15-Hydroxy-eicosatetraenoic acid (15-Hete) inhibits carragheenan-induced experimental arthritis and reduces synovial fluid leukotriene B4 (LTB4)

15-Hydroxy-eicosatetraenoic acid (15-HETE), a product of arachidonic acid, has no proinflammatory capacity, but can inhibit the formation and the chemotactic response of neutrophils to leukotriene B₄ (LTB₄), a potent mediator of inflammation. The purpose of the present study was to determine whether intraarticular administration of 15-HETE in carragheenan-induced acute arthritis might decrease the levels of LTB₄ in synovial fluid and modify the arthritis. A bilateral acute knee joint arthritis was established in 7 dogs by intraarticular injections of carragheenan every third day. To the right joints, 15-HETE was administered both concomitantly with the carragheenan injections and continously via an osmotic pump. In samples of synovial fluid obtained on day 0, 3 and 10 PGE₂ and LTB₄ were determined using reversed phase high performance liquid chromatography combined with radioimmunoassays and neutrophil chemokinesis. In the presence of 15-HETE the clinical severity of arthritis was significantly reduced and the volume synovial effusate was decreased on an average by 42%. Furthermore, the relative number of neutrophils in histological sections of synovial tissue was decreased by 58%. Intaarticular carragheenan injection induced LTB₄ formation, and maximum levels were obtained on day 3 (279.2 ± 148.2 pg/joint). PGE₂ was also present on a day 3, but maximum levels were detected on day 10 (9.5 ± 4.8 ng/joint). In joints injected with both carragheenan and 15-HETE the levels of LTB₄ on days 3 and 10 were inhibited by 90% and 83%, respectively. For PGE₂ a small but significant decrease was found on both day 3 and on day 10. These results show that LTB₄ may be an important mediator of acute arthritis induced by carragheenan in dogs, and that intraarticular administration of 15- HETE can modify this arthritis by inhibiting LTB₄ formation.     

Questionable role of leukotriene B4 in monosodium urate (MSU)-induced synovitis in the dog

Monosodium urate (MSU)-induced synovitis in the dog's stifle (knee joint) is similar to an acute gouty attack in man in which a loss of function of the joint correlates with massive influx of neutrophils and the release of an assortment of inflammatory mediators (e.g. histamine, bradykinin, lysosomal enzymes, complement and eicosanoids) into the synovial space. We found in the urate-induced inflammatory exudates 3 hr post MSU the following: 88 million leukocytes/ml (approximately 95% neutrophils) and eicosanoid concentrations of LTB4, LTC4, and PGE2 of less than 0.1, 1.4 and 20 ng/ml, respectively. Isotonic saline injected knee joints at 3 hr contained 5 million leukocytes/ml (approximately 95% neutrophils) and concentrations of LTB4, LTC4, and PGE2 of less than 0.1, 0.7 and 0.2 ng/ml, respectively. Intrasynovial injections of 1 microgram LTB4, 10 micrograms PGE2 or the combination of LTB4 and PGE2 produced no reduction of paw pressure for up to 3 hr. Leukocyte concentrations measured at 3 hr in joints injected with these arachidonic acids metabolites were similar to saline controls. These results question the role of LTB4 as a chemotactic and inflammatory mediator in urate-induced synovitis in the dog but confirm the importance of PGE2 and possibly LTC4 in this model.     

Enhanced levels of leukotriene B(4) in synovial fluid in Lyme disease

The purpose of this study was to evaluate the potential role of LTB(4) and cysteinyl leukotrienes in Lyme disease (LD). Therefore, a total number of 34 patients divided into four groups was studied. The patients were classified as having Lyme arthritis (n = 7) or Lyme meningitis (n = 10), and as control groups patients with a noninflammatory arthropathy (NIA) (n = 7) and healthy subjects (n = 10). LTB(4) as well as LTC(4) secretion from stimulated polymorphonuclear leukocytes (PMNL) from all groups of patients showed no statistical differences. LTB(4) levels in synovial fluid were significantly increased in patients with Lyme arthritis (median 142 ng/ml, range 88-296) when compared to the control subjects with NIA (median 46 ng/ml, range 28-72) (p < 0.05). No statistical difference of urinary LTE(4) levels between all the different groups of patients was observed. These results show that cysteinyl leukotrienes do not play an important role in the pathogenesis of LD. In contrast to previous findings in rheumatoid arthritis, LTB(4) production from stimulated PMNL was not found to be increased in LD. However, the significantly elevated levels of LTB(4) in synovial fluid of patients with Lyme arthritis underline the involvement of LTB(4) in the pathogenesis of this disease.     

