A-910823, a squalene-based emulsion adjuvant, induces T follicular helper cells and humoral immune responses via α-tocopherol component.

Background: Adjuvants are chemical or biological materials that enhance the efficacy of vaccines. A-910823 is a squalene-based emulsion adjuvant used for S-268019-b, a novel vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is currently in clinical development. Published evidence has demonstrated that A-910823 can enhance the induction of neutralizing antibodies against SARS-CoV-2 in humans and animal models. However, the characteristics and mechanisms of the immune responses induced by A-910823 are not yet known. Methods and Results: To characterize A-910823, we compared the adaptive immune response profile enhanced by A-910823 with that of other adjuvants (AddaVax, QS21, aluminum salt-based adjuvants, and empty lipid nanoparticle [eLNP]) in a murine model. Compared with other adjuvants, A-910823 enhanced humoral immune responses to an equal or greater extent following potent T follicular helper (Tfh) and germinal center B (GCB) cell induction, without inducing a strong systemic inflammatory cytokine response. Furthermore, S-268019-b containing A-910823 adjuvant produced similar results even when given as a booster dose following primary administration of a lipid nanoparticle-encapsulated messenger RNA (mRNA-LNP) vaccine. Preparation of modified A-910823 adjuvants to identify which components of A-910823 play a role in driving the adjuvant effect and detailed evaluation of the immunological characteristics induced by each adjuvant showed that the induction of humoral immunity and Tfh and GCB cell induction in A-910823 were dependent on α-tocopherol. Finally, we revealed that the recruitment of inflammatory cells to the draining lymph nodes and induction of serum cytokines and chemokines by A-910823 were also dependent on the α-tocopherol component. Conclusions: This study demonstrates that the novel adjuvant A-910823 is capable of robust Tfh cell induction and humoral immune responses, even when given as a booster dose. The findings also emphasize that α-tocopherol drives the potent Tfh-inducing adjuvant function of A-910823. Overall, our data provide key information that may inform the future production of improved adjuvants.

[Creation of Protective Equipment for Portable Radiography in Neonatal Intensive Care Unit].

Portable imaging in the NICU requires the assistance of a nurse, and the nurse is in close proximity to the X-ray tube, In all, 64 percent of our nurses thought that additional protective equipment was needed. Therefore, a radiation protection device was created and its usefulness was verified. A protective equipment of 0.13 mmPb with a width of 38 cm and a length of 70 cm was made and hung from the mono-tank X-ray unit of the mobile X-ray unit. The position of the nurse was set at 30 cm outward from the center of the irradiation field, and the protective effect was measured at three points: (a) the patient's height, (b) 30 cm above the patient, and (c) 60 cm above the patient. For the imaging conditions, a 2-liter plastic bottle filled with water was placed in the incubator, and measurements were taken with an SID of 100 cm, irradiated field of 20.3 cm×25.4 cm, tube voltage of 58 kV, and tube current-time product of 10 mAs, which was converted to the actual imaging condition of 1 mAs. Based on the results obtained, a questionnaire survey was conducted on nurses' thoughts for the protective equipment created for them. Only 3% reduction in height of (a) where no protective equipment is reached but (b) 50% and (c) 92%, respectively. In all, 82 percent of the nurses had a favorable impression of the new protective equipment. It is expected that the protective equipment designed to control lens dose and reduce anxiety of nurses will be useful.

Generation of Gαi knock-out HEK293 cells illuminates Gαi-coupling diversity of GPCRs.

G-protein-coupled receptors (GPCRs) are pivotal cell membrane proteins that sense extracellular molecules and activate cellular responses. The G-protein α subunit i (Gαi) family represents the most common GPCR-coupling partner and consists of eight subunits with distinct signaling properties. However, analyzing the coupling pattern has been challenging owing to endogenous expression of the Gαi subunits in virtually all cell lines. Here, we generate a HEK293 cell line lacking all Gαi subunits, which enables the measurement of GPCR-Gαi coupling upon transient re-expression of a specific Gαi subunit. We profile Gαi-coupling selectivity across 11 GPCRs by measuring ligand-induced inhibitory activity for cAMP accumulation. The coupling profiles are then classified into three clusters, representing those preferentially coupled to Gαz, those to Gαo, and those with unapparent selectivity. These results indicate that individual Gαi-coupled GPCRs fine-tune Gαi signaling by exerting coupling preference at the Gαi-subunit level.

