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ObjectivesSeveral 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.MethodsPeripheral 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.ResultsCD4+ 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.ConclusionNZB/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.

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 explored molecules associated with the pathophysiology of SLE. 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, and S100A8 on the cell surface was decreased after treatment; 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, 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.

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. Electronic supplementary material The online version of this article (10.1186/s13075-018-1511-5) contains supplementary material, which is available to authorized users.

BACKGROUND AND PURPOSE Intracranial aneurysm (IA), common in general public, causes lethal subarachnoid hemorrhage on rupture. Prevention of rupture of IA is therefore socially important. However, there is currently no medical treatment. Recent studies suggest that IA is a disease of chronic inflammation in the arterial wall caused by endothelial dysfunction and infiltrated macrophages. Sphingosine-1-phosphate receptor type 1 (S1P1) is present on the endothelium and promotes its barrier function. Here we have tested the potential of an S1P1 agonist, ASP4058, in prevention of IA in animal model. EXPERIMENTAL APPROACH A selective S1P1 agonist, ASP4058, was used. Its effects on endothelial permeability and migration of macrophages across endothelial cell monolayer were tested in vitro using a Transwell system, and effects of ASP4058 on size of IAs induced in a rat model was evaluated in rat IA model. KEY RESULTS S1P1 receptor was expressed in endothelial cells of human IA lesions and control arterial walls. ASP4058 significantly reduced FITC-dextran leakage through endothelial monolayer and suppressed migration of macrophages across the monolayer in vitro. Oral administration of ASP4058 suppressed vascular permeability, macrophage infiltration and size of IAs by acting as an S1P1 agonist in rat model. This effect was mimicked by two other structurally-unrelated S1P1 agonists. CONCLUSION AND IMPLICATIONS A selective S1P1 agonist is a strong drug candidate for IA treatment through promoting endothelial cell barrier and suppressing trans-endothelial migration of macrophages in IA lesions. Non-approved abbreviations anterior cerebral artery, ACA; 3-aminopropionitrile, BAPN; Chinese hamster ovary, CHO; diastolic blood pressures, DBP; geometric mean fluorescence intensity, geoMFI; human carotid artery endothelial cells, HCtAEC; Intracranial aneurysm, IA; mean blood pressures, MBP; methylcellulose, MC; olfactory artery, OA; α-smooth muscle actin, SMA; systolic blood pressures, SBP; Sphigosine-1-phosphate, S1P; Transmission Electron Microscopy, TEM; wall shear stress, WSS

The whole protein of osteopontin (OPN full) and its cleaved form (OPN N-half) are involved in the immune response and the migration of immune cells to an inflammatory lesion. We have reported that serum OPN full and urine OPN N-half are elevated in lupus nephritis (LN). Neuropsychiatric systemic lupus erythematosus (NPSLE) is a refractory complication of SLE. To investigate whether OPN full and OPN N-half could serve as diagnostic markers for NPSLE, and to elucidate their role in NPSLE pathogenesis, the concentrations of OPN full and OPN N-half in cerebrospinal fluid (CSF) were measured in NPSLE and non-NPSLE patients. We found that the concentration of OPN full in the CSF was significantly higher in NPSLE than in non-NPSLE, and it decreased after treatment. When the cutoff value of OPN full in CSF was set to 963.4 ng/ml, the sensitivity and specificity for the diagnosis of NPSLE were 70% and 100%, respectively. The correlation analysis of OPN full, OPN N-half and various cytokines/chemokines suggested that the cytokines/chemokines could be divided into two clusters: cluster A, which contains OPN full and cluster B, which contains interleukin-6. OPN full in CSF could be a novel diagnostic marker for NPSLE.

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.

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.

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.

To investigate whether FK506 (tacrolimus) can inhibit Fas- or A23187-induced interleukin (IL)-8 expression and cell death in A549 human alveolar epithelial cells, plus Fas-mediated acute lung injury in vivo.Assays for IL-8, cell death, and caspase-3 activity were performed. A549 cells were treated with 25 micromol A23187 or 0.2 microg/ml agonistic anti-Fas antibody plus 5 ng/ml interferon-gamma (IFN-gamma). Tacrolimus was treated at 0.1-10 ng/ml. For in vivo experiment, agonistic anti-Fas antibody (Jo2) at 2.5 microg/g was intratracheally instilled into C57BL/6 mice. Neutrophils and protein contents in bronchoalveolar lavage (BAL) fluid were measured within 24 h of instillation. Mice were orally treated with 32 mg/kg of tacrolimus 24 h and 1 h prior to instillation.Both Fas and A23187 caused significant IL-8 expression and cell death in A549 cells. Tacrolimus inhibited A23187-induced IL-8 expression alone while it protected all Fas-mediated responses. Mice instilled intratracheally with Jo2 at 2.5 microg/g had significant increases in neutrophils, protein contents in BAL fluid and in expression of chemoattractants for neutrophils. These increases were reversed by tacrolimus.Tacrolimus serves as a therapeutic option for improving lung injury through inhibition of Fas-mediated inflammation.

