Role of leukotriene B4 receptors in rheumatoid arthritis

The purpose of this review is to summarize the role that murine models of arthritis are playing in the understanding of human rheumatoid arthritis and how leukotriene B(4) (LTB(4)) is emerging as an important target in this field. Both the collagen-induced arthritis (CIA) model and the K/BxN serum transfer arthritis model have contributed to outline the potential mechanisms involved in inflammatory arthritis. Indeed, the CIA model has contributed to the development of effective anti-TNFalpha and anti-IL-1beta based treatments for RA that are currently in the clinic. Many recent studies in mouse models have suggested a critical role for LTB(4) and its receptors in the development of inflammatory arthritis. Inhibitors of LTB(4) biosynthesis as well as LTB(4) receptors are protective in mouse models of RA and mice deficient in the LTB(4) biosynthetic enzymes or LTB(4) receptors are resistant to disease development suggesting several promising targets for RA in this pathway.     

Relationship between leukotriene B4 and immunological parameters in rheumatoid synovial fluids

Leukotriene B₄ (LTB₄) was measured in synovial fluid from 20 patients with rheumatoid arthritis and 15 patients with osteoarthritis. The level of LTB₄ was significantly higher in synovial fluid from rheumatoid arthritis patients as compared with synovial fluid from osteoarthritis patients. LTB₄ levels also significantly correlated with cell numbers, rheumatoid factor, and immune complexes in synovial fluid from rheumatoid arthritis patients. There was an inverse correlation between LTB₄ levels and complement components. The high-pressure liquid chromatography peak of immunoreactivity extracted from the synovial fluid occurred at a retention volume identical to that of authentic LTB₄. These results suggest that the increased level of this mediator in synovial fluid may contribute to perpetuation of inflammation and tissue destruction in rheumatoid arthritis.     

钩吻碱通过下调circ_001680抑制类风湿关节炎滑膜成纤维细胞增殖和侵袭

目的探讨钩吻碱对类风湿关节炎滑膜成纤维细胞(RASFs)增殖、迁移和侵袭的影响及可能机制。方法体外分离培养RASFs。经50、100、200μg/mL的钩吻碱干预24 h或转染circ_001680的小干扰RNA或过表达载体至RASFs后,CCK-8法和克隆形成实验检测细胞增殖;划痕实验检测细胞迁移;Transwell小室法检测细胞侵袭;蛋白印迹法检测E-cadherin和N-cadherin蛋白表达;RT-qPCR检测circ_001680表达。结果钩吻碱或干扰circ_001680表达可降低RASFs的增殖、克隆形成数、划痕愈合率和侵袭细胞数及细胞中N-cadherin蛋白表达(P<0.05),而促进E-cadherin蛋白表达(P<0.05)。钩吻碱可降低RASFs中circ_001680的表达(P<0.05),过表达circ_001680减轻钩吻碱对RASFs增殖、迁移和侵袭的抑制作用。结论钩吻碱可抑制RASFs增殖、迁移和侵袭,其作用机制可能与下调circ_001680表达有关。     

Peripheral blood neutrophil leukotriene B4 release and migration in rheumatoid arthritis

The present study was designed to compare peripheral blood neutrophil migration and leukotriene (LT) release between patients with rheumatoid arthritis (RA) and healthy controls and to correlate the neutrophil functions with clinical disease activity. Nineteen patients with moderately active RA and 19 age and sex matched healthy volunteers participated in this study. Isolated peripheral blood neutrophils from RA patients released equal amounts of LTB₄ but their random migration was enhanced as compared with neutrophils from healthy controls. LTB₄ release in whole blood was significantly lower in samples from RA patients than in those from the healthy volunteers (13.5±1.4 and 19.1±1.4 ng/10⁶ neutrophils respectively; P<0.001). LTB₄ release from isolated RA neutrophils correlated with the levels of C-reactive protein, duration of morning stiffness and Ritchie articular swelling index. Concentrations of hyaluronate, cyclic AMP and 13,14-dihydro-15-keto-prostaglandin were not different between patients with RA and healthy volunteers. Neither was there any difference in TXB₂ production by platelets during blood clotting. In conclusion, peripheral blood neutrophils of RA patients seem to be primed and/or activated as their random migration is enhanced as compared with those of healthy volunteers. In RA, LTB₄ release from peripheral blood neutrophils seems to reflect the clinical activity of the disease. However, RA neutrophils released smaller (in whole blood) or equal (isolated cells) amounts of LTB₄ as compared with the respective controls. These contradictory findings suggest that LTB₄ release from peripheral blood neutrophils has no major role in the regulation of disease activity in rheumatoid arthritis.     

Mepacrine inhibits fMLP-induced activation of human neutrophil granulocytes, leukotriene B4 formation, and fMLP binding.

