Similarity and difference between systemic lupus erythematosus and NZB/W F1 mice by multi-omics analysis.

OBJECTIVES: Several animal disease models have been used to understand the mechanisms of systemic lupus erythematosus (SLE); however, the translation of findings from animals to humans has not been sufficiently examined in drug development. To confirm the validity of New Zealand black x New Zealand white (NZB/W) F1 mice as an SLE model, we extensively characterized SLE patients and NZB/W F1 mice by omics analysis. METHODS: Peripheral blood from patients and mice and spleen and lymph node tissue from mice were analysed using cell subset analysis, cytokine panel assays, and transcriptome analysis. RESULTS: CD4+ effector memory T cells, plasmablasts, and plasma cells were increased in both SLE patients and NZB/W F1 mice. Levels of tumor necrosis factor-α, interferon gamma induced protein-10, and B cell activating factor in plasma were significantly higher in SLE patients and NZB/W F1 mice than in their corresponding controls. Transcriptome analysis revealed an upregulation of genes involved in the interferon signalling pathway and T-cell exhaustion signalling pathway in both SLE patients and the mouse model. In contrast, death receptor signalling genes showed changes in the opposite direction between patients and mice. CONCLUSION: NZB/W F1 mice are a generally suitable model of SLE for analysing the pathophysiology and treatment response of T/B cells and monocytes/macrophages and their secreted cytokines.

Expression of S100A8 protein on B cells is associated with disease activity in patients with systemic lupus erythematosus.

BACKGROUND: Systemic lupus erythematosus (SLE) is an intractable disease characterized by autoantibody production and autoreactive B and T cell proliferation. Although several studies have revealed multiple genetic and environmental associations, the underlying mechanisms remain unknown. METHODS: We performed proteomics and transcriptomics using liquid chromatography-mass spectrometry and DNA microarray, using peripheral blood B cells from patients with SLE, and healthy controls (HC). We explored molecules associated with the pathophysiology of SLE by flow cytometry and B cell stimulation assay. RESULTS: We identified for the first time that expression of both S100A8 protein and mRNA were markedly upregulated in SLE B cells. The results obtained using flow cytometry showed that S100A8 was highly expressed on the surface of B cells of patients with active SLE (MFI; HC 102.5 ± 5.97, stable SLE 111.4 ± 12.87, active SLE 586.9 ± 142.9), and S100A8 on the cell surface was decreased after treatment (MFI; pre-treat 1094.5 ± 355.38, post-treat 492.25 ± 247.39); therefore, it is suggested that S100A8 may be a marker for disease activity. The mRNA expression of S100A8 was particularly upregulated in memory B cells of SLE (56.68 fold higher than HC), suggesting that S100A8 may be mainly secreted by memory B cells in the pathogenesis of SLE. CONCLUSIONS: Our results imply that the S100A8 proteins secreted from memory B cells may stimulate granulocytes and monocytes through pattern recognition receptors, activate the innate immune system, and are involved in the pathogenesis of SLE.

Dynamics of Type I and Type II Interferon Signature Determines Responsiveness to Anti-TNF Therapy in Rheumatoid Arthritis.

