 0.05). Moreover, an additive or synergistic effect was observed in the combined treatment of IL-27 and TNF-α or IL-1 β on the surface expression of ICAM-1 and VCAM-1 and the release of CXCL9 and CXCL10 of RA-FLS. Further investigations showed that the expression of ICAM-1, VCAM-1 and chemokines stimulated by IL-27 was differentially regulated by intracellular activation of phosphatidylinositol 3-OH kinase-AKT, c-Jun amino-terminal kinase and Janus kinase pathways.

CONCLUSIONS

Our results therefore provide a new insight into the IL-27-activated immunopathological mechanisms mediated by distinct intracellular signal transductions in joint inflammation of RA.

The immunological significance of IL-27 has been reported and discussed in various Th1/Th17-mediated inflammatory diseases. However, its importance in psoriasis is unknown. We investigated pathophysiological roles of IL-27 in psoriasis in this study. Serum IL-27 levels in psoriatic patients were significantly higher than those in healthy controls, and correlated with disease severity and serum IFN-gamma levels. An immunohistochemical analysis revealed the infiltration of IL-27-secreting cells in the papillary dermis of psoriatic skin lesions but not in skin lesions with atopic dermatitis or normal skin. Furthermore, IL-27 alone greatly induced in vitro CXCL9, CXCL10, and CXCL11 production and tyrosine phosphorylation of signal transducer and activator of transcription 1 in normal human keratinocytes, while it suppressed the tumor necrosis factor-alpha-induced production of IL-1alpha and CCL20. These results indicate that IL-27 may promote the onset of psoriasis, while it may simultaneously attenuate the expanded inflammation in this disease. Our results implicate potential therapeutic effects of IL-27 for psoriasis.

OBJECTIVE

Atherosclerosis is a chronic inflammatory disease with immune cell infiltration. Various cytokines and chemokines have been characterized as pro- or antiatherogenic factors. Interleukin-20 (IL-20) belongs to the IL-10 family and is a proinflammatory cytokine involved in the pathogenesis of psoriasis. However, the association between IL-20 and atherosclerosis is undetermined. Therefore, we sought to investigate whether IL-20 is associated with atherosclerosis.

RESULTS

We examined the expression of IL-20 and its receptor complex IL-20R1/IL-20R2 in atherosclerotic lesions of humans and mice using immunohistochemical staining. IL-20 was expressed in macrophage-rich areas. Both IL-20 and IL-20R1/IL-20R2 were expressed by endothelial cells lining the intimal microvessels, vasa vasorum, but rarely in nonatherosclerotic arteries. We used reverse-transcription polymerase chain reaction to analyze gene expression. IL-20 transcripts increased in hypoxic monocytes and monocytes treated with oxidized low-density lipoprotein. The expression of IL-20R1 and IL-20R2 was also upregulated by human umbilical vein endothelial cells in response to hypoxic treatment. Incubating IL-20 with human umbilical vein endothelial cells upregulated CXCL9 and CXCL11 transcripts. Furthermore, in vivo administration of IL-20 expression vector using intramuscular electroporation promoted atherosclerosis in apolipoprotein E-deficient mice.

CONCLUSIONS

Our data suggest that IL-20 is a proatherogenic cytokine that contributes to the progression of atherosclerosis.

BACKGROUND

Mucosal CXC chemokines recruit inflammatory cells to the infected urinary tract. The chemokine response repertoire of the urinary tract and the relationship to disease severity have not been examined, however.

METHODS

This study quantified CXC (CXCL1, CXCL3, CXCL5, CXCL8, CXCL9, and CXCL10) and CC (CCL2, CCL4, and CCL5) chemokines in sequential urine samples obtained from 50 patients with febrile urinary tract infections during 24 hours after diagnosis.

RESULTS

All patients had elevated chemokine levels, but bacteremic infections caused higher CXCL1, CXCL3, CXCL5, CXCL8, and CCL2 responses. CCL2 and CXCL8 levels were higher in patients with acute pyelonephritis symptoms and CCL2, CXCL3, CCL4, CXCL5, and CXCL10 were significantly correlated to C-reactive protein (CRP) and temperature. Women and men showed different chemokine responses.

CONCLUSIONS

Febrile urinary tract infections are accompanied by a complex chemokine response. The response magnitude reflects disease severity, and the repertoire is influenced by gender and underlying disease.

BACKGROUND

Two variants of the CXCR3 receptor exist, one (CXCR3-A) reactive with CXCL9, CXCL10, and CXCL11 and the other (CXCR3-B) also reactive with CXCL4. Both variants are contemporarily expressed by human T cells.

OBJECTIVE

We sought to investigate the in vitro effects of CXCL10 and CXCL4 on the production of TH1 or TH2 cytokines.