Ectodomain shedding of EGFR ligands serves as an activation readout for TRP channels.

Transient receptor potential (TRP) channels are activated by various extracellular and intracellular stimuli and are involved in many physiological events. Because compounds that act on TRP channels are potential candidates for therapeutic agents, a simple method for evaluating TRP channel activation is needed. In this study, we demonstrated that a transforming growth factor alpha (TGFα) shedding assay, previously developed for detecting G-protein-coupled receptor (GPCR) activation, can also detect TRP channel activation. This assay is a low-cost, easily accessible method that requires only an absorbance microplate reader. Mechanistically, TRP-channel-triggered TGFα shedding is achieved by both of a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10) and 17 (ADAM17), whereas the GPCR-induced TGFα shedding response depends solely on ADAM17. This difference may be the result of qualitative or quantitative differences in intracellular Ca2+ kinetics between TRP channels and GPCRs. Use of epidermal growth factor (EGF) and betacellulin (BTC), substrates of ADAM10, improved the specificity of the shedding assay by reducing background responses mediated by endogenously expressed GPCRs. This assay for TRP channel measurement will not only facilitate the high-throughput screening of TRP channel ligands but also contribute to understanding the roles played by TRP channels as regulators of membrane protein ectodomain shedding.

[Usefulness of Radiation Protection Cloths in Fluoroscopy with Clean Areas].

Lowering the dose limit for the lens of the eye incorporated into the Regulation on Prevention of Ionizing Radiation Hazards, effective on April 2021, and dose reduction will become more and more important in the field of radiation. Radiation protective cloth is used as a protective equipment in fluoroscopy rooms. Although it is usually used to protect staff from radiation exposure during endoscopic retrograde cholangiopancreatography, we investigated whether there is a way to use it for procedures in clean areas. Assuming ureterostomy fistula replacement in urology, the protective cloth was suspended on the side of the patient's head and posterior aspect of the tube, and the distance between the anterior aspect of the X-ray tube and the patient's foot was 55 cm. As a result of measuring the dose rate, a 10% dose reduction was obtained for the lens of the eye of the surgeon, and the distribution of air dose rate in the examination room was significantly reduced. Although scattered radiation from the radiation protection cloth appeared in some areas, the radiation dose to the patient was reduced throughout the body, and a high degree of radiation protection was obtained, especially for the lens of the eye. It is expected that the radiation protection cloths may be useful even when the length of the cloths is limited due to the cleanliness of the area.

[Comparison of Frontal and Lateral Measurements of Bone Mineral Density of the Lumbar Spine with Dual-energy X-ray Absorptiometry (DXA) in Spinal Deformity: a Phantom Study].

The bone mineral density (BMD) measurement of the lumbar spine with dual-energy X-ray absorptiometry (DXA) has the advantage of being able to detect early changes in BMD, which is usually used for the evaluation of drug therapy. However, DXA is not considered suitable for spinal deformity because it is a two-dimensional measurement. The aims of this study were to compare frontal and lateral measurements with a phantom and to examine the possibility of the evaluation of lumbar spine BMD in spinal deformity. The values of frontal and lateral measurements were compared when the lumbar phantom was tilted by 10 degrees from 0 to 40 degrees, assuming kyphosis, and when it was tilted by 5 degrees from 0 to 10 degrees to the right and left, assuming scoliosis. We revealed that in the case of kyphosis, the frontal is more accurate, and in the case of scoliosis, the lateral is more accurate; small rotation of subjects on the plane parallel to the image receiving surface could be acceptable. In general, the two-directional BMD measurement is useful for the improvement of the accuracy and may have a potential to measure patients with spinal deformity, which was previously thought to be impossible.

Low-Dose of Intrapulmonary Pirfenidone Improves Human Transforming Growth Factorß1-Driven Lung Fibrosis.