The factors influencing long-term responses to a tumor necrosis factor inhibitor (TNFi) in rheumatoid arthritis (RA) patients currently remain unknown. Therefore, we herein conducted a multi-omics analysis of TNFi responses in a Japanese RA cohort. Blood samples were collected from 27 biological disease-modifying antirheumatic drug (DMARD)-naive RA patients at the initiation of and after three months of treatment with TNFi. Treatment responses were evaluated at one year. Differences in gene expression levels in peripheral blood mononuclear cells (PBMCs), plasma protein levels, drug concentrations, and the presence/absence of anti-drug antibodies were investigated, and a cell phenotypic analysis of PBMCs was performed using flow cytometry. After one year of treatment, thirteen patients achieved clinical remission (responders), while the others did not or switched to other biologics (non-responders). Differentially expressed genes related to treatment responses were enriched for the interferon (IFN) pathway. The expression of type I IFN signaling-related genes was higher in non-responders than in responders before and after treatment (P = 0.03, 0.005, respectively). The expression of type II IFN signaling-related genes did not significantly differ before treatment; however, it increased in non-responders and decreased in responders, with a significant difference being observed after three months of treatment (P = 1.2×10-3). The total number of lymphocytes and C-X-C Motif Chemokine Ligand 10 (CXCL10) protein levels were associated with the type I IFN signature (P = 6.7×10-7, 6.4×10-3, respectively). Hepatocyte growth factor (HGF) protein levels before treatment predicted fold increases in type II IFN (P = 0.03). These IFN signature-related indices (the number of lymphocytes, CXCL10, and HGF) significantly differed between responders and non-responders (P = 0.01, 0.01, and 0.04, respectively). A single-cell analysis revealed that the type I IFN signature was more highly enriched in monocytes than in other cell types. A deconvolution analysis of bulk-RNA sequence data identified CD4+ and CD8+ T cells as the main sources of the type II IFN signature in non-responders. Collectively, the present results demonstrated that the dynamics of the type I and II IFN pathways affected long-term responses to TNFi, providing information on its biological background and potential for clinical applications.

Metabolomic approach to the exploration of biomarkers associated with disease activity in rheumatoid arthritis.

We aimed to investigate metabolites associated with the 28-joint disease activity score based on erythrocyte sedimentation rate (DAS28-ESR) in patients with rheumatoid arthritis (RA) using capillary electrophoresis quadrupole time-of-flight mass spectrometry. Plasma and urine samples were collected from 32 patients with active RA (DAS28-ESR≥3.2) and 17 with inactive RA (DAS28-ESR<3.2). We found 15 metabolites in plasma and 20 metabolites in urine which showed a significant but weak positive or negative correlation with DAS28-ESR. When metabolites between active and inactive patients were compared, 9 metabolites in plasma and 15 in urine were found to be significantly different. Consequently, we selected 11 metabolites in plasma and urine as biomarker candidates which significantly correlated positively or negatively with DAS28-ESR, and significantly differed between active and inactive patients. When a multiple logistic regression model was built to discriminate active and inactive cohorts, three variables-histidine and guanidoacetic acid from plasma and hypotaurine from urine-generated a high area under the receiver operating characteristic (ROC) curve value (AUC = 0.8934). Thus, this metabolomics approach appeared to be useful for investigating biomarkers of RA. Combination of plasma and urine analysis may lead to more precise and reliable understanding of the disease condition. We also considered the pathophysiological significance of the found biomarker candidates.

Increase of MZB1 in B cells in systemic lupus erythematosus: proteomic analysis of biopsied lymph nodes.

BACKGROUND: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease in which dysregulation of B cells has been recognized. Here, we searched for potential biomarkers of SLE using liquid chromatography-tandem mass spectrometry (LC-MS). METHODS: Lymph nodes from SLE patients and controls were analyzed by LC-MS. To validate the identified molecules, immunoblotting and immunohistochemistry were performed and B cells from SLE patients were analyzed by quantitative RT-PCR. B-cell subsets from NZB/W F1 mice, which exhibit autoimmune disease resembling human SLE, were analyzed by flow cytometry. Endoplasmic reticulum (ER) stress was induced by tunicamycin and the serum concentration of anti-dsDNA antibodies was determined by ELISA. TUNEL methods and immunoblotting were used to assess the effect of tunicamycin. RESULTS: MZB1, which comprises part of a B-cell-specific ER chaperone complex and is a key player in antibody secretion, was one of the differentially expressed proteins identified by LC-MS and confirmed by immunoblotting. Immunohistochemically, larger numbers of MZB1+ cells were located mainly in interfollicular areas and scattered in germinal centers in specimens from SLE patients compared with those from controls. MZB1 colocalized with CD138+ plasma cells and IRTA1+ marginal zone B cells. MZB1 mRNA was increased by 2.1-fold in B cells of SLE patients with active disease (SLE Disease Activity Index 2000 ≥ 6) compared with controls. In aged NZB/W F1 mice, splenic marginal zone B cells and plasma cells showed elevated MZB1 levels. Tunicamycin induced apoptosis of MZB1+ cells in target organs, resulting in decreased serum anti-dsDNA antibody levels. Additionally, MZB1+ cells were increased in synovial tissue specimens from patients with rheumatoid arthritis. CONCLUSIONS: MZB1 may be a potential therapeutic target in excessive antibody-secreting cells in SLE.