METHODS

The cytokine profile of antigen-specific human CD4+ T-cell lines obtained in the absence or presence of CXCL10 or CXCL4 was evaluated by means of quantitative RT-PCR, flow cytometry, and ELISA.

RESULTS

CXCL10 upregulated IFN-gamma and downregulated IL-4, IL-5, and IL-13 production, whereas CXCL4 downregulated IFN-gamma and upregulated TH2 cytokines. Similar effects were also observed on polyclonally activated pure naive CD4+ T cells. The opposite effects of CXCL10 and CXCL4 on TH1 and TH2 cytokine production were inhibited by an anti-CXCR3 antibody able to neutralize both CXCR3-A and CXCR3-B and were apparently related to the activation of distinct signal transduction pathways. Moreover, CXCL10 upregulated mRNA levels of T-box expressed in T cells and downregulated GATA-3 expression, whereas CXCL4 downregulated T-box expressed in T cells and upregulated GATA-3. Finally, CXCL4, but not CXCL10, induced direct activation of IL-5 and IL-13 promoters.

CONCLUSIONS

CXCL10 and CXCL4 exert opposite effects on the production of human TH1 and TH2 cytokines, likely through their respective interaction with CXCR3-A or CXCR3-B and the consequent activation of different signal transduction pathways. This might represent an internal regulatory pathway of TH cell responses and might contribute to the modulation of chronic inflammatory reactions, including allergy.

Citrobacter rodentium infection of mice induces cell-mediated immune responses associated with crypt hyperplasia and epithelial β-catenin signaling. Recent data suggest that phosphatidylinositol-3-kinase (PI3K)/Akt signaling cooperates with Wnt to activate β-catenin in intestinal stem and progenitor cells through phosphorylation at Ser552 (P-β-catenin(552)). Our aim was to determine whether epithelial PI3K/Akt activation is required for β-catenin signaling and host defense against C. rodentium. C57BL/6 mice were infected with C. rodentium and treated with dimethyl sulfoxide (DMSO) (vehicle control) or with the PI3K inhibitor LY294002 or wortmannin. The effects of infection on PI3K activation and β-catenin signaling were analyzed by immunohistochemistry. The effects of PI3K inhibition on host defense were analyzed by the quantification of splenic and colon bacterial clearance, and adaptive immune responses were measured by real-time PCR (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). Increased numbers of P-β-catenin(552)-stained epithelial cells were found throughout expanded crypts in C. rodentium colitis. We show that the inhibition of PI3K signaling attenuates epithelial Akt activation, the Ser552 phosphorylation and activation of β-catenin, and epithelial cell proliferative responses during C. rodentium infection. PI3K inhibition impairs bacterial clearance despite having no impact on mucosal cytokine (gamma interferon [IFN-γ], tumor necrosis factor [TNF], interleukin-17 [IL-17], and IL-1β) or chemokine (CXCL1, CXCL5, CXCL9, and CXCL10) induction. The results suggest that the host defense against C. rodentium requires epithelial PI3K activation to induce Akt-mediated β-catenin signaling and the clearance of C. rodentium independent of adaptive immune responses.

Murine cytomegalovirus (MCMV) brain infection induces a transient increase in chemokine production, which precedes the infiltration of CD3(+) lymphocytes. In this study, we hypothesized that an absence of anti-inflammatory cytokines would result in sustained proinflammatory neuroimmune responses. Direct intracerebroventricular injection of MCMV into IL-10 knockout (KO) mice produced an unexpected result: while wild-type animals controlled MCMV, the infection was lethal in IL-10 KO animals. Identical infection of IL-4 KO animals did not produce lethal disease. To further characterize the role of IL-10, infected brain tissue from both wild-type and IL-10 KO animals was assessed for cytokine and chemokine levels, as well as viral gene expression. These data show vastly elevated levels of interferon (IFN)-gamma, and the IFN-gamma-inducible chemokines CXCL9 and CXCL10, as well as IL-6 in brain homogenates obtained from IL-10 KO animals. However, MCMV viral load, glycoprotein B mRNA levels, and titers of infectious virus were similar in both IL-10 KO and wild-type animals. Separation of cells isolated from murine brain tissue into distinct populations using FACS, along with subsequent quantitative RT real-time PCR, showed that brain-infiltrating CD45(hi)/CD11b(-) and CD45(hi)/CD11b(int) were the cellular source of IL-10 in the brain. Taken together, these data demonstrate that MCMV brain infection of IL-10-deficient mice causes lethal disease, which occurs in the presence of a dysregulated IFN-gamma-mediated neuroimmune response.