Pulmonary sequestration and activation of polymorphonuclear leukocytes (PMNLs) are characteristic of many forms of acute lung injury. The present experiments were designed to study the effects of mepacrine on human neutrophils challenged with N-formylmethionyl-leucyl-phenylalanine (fMLP). Mepacrine inhibited fMLP-induced superoxide production and degranulation in a dose-dependent manner with Kd values of 2.3 +/- 0.5 x 10(-7) M and 5.7 +/- 1.3 x 10(-6) M, respectively. Stimulation of PMNLs by 10(-6) M fMLP provoked the formation of barely detectable amounts of leukotriene B4 (LTB4) (< 5 pg/10(7) cells). Pretreatment of the cells with cytochalasin B augmented generation of LTB4 in response to fMLP (339 +/- 79 pg/10(7) cells). LTB4 formation was also inhibited by mepacrine (50% inhibitory concentration 1.0 +/- 0.5 x 10(-6) M). Furthermore, mepacrine inhibited the specific binding of [3H]fMLP to neutrophils with a Ki value of 1.4 +/- 0.4 x 10(-5) M. Mepacrine decreased the receptor binding affinity without altering the number of receptors. These findings demonstrate that the inhibitory effect of mepacrine is response dependent and suggest that this action of mepacrine could, in part, be attributed to a decrease in fMLP receptor affinity.     

Induction of plasma exudation and inflammatory cell infiltration by leukotriene C4 and leukotriene B4 in mouse peritonitis

Leukotriene induction of the fluid and cellular phases of the inflammatory response in the mouse was evaluated. Intraperitoneal injection of leukotriene C₄ (LTC₄ 250 ng) led to dye extravasation but not polymorphonuclear leukocyte (PMN) infiltration, whereas injection of leukotriene B₄ (LTB₄ 250 ng), led to PMN infiltration but not dye extravasation. The injection of both leukotrienes did not result in synergy. LTC₄ did not appear to induce significant release or formation of chemotactic mediators, but the dye extravasation induced by LTC₄ was inhibited by the vasoactive amine antagonist cyproheptadine and not by the eicosanoid inhibitors phenidone or naproxen. The response was markedly inhibited by the cytokine and eicosanoid inhibitors SK&F 86002 and SK&F 104493. PMN infiltration induced by LTB₄ was not inhibited by SK&F 86002 or phenidone but was abrogated by colchicine treatment. LTB₄ in this model did not appear to cause release or formation of vasoactive mediators. These leukotrienes appeared to be independent, complementary, and sufficient to mount a complete inflammatory response in the mouse.     

Tolfenamic acid inhibits leukotriene B4-induced chemotaxis of polymorphonuclear leukocytes in vitro

Inhibition of prostanoid synthesis is usually regarded as the mode of action of nonsteroidal antiinflammatory drugs (NSAIDs). In addition, some NSAIDs have been reported to have prostanoid-independent inhibitory effects on neutrophil functions. In the present study, we examined the effects of acetylsalicylic acid, diclofenac, indomethacin, ketoprofen, piroxicam and tolfenamic acid on leukotriene B₄ (LTB₄)-induced chemotaxis of human polymorphonuclear leukocytes (PMNs) in vitro. Tolfenamic acid inhibited LTB₄-induced chemotaxis (IC₅₀ 59M), whereas the other compounds were ineffective. Tolfenamic acid inhibited also FMLP-induced chemotaxis at the same concentration range (IC₅₀ 46M). About 25% reduction in the chemotactic response was achieved with therapeutic concentrations of tolfenamic acid. We suggest that the inhibition of PMN chemotaxis is an additional mechanism in the antiinflammatory action of tolfenamic acid and that this action is not ligand specific.     

Blocking the leukotriene B4 receptor 1 inhibits late-phase airway responses in established disease

Most of the studies investigating the effectiveness of blocking the leukotriene B4 (LTB4) receptor 1 (BLT1) have been performed in models of primary or acute allergen challenge. The role of the LTB4-BLT1 pathway in secondary challenge models, where airway hyperresponsiveness (AHR) and airway inflammation have been established, has not been defined. We investigated the effects of blocking BLT1 on early- and late-phase development of AHR and airway inflammation in previously sensitized and challenged mice. Female BALB/c mice were sensitized (Days 1 and 14) and challenged (primary, Days 28-30) with ovalbumin. On Day 72, mice were challenged (secondary) with a single OVA aerosol, and the early and late phases of AHR and inflammation were determined. Specific blockade of BLT1 was attained by oral administration of a BLT1 antagonist on Days 70 through 72. Administration of the antagonist inhibited the secondary ovalbumin challenge-induced alterations in airway responses during the late phase but not during the early phase, as demonstrated by decreases in AHR and in bronchoalveolar lavage neutrophilia and eosinophilia 6 and 48 hours after secondary challenge. The latter was associated with decreased levels of KC protein, macrophage inflammatory protein 2, and IL-17 in the airways. These data identify the importance of the LTB4-BLT1 pathway in the development of late-phase, allergen-induced airway responsiveness after secondary airway challenge in mice with established airway disease.     