Transgelin-2 is upregulated on activated B-cells and expressed in hyperplastic follicles in lupus erythematosus patients.

Transgelin-2 (TAGLN2) is an actin-binding protein that controls actin stability and promotes T cell activation. TAGLN2 is also expressed on B-cells but its function in B-cells is unknown. We found that TAGLN2-expressing B-cells were localized in the germinal center (GC) of secondary lymphoid tissues and TAGLN2 mRNA was significantly upregulated after IgM+IgG stimulation in primary human B-cells, suggesting that TAGLN2 was upregulated upon B-cell activation. In support of this, lymph nodes (LNs) from patients with systemic lupus erythematosus (SLE), in which the intense GC activity have been recognized, showed increased TAGLN2 expression in B-cells compared to control LNs. Moreover, TAGLN2+B-cells were distributed widely not only in the GC but also in the perifollicular areas in SLE LNs. In contrast, CD19+ B-cells and CD19+CD27+ memory-B cells in peripheral blood of SLE patients showed no increase in TAGLN2 mRNA. Two-photon excitation microscopy of Raji cells demonstrated that TAGLN2 colocalized with F-actin and moved together to the periphery upon stimulation. TAGLN2-knockdown in Raji cells resulted in impaired phosphorylation of PLCγ2 leading to inhibition of cell migration. Microarray analysis of TAGLN2-knockdown Raji cells showed decreased expression of the genes associated with immune function including CCR6 and as well as of those associated with regulation of the actin cytoskeleton including ABI2, compared to controls. These results suggest that TAGLN2 might regulate activation and migration of B-cells, in particular, the entry of activated B-cells into the follicle. We also suggest that TAGLN2 could be used as a marker for activated B-cells.

A sphingosine-1-phosphate receptor type 1 agonist, ASP4058, suppresses intracranial aneurysm through promoting endothelial integrity and blocking macrophage transmigration.

BACKGROUND AND PURPOSE: Intracranial aneurysm (IA), common in the general public, causes lethal subarachnoid haemorrhage on rupture. It is, therefore, of utmost importance to prevent the IA from rupturing. However, there is currently no medical treatment. Recent studies suggest that IA is the result of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrating macrophages. The sphingosine-1-phosphate receptor type 1 (S1P1 receptor) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, to prevent IA in an animal model. EXPERIMENTAL APPROACH: The effects of a selective S1P1 agonist, ASP4058, on endothelial permeability and migration of macrophages across an endothelial cell monolayer were tested in vitro using a Transwell system, and its effects on the size of IAs were evaluated in a rat model of IA. KEY RESULTS: S1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC-dextran leakage through an endothelial monolayer and suppressed the migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 reduced the vascular permeability, macrophage infiltration and size of the IAs by acting as an S1P1 agonist in the rat model. This effect was mimicked by another two structurally-unrelated S1P1 agonists. CONCLUSION AND IMPLICATIONS: A selective S1P1 agonist is a strong drug candidate for IA treatment as it promotes the endothelial cell barrier and suppresses the trans-endothelial migration of macrophages in IA lesions.

Cleaved Form of Osteopontin in Urine as a Clinical Marker of Lupus Nephritis.

We assessed the utility of two forms of osteopontin (OPN), OPN full and its cleaved form (OPN N-half), in plasma and urine as markers of disease activity in lupus nephritis (LN). Samples were collected from patients with systemic lupus erythematosus (SLE) (LN: N = 29, non-LN: N = 27), IgA nephropathy (IgAN) (N = 14), minimal change nephrotic syndrome (MCNS) (N = 5), diabetic nephropathy (DN) (N = 14) and healthy volunteers (HC) (N = 17). While there was no significant difference in urine OPN full concentration between groups, urine OPN N-half concentration was significantly higher in patients with LN than HC (p < 0.05). Moreover, urine OPN N-half was higher in LN patients with overt proteinuria (urine protein/creatinine ratio: P/C > 0.5) than LN patients with minimal proteinuria (P/C < 0.5, p < 0.0001), and also higher than in DN patients with overt proteinuria (P/C > 0.5, p < 0.01). Urine thrombin activity correlated with urine OPN N-half concentration (p < 0.0001), but not with urine OPN full concentration. These results suggest that urine OPN N-half concentration reflects renal inflammation. Thus, urine OPN N-half may be a novel disease activity marker for LN.