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.

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.

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 (AUC0–24 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-1β 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.

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.

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 B2 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 ED50 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.

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.

Background: Atopic dermatitis (AD) is a chronic relapsing inflammation usually observed in patients with an individual or a familial history of atopic diseases, precipitated by environmental factors including mite antigens (Ag). However, the exact etiology of AD is unclear. To further explore the pathogenesis and treatment of AD, a suitable animal model is necessary. In this study, we developed a new animal model of AD induced by mite Ag in NC/Nga mice. Methods: We injected the extracts of mite Ag intradermally at the ventral side of the ear of SPF NC/Nga mice on days 0, 2, 4, 7, 9, 11, 14 and 16, and measured the clinical symptoms and the ear thickness. On day 18, we collected blood and submandibular lymph nodes (LN) of the immunized ear to perform a histochemical analysis, and to measure the plasma immunoglobulins and cytokines. Results: The NC/Nga mice immunized with mite Ag suffered from AD-like skin lesions including erythema followed by edema, excoriation and scaling. The histological and immunohistochemical examinations of the affected skin showed epidermal hyperplasia with hyperkeratosis, severe infiltration of CD4+ T lymphocytes, eosinophils and macrophages, and degranulation of mast cells. The total plasma IgE level was markedly elevated in mite Ag-treated mice. LN cells of mice immunized with mite Ag synthesized IgE in an Ag-dependent manner and secreted interleukin-4 (IL-4) and IL-5 but not interferon-γ. Conclusions: NC/Nga mice treated with mite Ag manifest clinical and immunological aspects similar to patients with AD, suggesting that this model is suitable for exploring the pathogenesis of human AD.

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.

1. We characterized the tachykinin receptor(s) mediating 'sensory-efferent' neural control of release of 35SO4-labelled macromolecules (mucus) from ferret trachea in vitro in Ussing chambers using selective tachykinin antagonists. Secretion was induced by substance P (SP), neurokinin A (NKA), capsaicin, the NK1 tachykinin receptor agonist [Sar9, Met(O2)11]substance P ([Sar9]SP), or acetylcholine (ACh), or by electrical stimulation of nerves. Antagonists used were FK888 and L-668,169, selective for the NK1 receptor, SR 48968, selective for the NK2 receptor, and FK224, a dual antagonist at NK1 and NK2 receptors. The selectivity of FK888 and SR 48968 was examined on NKA-induced contraction of ferret tracheal smooth muscle in vitro. 2. SP (1 microM) increased mucus secretion by 695% above vehicle controls. FK888 (0.1 microM-30 microM) inhibited SP-induced secretion in a dose-dependent manner, with complete inhibition at 10 microM and an IC50 of 1 microM. L-668,169 (1 microM) also completely inhibited SP-induced secretion. 3. NKA (1 microM) significantly increased mucus secretion by 271% above baseline, a response which was completely inhibited by FK888 (10 microM) or L-668,169 (microM). Secretion induced by ACh (10 microM: 317% above baseline) was not inhibited by FK888 but was inhibited by atropine. Capsaicin (10 microM)-induced secretion (456% above vehicle controls) was significantly inhibited by FK888 and by L-668,169 (111% and 103% inhibition respectively). 4. Electrical stimulation (50 V, 10 Hz, 0.5 ms, 5 min) increased mucus output above baseline (increased by 12 to 26 fold), a response blocked by tetrodotoxin (0.1 microM). FK888 (10 microM) inhibited the increase in secretion due to electrical stimulation by 47%. Atropine, propranolol and phentolamine in combination(APP) inhibited the response to electrical stimulation by 48%. The remaining NANC response, i.e. in the presence of APP, was further reduced by 66% with FK888. FK224 (10 microM) inhibited neurally evoked secretion by 73%. SR 48968 (0.1 fLM) had no effect on electrically-stimulated or [Sar9]SP-induced secretion.5. NKA (10nM- 1O microM: in the presence of DMSO control vehicle) induced tracheal smooth muscle contraction in a concentration-dependent manner with a maximal contraction of 30% of the maximal response to ACh (10 mM) and an ECm of 0.3 JAM. SR 48968 (0.1 microM in DMSO) inhibited the NKA induced contraction whereas FK888 did not. Neither antagonist had any inhibitory effect on ACh induced contraction.6. We conclude that 'sensory-efferent' neurogenic mucus secretion in ferret trachea in vitro is mediated via tachykinin NK, receptors with no involvement of NK2 receptors. Potent and selective tachykinin antagonists may have therapeutic potential in bronchial diseases such as asthma and chronic bronchitis in which neurogenic mucus hypersecretion may be aetiologically important.