Donor Ag-reactive CD4 and CD8 T cell production of IFN-gamma is a principal effector mechanism promoting tissue injury during allograft rejection. The CXCR3-binding chemokines CXCL9 and CXCL10 recruit donor-reactive T cells to the allograft, but their role during the priming of donor-reactive T cells to effector function is unknown. Using a murine model of MHC-mismatched cardiac transplantation, we investigated the influence of CXCL9 and CXCL10 during donor-reactive T cell priming. In allograft recipient spleens, CXCL9 and CXCL10 were expressed as early as 24 h posttransplant and increased with similar kinetics, concurrently with CXCR3 expression on T cells. CXCL9, but not CXCL10, expression required NK cell production of IFN-gamma. The absence of CXCL9 in donor allografts, recipients, or both significantly decreased the frequency of donor-reactive CD8 T cells producing IFN-gamma and increased the frequency of donor-reactive CD8 T cells producing IL-17A. In contrast, the absence of CXCL10 increased the frequency of IFN-gamma-producing CD8 T cells in a CXCL9-dependent manner. These data provide novel evidence that donor-reactive CD8 T cells use the CXCR3 chemokine axis as a costimulation pathway during priming to allografts where CXCL9 promotes the development of IFN-gamma-producing CD8 T cells, and CXCL10 antagonizes this skewing.

Microbial infections of the central nervous system (CNS) are often associated with local accumulation of antibody (Ab)-secreting cells (ASC). By providing a source of Ab at the site of infection, CNS-localized ASC play a critical role in acute viral control and in preventing viral recrudescence. Following coronavirus-induced encephalomyelitis, the CNS accumulation of ASC is chemokine (C-X-C motif) receptor 3 (CXCR3) dependent. This study demonstrates that CNS-expressed CXCR3 ligand CXCL10 is the critical chemokine regulating ASC accumulation. Impaired ASC recruitment in CXCL10(-/-) but not CXCL9(-/-) mice was consistent with reduced CNS IgG and κ-light chain mRNA and virus-specific Ab. Moreover, the few ASC recruited to the CNS in CXCL10(-/-) mice were confined to the vasculature, distinct from the parenchymal localization in wild-type and CXCL9(-/-) mice. However, neither CXCL9 nor CXCL10 deficiency diminished neutralizing serum Ab, supporting a direct role for CXCL10 in ASC migration. T cell accumulation, localization, and effector functions were also not affected in either CXCL9(-/-) or CXCL10(-/-) mice, consistent with similar control of infectious virus. There was also no evidence for dysregulation of chemokines or cytokines involved in ASC regulation. The distinct roles of CXCL9 and CXCL10 in ASC accumulation rather coincided with their differential localization. While CXCL10 was predominantly expressed by astrocytes, CXCL9 expression was confined to the vasculature/perivascular spaces. These results suggest that CXCL10 is critical for two phases: recruitment of ASC to the CNS vasculature and ASC entry into the CNS parenchyma.

Transmigration through the liver endothelium is a prerequisite for the homeostatic balance of intrahepatic T cells and a key regulator of inflammatory processes within the liver. Extravasation into the liver parenchyma is regulated by the distinct expression patterns of adhesion molecules and chemokines and their receptors on the lymphocyte and endothelial cell surface. In the present study, we investigated whether liver sinusoidal endothelial cells (LSEC) inhibit or support the chemokine-driven transmigration and differentially influence the transmigration of pro-inflammatory or anti-inflammatory CD4(+) T cells, indicating a mechanism of hepatic immunoregulation. Finally, the results shed light on the molecular mechanisms by which LSEC modulate chemokine-dependent transmigration. LSEC significantly enhanced the chemotactic effect of CXC-motif chemokine ligand 12 (CXCL12) and CXCL9, but not of CXCL16 or CCL20, on naive and memory CD4(+) T cells of a T helper 1, T helper 2, or interleukin-10-producing phenotype. In contrast, brain and lymphatic endothelioma cells and ex vivo isolated lung endothelia inhibited chemokine-driven transmigration. As for the molecular mechanisms, chemokine-induced activation of LSEC was excluded by blockage of G(i)-protein-coupled signaling and the use of knockout mice. After preincubation of CXCL12 to the basal side, LSEC took up CXCL12 and enhanced transmigration as efficiently as in the presence of the soluble chemokine. Blockage of transcytosis in LSEC significantly inhibited this effect, and this suggested that chemokines taken up from the basolateral side and presented on the luminal side of endothelial cells trigger T cell transmigration.

CONCLUSIONS

Our findings demonstrate a unique capacity of LSEC to present chemokines to circulating lymphocytes and highlight the importance of endothelial cells for the in vivo effects of chemokines. Chemokine presentation by LSEC could provide a future therapeutic target for inhibiting lymphocyte immigration and suppressing hepatic inflammation.