Prevention of progressive joint destruction in collagen-induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR255031.

FR255031 (2-[(7S)-7-[5-(4-ethylphenyl)-2-thienyl]-1,1-dioxido-4-(2-pyridinylcarbonyl)hexahydro-1,4-thiazepin-7-yl]-N-hydroxyacetamide) is a novel synthetic matrix metalloproteinase (MMP) inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type 1 MMP (MT1-MMP/MMP-14). FR255031 also inhibits rat collagenase and gelatinase. We studied the effect of FR255031 and Trocade, an inhibitor of collagenase and MMP-14, on a rat collagen-induced arthritis (CIA) model. Rat CIA was induced by intradermal injection of type II collagen (IIC) and oral administration of FR255031 or Trocade was performed for 28 days. Body weight loss, hind paw swelling, elevation of serum anti-IIC antibody, and histological and radiographic scores were evaluated. FR255031 markedly inhibited cartilage degradation in a dose-dependent manner in the CIA model, but Trocade failed to prevent the degradation. FR255031 at a dose of 100 mg kg(-1) also had statistically significant effects on bone destruction and pannus formation and on the recovery of body weight loss on day 28. These results indicate that FR255031 is effective for rat CIA, especially on joint cartilage destruction. These data suggest that as well as collagenases or MT-MMP, gelatinases are also involved in joint destruction in arthritis.

Effects of inhaled FK 506 on the suppression of acute rejection after lung transplantation: use of a rat orthotopic lung transplantation model.

BACKGROUND: FK 506 inhalant was recently developed for localized administration. We investigated its effects on acute lung allograft rejection and compared its efficacy with that of intramuscular administration of FK 506. METHODS: Rats (n = 123) with orthotopic left lung transplantation were divided into 9 groups. Six groups inhaled FK 506 (5 puffs, 10 puffs or 20 puffs per day), or were given intramuscular administration of FK 506 (0.05, 0.1 or 1.0 mg/kg/day). The other groups included rats receiving an isograft, rats with an untreated allograft, and a placebo group. All groups (n = 6 each) were monitored for 14 days post-operatively as an end-point and graft survival time was determined. The remaining animals were killed 4 days after transplantation. The histologic grade of rejection was determined for all groups (n = 6 each). With both (n = 3 each) inhalation therapy and intramuscular administration of FK 506, which showed similar degrees of effectiveness, both blood FK 506 concentration and cytokine expression in the graft and spleen were evaluated. RESULTS: FK 506 inhalation therapy extended allograft survival time and reduced histologic rejection on Day 4 in all groups. Graft survival time and histologic rejection scores at a dose of 10 puffs/day were comparable to those with 0.1 mg/kg/day of intramuscular FK 506. Trough concentrations of FK 506 in blood were detectable with 0.1 mg/kg/day of intramuscular FK 506, but not with 10 puffs/day. The messenger RNA expression levels of interferon-gamma in the lung allograft was suppressed significantly at a dose of 10 puffs/day. CONCLUSIONS: FK 506 inhalant enhances acute lung allograft survival with lower blood concentrations than when using comparable intramuscular administration.

Tacrolimus (FK506) has protective actions against murine bleomycin-induced acute lung injuries.

The effects of tacrolimus on murine acute lung injury were tested, especially in comparison to dexamethasone. Acute lung injury was induced by intratracheal instillation of bleomycin. Oral tacrolimus significantly improved survival rates of bleomycin-exposed mice, while cyclosporin A or dexamethasone did not. After instillation of bleomycin (day 0), a migration of neutrophils into alveolar spaces peaked on day 3, with concomitant increases of chemokines. On day 6, marked morphological changes in the lungs were observed. All these changes were significantly inhibited by tacrolimus. Furthermore, DNA ladder and immunohistochemical analyses of lungs showed that apoptosis of lung cells appeared on day 6 and was abolished only by the treatment of tacrolimus. These results suggest that both anti-inflammatory and anti-apoptotic action of tacrolimus contribute to improvement of bleomycin-induced acute lung injury.