Eicosanoid synthesis by alveolar macrophages in rats with malignant mammary tumors: differences in rats treated with and without carrageenan implants

Eicosanoid synthesis by alveolar macrophages (AM), harvested from tumor bearing animals, was measured after tumor inoculation in rats treated with or without carrageenan (carra), an immunomodulating agent. After incubation of the cells with [14]C-arachidonic acid and the Ca-ionophore A23187, samples were measured by high pressure liquid chromatography (HPLC). From the HPLC profiles the lypoxygenase products, 12-hydroxyeicosatetraenoic acid (12-HETE), 15-HETE, and leukotriene-B4 (LTB4) were determined as well as the cyclooxygenase products, prostaglandin (PG)E2, PGF2 alpha and TXB2. After tumor inoculation AM-synthesis of lipoxygenase products tended to increase to values twice those of the base line values, whereas cyclooxygenase products showed subnormal values. In the non treated animals, 10 days after tumor inoculation, statistically significant increases in 12- and 15-HETE, LTB4 and PGE2 were observed when compared with carra treated animals. Later measurements did not show these differences in AM metabolism. AM metabolism was (negatively) correlated with the number of macrophages, which was particularly evident in the correlation with 12-HETE synthesis.     

Pharmacological characterisation of arthritis induced by Bothrops jararaca venom in rabbits: a positive cross talk between bradykinin, nitric oxide and prostaglandin E2

BACKGROUND: Our previous results showed that nitric oxide (NO) and bradykinin (BK) mediate the arthritis induced by Bothrops jararaca venom (BjV) in rabbits. In this study, we investigated the contribution of each receptor of BK as well as the inter-relationship between NO and eicosanoids in BjV-induced arthritis. METHODS: The arthritis was induced in rabbits with 16 microg of BjV injected intra-articularly. Prostaglandin E2 (PGE2), thromboxane B2 (TxB2), leukotriene B4 (LTB4) (radioimmunoassay) and nitrite/nitrate concentrations (NO2/NO3) (Griess reaction) were evaluated in the synovial fluid 4 h later. The animals were prior treated with NO synthase inhibitor (L-NAME; 20 mg/kg/day for 14 days), the B2 antagonist of BK (HOE-140) and the B1 antagonist of BK (des-Arg9[Leu8]-bradykinin), both at a dose of 0.3mg/kg, 30 min prior to the venom injection. RESULTS: Data show that L-NAME and HOE-140 treatment were equally able to reduce PGE2 and NO2/NO3 levels without interfering with TxB2 and LTB4 production. On the contrary, the B1 antagonist of BK inhibited TxB2 and LTB4 production, and did not alter PGE2 and NO metabolites levels in the inflamed joint. DISCUSSIONS: The results presented clarify the contribution of the kinin system, mainly through the B2 receptor, to the local inflammatory response induced by BjV, as well as its positive interaction with PGE2 and NO production.     

12(S)-hydroxy-5,8,10,14-eicosatetraenoic acid is a more potent neutrophil chemoattractant than the 12(R) epimer in the rat cornea

12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid [12(R)-HETE] is reported to be more potent than its epimer 12(S)-HETE as a chemoattractant for human neutrophils in vitro and following topical application to the skin. To assess the in vivo neutrophil chemoattractant potencies of 12(S)-HETE and 12(R)-HETE in the rat, we injected 1 microgram, 5 micrograms, or 10 micrograms of these eicosanoids into the corneal stroma. Rats were killed 12-15 hours after injection, and the number of neutrophils in the stroma was counted in a histological section of the cornea including the injection site. The number of neutrophils was significantly increased in corneas injected with 5 micrograms (+103% of control) or 10 micrograms (+456% of control) of 12(S)-HETE and in those injected with 10 micrograms of 12(R)-HETE (+111% of control). The neutrophilic infiltrate in corneas injected with 1 microgram or 5 micrograms of 12(S)-HETE was not significantly different from that in corneas injected with 1 microgram of leukotriene B4. The data for the 10 micrograms injections indicate that 12(S)-HETE is a more potent neutrophil chemoattractant than 12(R)-HETE in the rat cornea. Our results suggest that species or tissue specificity may determine the relative potencies of 12-HETE epimers as chemoattractants for neutrophils, and that 12(S)-HETE may be an important inflammatory mediator in the rat cornea.     

15-Hydroxyeicosatetraenoic acid (15-HETE) receptors. Involvement in the 15-HETE-induced stimulation of the cryptic 5-lipoxygenase in PT-18 mast/basophil cells.