Idiopathic pulmonary fibrosis is a chronic, progressive, and lethal lung disease of unknown etiology. Antifibrotic drugs, including pirfenidone, are currently used for the treatment of the disease. The oral administration of pirfenidone is an effective therapy, as demonstrated by several clinical trials, although it causes severe adverse events in some patients. We hypothesized that low-dose intrapulmonary delivery of pirfenidone is effective in human transforming growth factorß1-driven pulmonary fibrosis. To demonstrate our hypothesis, we compared the therapeutic efficacy of varying doses of pirfenidone administered by oral and intranasal routes in a human transforming growth factor-ß1 transgenic mouse with established pulmonary fibrosis. We found similar amelioration of lung cell infiltration, inflammatory and fibrotic cytokines, lung fibrosis score, and hydroxyproline content in mice with human transforming growth factor-ß1-mediated pulmonary fibrosis treated with low-dose intranasal pirfenidone and high-dose oral pirfenidone. This study showed that pirfenidone is a potent inhibitor of human transforming growth factor-ß1-driven lung fibrosis and that intrapulmonary delivery of low-dose pirfenidone produces therapeutic responses equivalent to high-dose of oral pirfenidone.

Illuminating G-Protein-Coupling Selectivity of GPCRs.

Heterotrimetic G proteins consist of four subfamilies (Gs, Gi/o, Gq/11, and G12/13) that mediate signaling via G-protein-coupled receptors (GPCRs), principally by receptors binding Gα C termini. G-protein-coupling profiles govern GPCR-induced cellular responses, yet receptor sequence selectivity determinants remain elusive. Here, we systematically quantified ligand-induced interactions between 148 GPCRs and all 11 unique Gα subunit C termini. For each receptor, we probed chimeric Gα subunit activation via a transforming growth factor-α (TGF-α) shedding response in HEK293 cells lacking endogenous Gq/11 and G12/13 proteins, and complemented G-protein-coupling profiles through a NanoBiT-G-protein dissociation assay. Interrogation of the dataset identified sequence-based coupling specificity features, inside and outside the transmembrane domain, which we used to develop a coupling predictor that outperforms previous methods. We used the predictor to engineer designer GPCRs selectively coupled to G12. This dataset of fine-tuned signaling mechanisms for diverse GPCRs is a valuable resource for research in GPCR signaling.

Lysolipid receptor cross-talk regulates lymphatic endothelial junctions in lymph nodes.

Sphingosine 1-phosphate (S1P) and lysophosphatidic acid (LPA) activate G protein-coupled receptors (GPCRs) to regulate biological processes. Using a genome-wide CRISPR/dCas9-based GPCR signaling screen, LPAR1 was identified as an inducer of S1PR1/ß-arrestin coupling while suppressing Gαi signaling. S1pr1 and Lpar1-positive lymphatic endothelial cells (LECs) of lymph nodes exhibit constitutive S1PR1/ß-arrestin signaling, which was suppressed by LPAR1 antagonism. Pharmacological inhibition or genetic loss of function of Lpar1 reduced the frequency of punctate junctions at sinus-lining LECs. Ligand activation of transfected LPAR1 in endothelial cells remodeled junctions from continuous to punctate structures and increased transendothelial permeability. In addition, LPAR1 antagonism in mice increased lymph node retention of adoptively transferred lymphocytes. These data suggest that cross-talk between LPAR1 and S1PR1 promotes the porous junctional architecture of sinus-lining LECs, which enables efficient lymphocyte trafficking. Heterotypic inter-GPCR coupling may regulate complex cellular phenotypes in physiological milieu containing many GPCR ligands.

Lack of beta-arrestin signaling in the absence of active G proteins.

G protein-independent, arrestin-dependent signaling is a paradigm that broadens the signaling scope of G protein-coupled receptors (GPCRs) beyond G proteins for numerous biological processes. However, arrestin signaling in the collective absence of functional G proteins has never been demonstrated. Here we achieve a state of "zero functional G" at the cellular level using HEK293 cells depleted by CRISPR/Cas9 technology of the Gs/q/12 families of Gα proteins, along with pertussis toxin-mediated inactivation of Gi/o. Together with HEK293 cells lacking ß-arrestins ("zero arrestin"), we systematically dissect G protein- from arrestin-driven signaling outcomes for a broad set of GPCRs. We use biochemical, biophysical, label-free whole-cell biosensing and ERK phosphorylation to identify four salient features for all receptors at "zero functional G": arrestin recruitment and internalization, but-unexpectedly-complete failure to activate ERK and whole-cell responses. These findings change our understanding of how GPCRs function and in particular of how they activate ERK1/2.

Hyper-reactive cloned mice generated by direct nuclear transfer of antigen-specific CD4+ T cells.