Prevention of progressive joint destruction in adjuvant induced arthritis in rats by a novel matrix metalloproteinase inhibitor, FR217840.

Matrix metalloproteinase (MMP) has been implicated in joint destruction of chronic arthritis diseases, such as rheumatoid arthritis. FR217840 (2R)-1-([5-(4-fluorophenyl)-2-thienyl]sulfonyl)-N-hydroxy-4-(methylsulfonyl)-2-piperazinecarboxamide is a potent, orally active synthetic MMP inhibitor that inhibits human collagenases (MMP-1, MMP-8 and MMP-13), gelatinases (MMP-2 and MMP-9) and membrane type MMP (MT-MMP) (MT1-MMP/MMP-14). FR217840 also inhibits rat collagenase and gelatinase. We studied the effect of FR217840 on a rat adjuvant induced arthritis model. Although oral administration (days 1-21) of FR217840 (3.2, 10, 32 mg/kg) to adjuvant injected Lewis rats did not affect inflammation, as indicated by both hind paw swelling and histological inflammatory infiltration, FR217840 suppressed both bone destruction and serum pyridinoline content in a dose-dependent manner. Also, FR217840 (32 mg/kg) reduced tartrate-resistant acid phosphatase (TRAP) cell number in the ankle joints of rats with arthritis. These results indicate that FR217840 successfully suppressed joint destruction and suggest that FR217840 may have potential as a novel anti-rheumatic drug.

Effect of FK3657, a non-peptide bradykinin B2 receptor antagonist, on allergic airway disease models.

Bradykinin has been suggested to be involved in allergic diseases. In this study, we tested the effect of FK3657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)-oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide), an orally active non-peptide bradykinin B(2) receptor antagonist, on allergic airway disease models in guinea pigs. FK3657 given orally inhibited bradykinin-induced or dextran sulfate (an activator of kinin-kallikrein cascade)-induced bronchoconstriction and plasma extravasation in the lower airways (trachea and main bronchi) and nasal mucosa of guinea pigs with ED(50) of 0.04-0.23 mg/kg. In the antigen-induced dual asthmatic response model of guinea pigs, FK3657 significantly attenuated the late phase asthmatic response, but not the immediate asthmatic response. FK3657 also significantly inhibited the 2,4-tolylene diisocyanate (TDI)-induced plasma extravasation in nasal mucosa of TDI-sensitized guinea pigs. These results suggest that oral FK3657 may be useful for asthma or allergic rhinitis as a therapeutic drug.

Human T cells expressing BEND3 on their surface represent a novel subpopulation that preferentially produces IL-6 and IL-8.

BEN domain-containing protein 3 (BEND3) has no transmembrane region, is localized in the cytoplasm, and is involved in chromatin function and transcription. We here identified a novel subpopulation of human T cells that expressed BEND3 on their cell surface (BEND3(+) T cells). BEND3(+) T cells consisted of approximately 3% of T cells in the peripheral blood, were present in both CD4(+) and CD8(+) T cells, and were also observed in cord blood. The stimulation of BEND3(+) T cells through the TCR/CD3 complex led to the production of various kinds of cytokines; however, the levels of IL-6 and IL-8 produced by BEND3(+) T cells were higher than those by BEND3(-) T cells. The proportion of BEND3(+) T cells was also increased in some patients with inflammatory diseases. Taken together, these results indicate that BEND3(+) T cells are a new subpopulation of T cells in terms of their cytokine profile. Further analyses on BEND3(+) T cells may be of importance and useful in understanding human T cell immunology.

Inhibition of antigen-induced airway inflammation and hyperresponsiveness in guinea pigs by a selective antagonist of "chemoattractant receptor homologous molecule expressed on Th2 cells" (CRTH2).