The mechanisms of stimulation of the inactive 5-lipoxygenase in mast/basophil PT-18 cells by microM 15-hydroxyeicosatetraenoic acid (15-HETE) was investigated. Treatment of PT-18 cells with pM 15-[3H]HETE at 4 degrees for 3 h resulted in the cell association of 10% of the ligand: two-thirds was incorporated into cellular lipids and a third was bound to specific 15-HETE cellular binding sites. Binding data analysis indicated a single class of 15-HETE binding sites with a Kd of 162 nM and a Bmax of 7.1 x 10(5) sites/cell. Unlabeled 15-HETE, 12-HETE, and 5,15-diHETE inhibited the binding of 15-[3H]HETE to cells, whereas LTB4 and PGF2 alpha were relatively ineffective. 2.4 microM 15-HETE (unlabeled) prevented 50% 15-[3H]HETE incorporation. Examination of the effects of 15-HETE methyl ester, 12-HETE, 5,15-diHETE, and pertussis toxin on both the 15-HETE-induced 5-lipoxygenase activation and 15-HETE cell association processes indicated a preponderant correlation of this activation process with specific 15-HETE binding rather than 15-HETE incorporation into phospholipids. In addition, 5,15-diHETE itself stimulated the inactive 5-lipoxygenase and eight times more [3H]diHETE was bound to cells than became incorporated into cellular lipids. The results support the involvement of low affinity 15-HETE receptors, rather than 15-HETE incorporation into cellular lipids, in the 15-HETE-induced stimulation of the 5-lipoxygenase in PT-18 cells.     

SC-41930 inhibits neutrophil infiltration of the cavine dermis induced by 12(R)-hydroxyeicosatetraenoic acid

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4 (LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000X less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the cavine dermis. At a dose of 25 micrograms m per intradermal site, 12(R)-HETE was a significant chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels). SC-41930, 7-[3-(4-acetyl-3-methoxy-2-propylphenoxy)-propoxy]- 3,4-dihydro-8-propyl-2H-1-benzopyran-2-carboxylic acid, given intragastrically inhibited 12(R)-HETE-induced neutrophil infiltration of the cavine dermis with an ED50 value of 13.5 mg/kg. Compounds such as SC-41930 may well have utility for treating human psoriasis.     

12(R)-hydroxyeicosatetraenoic acid is a neutrophil chemoattractant in the cavine, lapine, murine and canine dermis

Psoriasis is a disease state characterized by epidermal proliferation, neutrophil infiltration, along with release of the proinflammatory mediators leukotriene-B4(LTB4) and 12(R)-hydroxyeicosatetraenoic acid [12(R)-HETE]. LTB4 and 12(R)-HETE are chemoattractant to the neutrophil, the latter approximately 1000x less potent. LTB4 and 12(R)-HETE are present in psoriatic scale, the latter in quantities so much greater than LTB4 that it is proposed as a primary mediator of neutrophil infiltration in psoriasis. 12(R)-HETE, synthesized in optically pure form by a new, shorter route, was injected into the dermis of the cavine, lapine, canine, mouse and rat. At doses up to 50 mu gm per intradermal site, 12(R)-HETE was chemoattractant to the neutrophil (as assessed by dermal myeloperoxidase levels) with response in the cavine greater than canine greater than lapine greater than mouse greater than rat.     

1-[4-[3-[4-[bis(4-fluorophenyl)hydroxymethyl]-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone(AHR-5333): a selective human blood neutrophil 5-lipoxygenase inhibitor

In this study we report the in vitro inhibition of leukotriene synthesis in calcium ionophore (A23187)-stimulated, intact human blood neutrophils by AHR-5333. The results showed that AHR-5333 inhibits 5-HETE, LTB4 and LTC4 synthesis with IC50 values of 13.9, 13.7 and 6.9 microM, respectively. Further examination of the effect of AHR-5333 on individual reactions of the 5-lipoxygenase pathway (i.e. conversion of LTA4 to LTB4, LTA4 to LTC4, and arachidonic acid to 5-HETE) showed that this agent was not inhibitory to LTA4 epoxyhydrolase and glutathione-S-transferase activity in neutrophil homogenates. However, conversion of arachidonic acid (30 microM) to 5-HETE was half maximally inhibited by 20 microM AHR-5333 in the cell-free system. The inhibition of LTB4 and LTC4 formation in intact neutrophils by AHR-5333 appears to be entirely due to a selective inhibition of 5-lipoxygenase activity and an impaired formation of LTA4, which serves as substrate for LTA4 epoxyhydrolase and glutathione-S-transferase. AHR-5333 did not affect the transformation of exogenous arachidonic acid to thromboxane B2, HHT and 12-HETE in preparations of washed human platelets, indicating that this agent has no effect on platelet prostaglandin H synthase, thromboxane synthase and 12-lipoxygenase activity. The lack of inhibitory activity of AHR-5333 on prostaglandin H synthase activity was confirmed with microsomal preparations of sheep vesicular glands.     