T-cell receptor (TCR)-transgenic mice have been employed for evaluating antigen-response mechanisms, but their non-endogenous TCR might induce immune response differently than the physiologically expressed TCR Nuclear transfer cloning produces animals that retain the donor genotype in all tissues including germline and immune systems. Taking advantage of this feature, we generated cloned mice that carry endogenously rearranged TCR genes from antigen-specific CD4+ T cells. We show that T cells of the cloned mice display distinct developmental pattern and antigen reactivity because of their endogenously pre-rearranged TCRα (rTα) and TCRß (rTß) alleles. These alleles were transmitted to the offspring, allowing us to establish a set of mouse lines that show chronic-type allergic phenotypes, that is, bronchial and nasal inflammation, upon local administrations of the corresponding antigens. Intriguingly, the existence of either rTα or rTß is sufficient to induce in vivo hypersensitivity. These cloned mice expressing intrinsic promoter-regulated antigen-specific TCR are a unique animal model with allergic predisposition for investigating CD4+ T-cell-mediated pathogenesis and cellular commitment in immune diseases.

Purinergic Receptor Transactivation by the ß2-Adrenergic Receptor Increases Intracellular Ca2+ in Nonexcitable Cells.

The ß2 adrenergic receptor (ß2AR) increases intracellular Ca2+ in a variety of cell types. By combining pharmacological and genetic manipulations, we reveal a novel mechanism through which the ß2AR promotes Ca2+ mobilization (pEC50 = 7.32 ± 0.10) in nonexcitable human embryonic kidney (HEK)293S cells. Downregulation of Gs with sustained cholera toxin pretreatment and the use of Gs-null HEK293 (∆Gs-HEK293) cells generated using the clustered regularly interspaced short palindromic repeat-associated protein-9 nuclease (CRISPR/Cas9) system, combined with pharmacological modulation of cAMP formation, revealed a Gs-dependent but cAMP-independent increase in intracellular Ca2+ following ß2AR stimulation. The increase in cytoplasmic Ca2+ was inhibited by P2Y purinergic receptor antagonists as well as a dominant-negative mutant form of Gq, a Gq-selective inhibitor, and an inositol 1,4,5-trisphosphate (IP3) receptor antagonist, suggesting a role for this Gq-coupled receptor family downstream of the ß2AR activation. Consistent with this mechanism, ß2AR stimulation promoted the extracellular release of ATP, and pretreatment with apyrase inhibited the ß2AR-promoted Ca2+ mobilization. Together, these data support a model whereby the ß2AR stimulates a Gs-dependent release of ATP, which transactivates Gq-coupled P2Y receptors through an inside-out mechanism, leading to a Gq- and IP3-dependent Ca2+ mobilization from intracellular stores. Given that ß2AR and P2Y receptors are coexpressed in various tissues, this novel signaling paradigm could be physiologically important and have therapeutic implications. In addition, this study reports the generation and validation of HEK293 cells deleted of Gs using the CRISPR/Cas9 genome editing technology that will undoubtedly be powerful tools to study Gs-dependent signaling.

Peanut oral immunotherapy results in increased antigen-induced regulatory T-cell function and hypomethylation of forkhead box protein 3 (FOXP3).

BACKGROUND: The mechanisms contributing to clinical immune tolerance remain incompletely understood. This study provides evidence for specific immune mechanisms that are associated with a model of operationally defined clinical tolerance. OBJECTIVE: Our overall objective was to study laboratory changes associated with clinical immune tolerance in antigen-induced T cells, basophils, and antibodies in subjects undergoing oral immunotherapy (OIT) for peanut allergy. METHODS: In a phase 1 single-site study, we studied participants (n = 23) undergoing peanut OIT and compared them with age-matched allergic control subjects (n = 20) undergoing standard of care (abstaining from peanut) for 24 months. Participants were operationally defined as clinically immune tolerant (IT) if they had no detectable allergic reactions to a peanut oral food challenge after 3 months of therapy withdrawal (IT, n = 7), whereas those who had an allergic reaction were categorized as nontolerant (NT; n = 13). RESULTS: Antibody and basophil activation measurements did not statistically differentiate between NT versus IT participants. However, T-cell function and demethylation of forkhead box protein 3 (FOXP3) CpG sites in antigen-induced regulatory T cells were significantly different between IT versus NT participants. When IT participants were withdrawn from peanut therapy for an additional 3 months (total of 6 months), only 3 participants remained classified as IT participants, and 4 participants regained sensitivity along with increased methylation of FOXP3 CpG sites in antigen-induced regulatory T cells. CONCLUSION: In summary, modifications at the DNA level of antigen-induced T-cell subsets might be predictive of a state of operationally defined clinical immune tolerance during peanut OIT.