Chemoattractant receptor homologous molecule expressed on T helper type 2 cells (CRTH2) is a PGD2 receptor found on eosinophils, basophils, and Th2 type T cells which exhibits chemotaxis and functions in activation cascades. However, while a number of CRTH2 antagonists, including ramatroban, are known to exert activity in certain animal models, activity in a guinea pig model of EA-induced airway hyperresponsiveness has not been demonstrated. The newly developed CRTH2 antagonist ASP5642 has shown antagonistic activity against human and guinea pig CRTH2 in previous studies and has also been found effective in treating guinea pig models of airway inflammation and airway hyperresponsiveness. While previous studies have used animals such as rats and mice to evaluate CRTH2 antagonist effects, ours is the first attempt to evaluate CRTH2 function in a guinea pig asthma model, which may prove useful in evaluating the compound's effects in humans, given the comparable airway function between the two species taken together, these data from the present study strongly suggest the utility of ASP5642 in investigating the role of CRTH2 in inflammatory responses and as a drug treatment for human asthma.

FK506 aerosol locally inhibits antigen-induced airway inflammation in Guinea pigs.

BACKGROUND: Eosinophilic airway inflammation is a common pathological feature of asthma. It has been shown that FK506 given systemically suppresses antigen-induced airway inflammation in animal models. However, it is unknown whether inhaled FK506 can suppress the airway allergic inflammation/immune response and whether it acts locally or systemically. METHODS: We tested the effects of oral FK506 and inhaled FK506 on antigen-induced airway inflammation in guinea pigs. The tissue and blood concentrations of FK506 given via both routes were compared. The effect of inhaled FK506 on the expression of cytokine mRNA in lung and bronchoalveolar lavage fluid (BALF) cells was also tested. RESULTS: Both routes of administration of FK506 suppressed the airway eosinophilia in egg albumin (EA)-sensitized and -challenged animals. The effect of three inhaled puffs was almost equal to that of 1 mg/kg administered by the oral route. Following inhalation of three puffs, FK506 concentration in blood (AUC(0-24 h)) was approximately 1/21 of that following oral FK506 (1 mg/kg). After EA challenge, mRNA expression of interleukin (IL)-5, eotaxin and IL-1beta in BALF cells and IL-5 in the lung increased significantly. FK506 aerosol markedly inhibited IL-5 mRNA expression in the lung. In situ hybridization indicated that in the BALF IL-5 mRNA expression by lymphocyte-like cells was inhibited by FK506 aerosol. In addition, anti-IL-5 antibody injected intratracheally almost completely abolished eosinophilia in this model. CONCLUSION: Inhaled FK506 can suppress airway inflammation in guinea pigs, where the local action, presumably the inhibition of T-cell activation/function in the lung and airways, was primarily important.

FK506 suppresses E-selectin, ICAM-1 and VCAM-1 expression on vascular endothelial cells by inhibiting tumor necrosis factor alpha secretion from peripheral blood mononuclear cells.

FK506 suppresses activation of T cells; however, it down-regulates E-selectin, ICAM-1 and VCAM-1 expression in inflamed tissues. In this study, we investigated the effect of FK506 on expression of those adhesion molecules on human vascular endothelial cells (HMVEC). Culture supernatant from peripheral blood mononuclear cells (PBMC) stimulated with anti-CD3 plus anti-CD2 antibodies effectively induced the expression of E-selectin, ICAM-1 and VCAM-1 on HMVEC, and treatment with FK506 down-regulated their expression. Culture supernatant contained tumor necrosis factor (TNF) alpha and interleukin (IL)-1beta, which effectively induced adhesion molecules, and FK506 suppressed both cytokine secretions. TNFalpha content in culture supernatant was parallel to the induction of adhesion molecules by the culture supernatant. IL-1beta content was not enough to induce those adhesion molecules. Anti-TNFalpha antibody completely inhibited those expressions. FK506 did not inhibit either TNFalpha- or IL-1beta-induced expression of adhesion molecules, or viability of HMVEC. These results indicate that FK506 suppresses migration of inflammatory cells through the inhibition of TNFalpha secretion from leukocytes.

[Bradykinin antagonist: current status and perspective].