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.

BACKGROUND: Angiotensin converting enzyme (ACE) inhibitors cause coughing in 5-10% of patients, but the exact mechanisms of this effect are still unclear. In the airways ACE degrades substance P so the cough mechanism may be related to this peptide. METHODS: Nine patients who developed a cough and five patients who did not develop a cough when taking the ACE inhibitor enalapril (2.5 or 5.0 mg/day) for hypertension were enrolled in the study. No subjects had respiratory disease and the respiratory function of all subjects was normal. One month after stopping enalapril, inhalation of hypertonic saline (4%) was performed using an ultrasonic nebuliser for 15-30 minutes to induce sputum. The concentration of substance P in the sputum sample was measured by radioimmunoassay. In four of the nine cases with a cough enalapril was given again for 1-2 weeks and the concentration of substance P in the induced sputum was again measured. RESULTS: One month after stopping enalapril the mean (SE) concentration of substance P in the sputum of the group with a cough was 16.6 (3.0) fmol/ml, significantly higher than that in the subjects without a cough (0.9 (0.5) fmol/ml). All four subjects in the group with a cough who were given a repeat dose of enalapril developed a cough again, but the concentrations of substance P in the induced sputum while taking enalapril (17.9 (3.2) fmol/ml) were similar to the values whilst off enalapril (20.0 (2.5) fmol/ml). CONCLUSIONS: The mechanisms of ACE inhibitor-induced coughing may involve substance P mediated airway priming. However, the final triggering of the ACE inhibitor-induced coughing is unlikely to be due to this peptide.

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.

To assess the ability of a marker of collagen type II degradation (CTX-II) to quantify cartilage turnover in vitro in cartilage explants and in vivo in rats with collagen induced arthritis (CIA).Bovine articular cartilage explants were cultured in the presence of interleukin 1a, oncostatin M, and plasminogen to induce cartilage degradation. CTX-II, CTX-I (C-telopeptide fragment of collagen type I), glycosaminoglycan, and hydroxyproline contents in culture supernatants were measured. CIA was induced in 12-week-old female Lewis rats by immunization with bovine type II collagen. The incidence and severity of arthritis were monitored by measuring paw swelling, and urinary levels of CTX-II and CTX-I were determined. The knee joints of rats were histopathologically examined after sacrifice. Results. CTX-II but not CTX-I levels correlated well with collagen degradation in bovine articular cartilage in vitro quantified by hydroxyproline release. Urinary CTX-II levels as well as paw volume of CIA rats were significantly higher than normal rats on Days 21, 28, and 42 and were apparently correlated with cartilage destruction, assessed histopathologically. Urinary CTX-I level began to increase on Day 21, but only on Day 42 was it significantly different between CIA and normal rats. The elevation in CTX-I level appeared to occur later than that of CTX-II, in accord with the more delayed onset of bone erosion in the CIA model of rheumatoid arthritis.Urinary CTX-II may be a useful marker for evaluation of dynamics of cartilage destruction in CIA rats.

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-γ 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.

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)-γ were detected, even though the IL-4+/IFN-γ– 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-γ 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.