αvß5 integrin promotes dedifferentiation of monolayer-cultured articular chondrocytes.

OBJECTIVE: When cultured in monolayers, articular chondrocytes undergo an obvious phenotypic change. Although the involvement of integrins has been suggested, the exact mechanisms of the change have not been determined. This study was undertaken to clarify the mechanisms underlying the loss of chondrocyte phenotype early after plating. METHODS: Primary cultured human articular chondrocytes were used for the experiments. Involvement of respective integrins in the phenotypic change was investigated in RNA interference (RNAi) experiments. A signaling pathway involved in the change was identified in experiments using specific inhibitors and adenoviruses encoding mutated genes involved in the pathway. Adenoviruses carrying mutated GTPases were used to determine the involvement of small GTPases in the process. RESULTS: In monolayer-cultured chondrocytes, suppression of αv or ß5 integrin expression by RNAi inhibited morphologic changes in the cells and increased (or prevented a reduction in) the expression of various cartilage matrix genes. Consistent results were obtained in experiments using a blocking antibody and a synthetic inhibitor of αvß5 integrin. The decrease in cartilage matrix gene expression in chondrocytes after plating was mediated by ERK signaling, which was promoted primarily by αvß5 integrin. In articular chondrocytes, the affinity of αvß5 integrin for ligands was regulated by the small GTPase R-Ras. R-Ras was gradually activated in monolayer-cultured chondrocytes after plating, which caused a gradual decline in cartilage matrix gene expression through enhanced αvß5 integrin activation and the subsequent increase in ERK signaling. CONCLUSION: Our findings indicate that αvß5 integrin may be involved in the change that occurs in monolayer-cultured chondrocytes after plating.

Relationship between radiographic changes and symptoms or physical examination findings in subjects with symptomatic medial knee osteoarthritis: a three-year prospective study.

BACKGROUND: Although osteoarthritis (OA) of the knee joints is the most common and debilitating joint disease in developed countries, the factors that determine the severity of symptoms are not yet understood well. Subjects with symptomatic medial knee OA were followed up prospectively to explore the relationship between radiographic changes and symptoms or physical examination findings. METHODS: One-hundred six OA knees in 68 subjects (mean age 71.1 years; 85% women) were followed up at 6-month intervals over 36 months. At each visit, knee radiographs were obtained, symptoms were assessed by a validated questionnaire, and the result of physical examination was recorded systematically using a specific chart. Correlations between the change of radiographs and clinical data were investigated in a longitudinal manner. RESULTS: During the study period, the narrowing of joint space width (JSW) was observed in 34 joints (32%). Although those knees were clinically or radiographically indistinguishable at baseline from those without JSW narrowing, differences became apparent at later visits during the follow-up. The subjects with knees that underwent JSW narrowing had severer symptoms, and the symptoms tended to be worse for those with higher rates of narrowing. A significant correlation was not found between the severity of symptoms and the growth of osteophytes. For the knees that did not undergo radiographic progression, the range of motion improved during the follow-up period, possibly due to the reduction of knee pain. Such improvement was not observed with the knees that underwent JSW narrowing or osteophyte growth. CONCLUSION: The result of this study indicates that the symptoms of knee OA patients tend to be worse when JSW narrowing is underway. This finding may explain, at least partly, a known dissociation between the radiographic stage of OA and the severity of symptoms.

The interaction of monocytes with rheumatoid synovial cells is a key step in LIGHT-mediated inflammatory bone destruction.

Formation of osteoclasts and consequent joint destruction are hallmarks of rheumatoid arthritis (RA). Here we show that LIGHT, a member of the tumour necrosis factor (TNF) superfamily, induced the differentiation into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) of CD14(+) monocytes cocultured with nurse-like cells isolated from RA synovium, but not of freshly isolated CD14(+) monocytes. Receptor activator of nuclear factor-kappaB ligand (RANKL) enhanced this LIGHT-induced generation of TRAP-positive MNCs. The MNCs showed the phenotypical and functional characteristics of osteoclasts; they showed the expression of osteoclast markers such as cathepsin K, actin-ring formation, and the ability to resorb bone. Moreover, the MNCs expressed both matrix metalloproteinase 9 (MMP-9) and MMP-12, but the latter was not expressed in osteoclasts induced from CD14(+) monocytes by RANKL. Immunohistochemical analysis showed that the MMP-12-producing MNCs were present in the erosive areas of joints in RA, but not in the affected joints of osteoarthritic patients. These findings suggested that LIGHT might be involved in the progression of inflammatory bone destruction in RA, and that osteoclast progenitors might become competent for LIGHT-mediated osteoclastogenesis via interactions with synoviocyte-like nurse-like cells.