The kallikrein-kinin system plays an important role in many physiological and pathophysiological conditions such as homeostasis of circulation, inflammation/allergy, pain, shock, etc. Two types of kinin receptor are known, bradykinin (BK) B1 receptor and BK B2 receptor. B2 receptors are constitutively expressed and mediate most physiological actions of kinins, whereas B1 receptors are highly inducible upon inflammatory stimulation or tissue injury, suggesting that they are involved in inflammation and/or nociception. Only three peptide type B2 antagonists, NPC 567, CP-0127 and HOE-140, have been evaluated in clinical studies so far, and some beneficial effects of B2 antagonists have been shown for rhinitis, asthma, systemic inflammatory response syndrome/sepsis and brain injury. However, the results were less convincing than expected. Now several potent and orally active nonpeptide B2-receptor antagonists have been found, which are expected to overcome the weak point of the peptide type antagonists and clarify the therapeutic potential of the B2-receptor antagonist for novel indications as well as those mentioned above. As for B1 receptors, no antagonist has been tested in a clinical trial. The important role of B1 receptors is just being elucidated by use of peptide type antagonists or B1 receptor gene knockout mice. The further development of newer B1 antagonists and clinical evaluation is desired.

Topical application of FK506 (tacrolimus) ointment inhibits mite antigen-induced dermatitis by local action in NC/Nga mice.

BACKGROUND: FK506 ointment (tacrolimus ointment, protopic) is a new drug therapeutically effective for patients with atopic dermatitis (AD). However, the mechanism of action of FK506 ointment on AD is not fully understood. METHODS: We examined the effect of FK506 ointment on mite antigen-induced dermatitis in NC/Nga mice. Clinical symptoms and ear thickness were recorded, and histopathological studies and in vitro analyses were performed. RESULTS: Topical application of FK506 ointment (0.03-0.3%) suppressed the development of dermatitis. In the lesional skin, both interleukin (IL)-4 and interferon (IFN)-gamma were detected, even though the IL-4+/IFN-gamma- T helper 2 (Th2) population was predominant in the regional lymph nodes (LNs). Topical application of FK506 treatment reduced the elevated level of both IL-4 and IFN-gamma in the skin, but did not decrease the expansion of the Th2 population in the LNs. CONCLUSIONS: Topical application of FK506 ointment suppresses dermatitis by inhibiting the activation of inflammatory cells locally, without systemic immune suppression, in this AD model.

Hyperoxia-induced emphysematous changes in subacute phase of endotoxin-induced lung injury in rats.

We examined the effects of prolonged hyperoxia (75% O(2)) on lung structure and collagen metabolism in the subacute phase of lung injury induced by continuous infusion of endotoxin (LPS) in a rat model. Experimental groups included control, endotoxin alone, endotoxin plus hyperoxia, and hyperoxia alone. Endotoxin-treated rats received a bolus of LPS (10 mg/kg i.v.) followed by 500 microg.kg(-1).day(-1) in continuous infusion for 10 days. The bronchoalveolar lavage (BAL) fluid/plasma albumin concentration ratio, an index of capillary permeability, and neutrophil and macrophage counts in BAL fluid were highest in the endotoxin plus hyperoxia group. On pathological examination, prolonged hyperoxia exacerbated destruction of the alveolar wall and caused most prominent emphysematous changes in the endotoxin plus hyperoxia group. Lung tissue hydroxyproline concentration was significantly decreased in the hyperoxia group and increased in the endotoxin group. The latent forms of MMP-2 and MMP-9 increased in BAL fluid of the endotoxin- and/or hyperoxia-treated groups, whereas the activities of collagenase and gelatinase, and the active form of MMP-2 were all increased in the hyperoxia-treated groups. Added to endotoxin, prolonged hyperoxia degraded collagen, the major structural component of basement membranes, and caused emphysematous changes associated with activation of collagenase and MMP-2. Our observations suggest that, in the subacute phase of endotoxin-induced lung injury, prolonged hyperoxia causes pulmonary emphysematous changes with persistent injury to the alveolar capillary barrier. Collagenase and MMP-2 activated by hyperoxia, together with MMP-9, may play prominent roles in disruption of the alveolar basement membranes and degradation of collagen lining the alveolar walls.