SC-41930, a leukotriene B4 receptor antagonist,inhibits 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) binding to epidermal cells

SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxyl]-3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, a potent leukotriene-B₄ (LTB₄) receptor antagonist, inhibitsin vivo 12-hydroxyei-cosatetraenoic acid (12-HETE)-induced neutrophil infiltration, suggesting a potential 12-HETE receptor antagonist effect, as well. Since 12-HETE is assumed to have a pathophysiological role in inflammatory skin diseases, and epidermal cells possess high affinity binding sites for 12(S)-HETE, we studied the effect of SC-41930 on 12(S)-HETE binding to the human epidermal cell line, SCL-II. SC-41930 antagonized the 12(S)-HETE binding to SCL-II cells with a Ki of 480 nM. This Ki value is similar to that obtained for the inhibition of LTB₄ binding to human neutrophils. Our results show that SC-41930, in addition to its LTB₄ receptor antagonist effect, exhibits 12-HETE receptor antagonist effect as well, and therefore may be of benefit in skin diseases with elevated 12-HETE levels.Dr. Kemény is on leave from the Department of Dermatology, Albert Szent-Györgyi Medical University, Szeged, Hungary, as a recipient of the Humboldt Fellowship, and his work was supported by the Alexander von Humboldt Foundation.     

Role of phagocytic synovial lining cells in experimental arthritis

We investigated thein vivo role of phagocytic synovial lining cells (SLC) in the onset of experimental arthritis by depleting phagocytic SLC prior to arthritis induction. Phagocytic SLC were depleted by a single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL₂MDP). Seven days after injection optimal depletion was observed and this time point was chosen for induction of arthritis in SLC depleted joints. Joint swelling was highly reduced after elicitation with either zymosan, immune complexes or antigen, as compared to observations in normal non-depleted joints. In addition cellular infiltration was markedly reduced. Further study in the immune complex mediated arthritis revealed that reduced cell influx in SLC depleted knee joints was correlated to lowered chemotactic activity and IL-1 levels as measured in washouts of joint tissues. This indicates that IL-1 driven chemotactic factors might be involved. Furthermore reduced cell influx was also correlated to significantly diminished loss of³⁵S-prelabeled PG from the cartilage. Out data indicate that SLC are directly involved in the onset of joint inflammation.

     

Role of phagocytic synovial lining cells in experimental arthritis

We investigated thein vivo role of phagocytic synovial lining cells (SLC) in the onset of experimental arthritis by depleting phagocytic SLC prior to arthritis induction. Phagocytic SLC were depleted by a single intra-articular injection of liposomes encapsulating the drug dichloromethylene diphosphonate (CL₂MDP). Seven days after injection optimal depletion was observed and this time point was chosen for induction of arthritis in SLC depleted joints. Joint swelling was highly reduced after elicitation with either zymosan, immune complexes or antigen, as compared to observations in normal non-depleted joints. In addition cellular infiltration was markedly reduced. Further study in the immune complex mediated arthritis revealed that reduced cell influx in SLC depleted knee joints was correlated to lowered chemotactic activity and IL-1 levels as measured in washouts of joint tissues. This indicates that IL-1 driven chemotactic factors might be involved. Furthermore reduced cell influx was also correlated to significantly diminished loss of³⁵S-prelabeled PG from the cartilage. Out data indicate that SLC are directly involved in the onset of joint inflammation.     

B effector cells in rheumatoid arthritis and experimental arthritis

Rheumatoid arthritis is a chronic autoimmune immune disease affecting approximately 1% of the population. There has been a renewed interest in the role of B cells in rheumatoid arthritis based on the evidence that B cell depletion therapy is effective in the treatment of disease. This review summarizes the current knowledge of the mechanisms by which B cells contribute to autoimmune arthritis including roles as autoantibody producing cells, antigen-presenting cells, cytokine producing cells, and regulatory cells.     

Inhibition of leukotriene B4 in neutrophils by lipoxins A4 and B4

Lipoxins are derived from the oxygenation products of arachidonic acid in human leukocytes. They have exhibited selective biological effects different from those of other eicosanoids. We have examined the effect of lipoxin A₄ and B₄ (LXA₄, LXB₄) on the production of leukotriene B₄ (LTB₄) in human neutrophils. Cultured human polymorphonuclear leukocytes were preincubated with LXA and B and their ability to inhibit LTB₄ generation was assessed after incubation with calcium ionophore A23187. We found that the pretreatment of neutrophils with lipoxins inhibit the release of LTB₄ by A23187 stimulated PMNs. Our data suggests that LXA₄ and B₄ can contribute to immunosuppression in an inflammatory state via the inhibition of LTB₄ synthesis.