Proinflammatory role of amphiregulin, an epidermal growth factor family member whose expression is augmented in rheumatoid arthritis patients.

BACKGROUND: The epidermal growth factor (EGF) and EGF receptor (EGFR) families play important roles in the hyperplastic growth of several tissues as well as tumor growth. Since synovial hyperplasia in rheumatoid arthritis (RA) resembles a tumor, involvement of the EGF/EGFR families in RA pathology has been implied. Although several reports have suggested that ErbB2 is the most important member of the EGFR family for the synovitis in RA, it remains unclear which members of the EGF family are involved. To clarify the EGF-like growth factors involved in the pathology of RA, we investigated the expression levels of seven major EGF-like growth factors in RA patients compared with those in osteoarthritis (OA) patients and healthy control subjects. METHODS: The expression levels of seven EGF-like growth factors and four EGFR-like receptors were measured in mononuclear cells isolated from bone marrow and venous blood, as well as in synovial tissues, using quantitative RT-PCR. Further evidence of gene expression was obtained by ELISAs. The proinflammatory roles were assessed by the growth-promoting and cytokine-inducing effects of the corresponding recombinant proteins on cultured fibroblast-like synoviocytes (FLS). RESULTS: Among the seven EGF-like ligands examined, only amphiregulin (AREG) was expressed at higher levels in all three RA tissues tested compared with the levels in OA tissues. The AREG protein concentration in RA synovial fluid was also higher than that in OA synovial fluid. Furthermore, recombinant human AREG stimulated FLS to proliferate and produce several proinflammatory cytokines, including angiogenic cytokines such as interleukin-8 and vascular endothelial growth factor (VEGF), in a dose-dependent manner. The VEGF mRNA levels in RA synovia and VEGF protein concentrations in RA synovial fluid were significantly higher than those in the corresponding OA samples and highly correlated with the levels of AREG. CONCLUSION: The present findings suggest that AREG functions to stimulate synovial cells and that elevated levels of AREG may be involved in the pathogenesis of RA.

LIGHT induces cell proliferation and inflammatory responses of rheumatoid arthritis synovial fibroblasts via lymphotoxin beta receptor.

OBJECTIVE: To investigate the effects of LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) on the proliferation and gene expression of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA). METHODS: We measured LIGHT levels in RA synovial fluids (SF) by ELISA, and compared them with those in osteoarthritis (OA) SF. Levels of LIGHT and its receptors in RA-FLS and synovium were assessed using real-time quantitative polymerase chain reaction (PCR). RA-FLS proliferation was examined by a bromodeoxyuridine assay. Expression of intercellular adhesion molecule-1 (ICAM-1) and several chemokines, such as interleukin 8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1alpha (MIP-1alpha), was examined by real-time quantitative PCR, ELISA, and flow cytometry. The effects of LIGHT on nuclear factor-kappaB (NF-kappaB) activation were investigated using immunofluorescence and Western blotting. RESULTS: LIGHT was upregulated in both SF and synovium of RA patients compared with OA patients. Herpes virus entry mediator (HVEM) and lymphotoxin beta receptor (LTbetaR), but not LIGHT, were detected in RA-FLS. LIGHT significantly promoted RA-FLS proliferation and induced expression of MCP-1, IL-8, MIP-1alpha, and ICAM-1 by RA-FLS. As well, LTbetaR small interfering RNA (siRNA), but not HVEM siRNA, inhibited these effects of LIGHT. LIGHT induced IkappaBa degradation and NF-kappaB translocation, and a NF-kappaB inhibitor suppressed the effects of LIGHT on RA-FLS. CONCLUSION: Our findings suggest that LIGHT signaling via LTbetaR plays an important role in the pathogenesis of RA by affecting key processes such as the proliferation and activation of RA-FLS. Regulation of LIGHT-LTbetaR signaling may represent a new therapeutic target for RA treatment.