Objective and Design: Recently, Ogawa et al. [17] reported that the peritoneal mast cells (PMCs) of rats can release histamine by substance P (SP) in a receptor-dependent manner. In the present study, we confirmed and extended their findings.¶Material: PMCs were isolated from six strains of rats. In some experiments, peritoneal cells in the non-MC fraction were used.¶Methods: PMCs were incubated with SP, neurokinin (NK) receptor agonists or antagonists, and histamine content in the supernatant was measured. In the binding assay, PMCs were incubated with [125I]BH-SP together with SP or NK receptor antagonists. NK-1 receptor mRNA was detected using a reverse transcription-polymerase chain reaction (RT-PCR) assay.¶Results: PMCs from Slc:Wistar and F344/NSlc were highly sensitive to SP, leading to histamine release, whereas those from Slc:SD and three other strains were not. PMCs from Slc:Wistar and F344/NSlc also released histamine in the presence of an NK-1 agonist. The histamine release induced by SP and the NK-1 agonist was inhibited by the NK-1 receptor antagonists, FK888 and CP-99,994. [125I]BH-SP binding experiments revealed that PMCs from Slc:Wistar rats possessed a single high affinity binding site for SP and that the binding was blocked by NK-1 receptor antagonists. Peritoneal cells in the non-MC fraction exhibited no appreciable binding. In the RT-PCR assay, expression of NK-1 receptor mRNA was evident in Slc:Wistar PMCs, but not in the non-MC fraction from Slc:Wistar or Slc:SD PMCs.¶Conclusion: These data demonstrate the existence of functional NK-1 receptors on freshly isolated PMCs in at least some strains of rats.

The sensory neuropeptide, substance P (SP), is present in human airway nerves, beneath and within the epithelium where the condensed localization of neutral endopeptidase (NEP), the major enzyme degrading SP, is observed. To test the hypothesis whether SP stimulates the cough reflex and NEP modifies the cough reflex, we studied the cough response to various stimuli in awake guinea-pigs. Inhibition of NEP with phosphoramidon caused cough, which was inhibited by systemic capsaicin treatment and by aerosols of a specific NK1receptor antagonist FK 888. Aerosols of FK 888 also inhibited cough induced by bronchoconstricting agents such as acetylcholine and histamine in non-sensitized animals and by ovalbumin antigen in animals sensitized to ovalbumin. The number of coughs induced by histamine aerosols was inhibited by systemic capsaicin treatment and enhanced by pretreatment with a NEP inhibitor phosphoramidon. Likewise, FK 888 inhibited the augumented cough response to aerosolized capsaicin in female guinea-pigs treated with a long-term medication of an angiotensin-converting enzyme inhibitor, cilazapril. In humans, aerosols of SP did not cause cough in normal subjects, whereas it did in patients with common colds. The SP fragment1–9a major metabolite of SP produced by NEP, was less effective compared with SP in these patients, suggesting that damaged epithelium may facilitate the penetration of SP. These findings suggest that SP released from sensory nerves in response to stimuli may mediate cough and NEP may have a role in modulating SP-induced effects.

We compared the effects of a tachykinin NK1 receptor antagonist, FK888 (N2-[(4R)-4-hydroxy-1-(1-methyl-1H-indol-3-yl)carbonyl-L-prolyl]-N-methy l-N -phenylmethyl-3-(2-naphthyl)-L-alaninamide), and a tachykinin NK2 receptor antagonist, SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]benzamide]), on citric acid-induced cough and bronchoconstriction in conscious guinea pigs. FK888 and SR48968 inhibited the cough dose dependently. Combination of FK888 and SR48968 showed a small additive effect compared with that of FK888 or SR48968 alone. SR48968 but not FK888 inhibited the bronchoconstriction dose dependently. These results indicate that tachykinin NK1 receptors as well as tachykinin NK2 receptors are involved in the citric acid-induced cough response. The antitussive activity of the tachykinin NK1 receptor antagonist appeared not to depend on the anti-bronchoconstrictor effects.

In experimental studies, tachykinins, especially substance P (SP), cause many of the pathophysiological features of neurogenic inflammation. It is unclear whether these peptides are involved in human airway inflammation in diseases such as asthma and chronic bronchitis. To elucidate the relation between neurogenic inflammation and airway inflammatory diseases, we examined the SP concentration in sputum after hypertonic saline inhalation challenge in patients with asthma, patients with chronic bronchitis, and normal volunteers. SP concentration was measured by radioimmunoassay. The sputum SP concentration was significantly higher in patients with asthma (mean +/- SEM, 17.7 +/- 2.4 fmol/ml; p0.01) and patients with chronic bronchitis (25.6 +/- 5.5 fmol/ml; p0.01) than in normal volunteers (1.1 +/- 0.4 fmol/ml). In patients with asthma, the SP concentration was significantly related to the eosinophil cell count in induced sputum. In all subjects, the SP concentration in induced sputum correlated with FEV1/FVC. These data suggest that neurogenic inflammation may be involved in the airway inflammatory process and subsequent airway narrowing not only in asthma but also in chronic bronchitis.

The tachykinin receptor type that medicates tachykinin-induced plasma extravasation in the rat knee joint was identified by using selective antagonists as well as natural or synthetic agonists. Substance P (SP) and neurokinin (NK) A induced plasma extravasation with almost the same potency and the maximum response was obtained at 5 nmol/knee. NKB was about ten times less potent than SP or NKA. The NK1 selective agonist, [Sar9, Met(O2)11]-SP, was about ten times more potent than SP, and the NK2 selective agonist, [Nle10]-NKA4–10, was about fifty times less potent than NK1 agonist. The NK3 agonist, Senktide, was totally ineffective at 0.5–50 nmol/knee. All responses induced by SP (5 nmol/knee), NKA (5 nmol/knee), NKB (50 nmol/knee), NK1 agonsit (0.5 nmol/knee) or NK2 agonist (25 nmol/knee) were significantly and profoundly inhibited by the NK1 selective antagonist, RP67580, but not by the NK2 selective antagonist, SR48968. Taken together, we conclude that tachykinin-induced plasma extravasation in the rat knee joint is mediated via NK1 receptors.

1 We compared the effects of two novel tachykinin receptor antagonists, FK888 (selective at the tachykinin NK1 receptor) and FK224 (dual antagonist at NK1 and NK2 tachykinin receptors) on stimulus-evoked airway plasma exudation, bronchoconstriction and systemic hypotension in guinea-pigs in vivo. Plasma exudation was induced by substance P (SP), synthetic tachykinin receptor agonists, platelet activating factor (PAF), electrical stimulation of the cervical vagus nerves or by inhalation of cigarette smoke. Changes in airway tone and in carotid artery blood pressure (BP) were induced by synthetic tachykinin agonists, PAF and vagal stimulation. 2 Both FK224 and FK888 dose-dependently inhibited SP-induced plasma exudation in the lower trachea and main bronchi (ID50 values respectively of 1.1 and 0.1 μmol kg−1 in lower trachea, and of 0.5 and 0.1 μmol kg−1 in main bronchi) with complete inhibition at both airway levels at 10 μmol kg−1 for FK224 and at 2 μmol kg−1 for FK888. 3 The NK1-selective tachykinin receptor agonist, [Sar9,Met(O2)11]substance P ([Sar]SP), induced plasma exudation, a response which was blocked by both FK888 and FK224. The NK2-selective agonist, [β-Ala8]neurokinin A-(4–10) ([β-Ala]NKA), did not induce plasma exudation: neither FK888 nor FK224 affected this lack of response to [β-Ala]NKA. 4 [β-Ala]NKA induced bronchoconstriction, a response which was blocked by FK224 but which was completely unaffected by FK888. [Sar]SP induced a small but significant bronchoconstriction which was completely inhibited by both tachykinin antagonists. 5 In animals pretreated with capsaicin to deplete sensory neuropeptides, PAF induced both plasma exudation and bronchoconstriction. Neither response to PAF was inhibited by either FK888 or FK224. 6 Both FK888 and FK224 inhibited plasma exudation induced by vagus nerve stimulation or by cigarette smoke, with FK888 more potent than FK224. 7 FK224 inhibited non-cholinergic bronchoconstriction induced by vagal stimulation, whereas FK888, at doses inhibiting vagally-induced plasma exudation, did not. 8 Decreases in BP induced by SP or [Sar]SP were blocked by both FK888 and FK224. In contrast, neither antagonist had any significant inhibitory effect on the decrease in BP induced by vagal stimulation (in the presence of atropine) or PAF. [β-Ala]NKA did not decrease BP and neither tachykinin antagonist had any significant effect on this lack of response. 9 We conclude that in guinea-pig airways, plasma leakage induced by endogenous tachykinins is mediated predominantly via NK1-receptors, whereas bronchoconstriction is mediated predominantly via NK2-receptors. In addition, SP-evoked decreases in BP are also mediated via NK1 receptors, whereas the contribution of endogenous tachykinins to vagally-induced decreases in BP appears to be minimal. Development of selective tachykinin receptor antagonists will be important in understanding the involvement of tachykinins in airway physiology and pathophysiology, whereas potent dual tachykinin receptor antagonists such as FK224 may have greater therapeutic potential in certain airway diseases in which tachykinins have been implicated in pathogenesis, including asthma and chronic bronchitis associated with cigarette smoking.