Involvement of 15-lipoxygenase in the inflammatory arthritis

15-Lipoxygenase (15-LOX) is involved in many pathological processes. The aim of this study is to examine the role of 15-LOX in the matrix metalloproteinase (MMP) expression and inflammatory arthritis. It was found that treatment of 15-LOX downstream product of 15-(S)-HETE (15-S-hydroxyeicosatetraenoic acid) increased the mRNA and protein levels of MMP-2 in rheumatoid arthritis synovial fibroblast (RASF) derived from rheumatoid arthritis patients. The enhancement effect of 15-(S)-HETE was antagonized by the addition of LY294002 (PI3K inhibitor) and PDTC (NF-κB inhibitor). Treatment of 15-(S)-HETE increased the phosphorylation of AKT, nuclear translocation of p65 and the breakdown of IκBα. TNF-α and IL-1β are the key cytokines involved in arthritis and also increase the activity of MMP-2 in RASF, which was antagonized by pretreatment with 15-LOX inhibitor PD146176 or knockdown of 15-LOX. It was also found that these two cytokines increased the expression of 15-LOX in RASF. Treatment of glucocorticoid but not NSAIDs inhibited 15-(S)-HETE-induced expression of MMP-2. In comparison with wild-type mice, adjuvant-induced arthritis and MMP-2 expression in synovial membrane were markedly inhibited in 15-LOX knockout (KO) mice. These results indicate that 15-LOX plays an important role in the disease progression of arthritis and may be involved in the inflammatory action induced by TNF-α and IL-1β. 15-LOX is thus a good target for developing drugs in the treatment of inflammatory arthritis.     

Quantitation of arachidonic acid metabolites in small tissue biopsies by reversed-phase high-performance liquid chromatography

Arachidonic acid metabolites exert a variety of distinct biological effects on the initiation and resolution of inflammatory diseases and their measurements in tissue can be critical to evaluate their regulatory function during the course of inflammation and to supplement in vitro experiments. The aim of this study was the detection and quantitative analysis of four arachidonic acid metabolites in small-sized biopsies of human periodontal tissues. The biopsies were homogenized and injected directly into a single analytical column of a RP-HPLC system. Detection was performed by a photodiode array detector. Calibration was established by dilutions of authentic standards of prostaglandin E2 (PGE2), leukotriene B4 (LTB4), 12(R)-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE), and 15(S)-hydroxy-5,8,11,13-eicosatetraenoic acid (15-HETE). A total of 38 specimens weighing between 19 and 191 mg (wet tissue) were analyzed (mean = 59.9 +/- 30.2 mg). The detection limits were 1 pg for LTB4 and 12-HETE, 0.5 pg for 15-HETE, and 10 ng for PGE2. The concentrations of PGE2 and LTB4 were significantly higher in inflamed than in healthy periodontal tissues (P = 0.0079; P = 0. 0114). 12-HETE was detected in one biopsy (30 pg/g); 15-HETE was not detected. This method of homogenization, extraction, and analysis of arachidonic acid metabolites by RP-HPLC appears to be well suited for studies of human oral biopsies. Only small tissue samples and minimal laboratory equipment were required for a sensitive analysis.     

Amniotic fluid lipoxygenase metabolites during spontaneous labor and after RU486 treatment during late pregnancy in rhesus macaques

To determine changes in amniotic fluid (AF) lipoxygenase metabolites prior to spontaneous labor and after RU486 administration, we implanted AF and vascular catheters and myometrial electromyographic (EMG) electrodes in 8 rhesus macaques at 120-130 days of pregnancy (term = 167 days). Four animals had AF samples taken serially until they delivered their infants normally at term. The other four animals received RU486 (20 mg/kg/day) for 3 days. AF samples were collected every 2-3 days and at 12 hour intervals for 72 hours before and after treatment with RU486. Uterine activity was monitored continuously. LTB4, 5-HETE and 15-HETE were measured by radioimmunoassay. In untreated animals, LTB4 and 5-HETE concentrations in AF increased significantly (P less than 0.05) 4 days before delivery with no change in 15-HETE. After RU486, mean levels of LTB4 and 5-HETE were increased although the difference was not statistically significant. No change in 15-HETE levels was observed. In conclusion, LTB4 and 5-HETE increase in AF before the onset of spontaneous labor. Progesterone receptor blockade by RU486 does not reproduce the changes in AF lipoxygenase metabolites observed during normal parturition.     

In vivo release of 15-HETE and other arachidonic acid metabolites in nasal secretions during early allergic reactions.

The purpose of this study is to examine the "in vivo" release of 15-HETE and other arachidonic acid metabolites in nasal secretions following a challenge with "Dermatophagoides Pteronyssinus" in patients with allergic rhinitis and non-allergic controls. In addition, we examine the effects of a membrane stabilizer, such as sodium cromoglycate, on these metabolites. Thirteen allergic subjects and seven healthy controls are studied. 15-HETE, peptide leukotrienes, LTB4, PGD2, PGE2 and PGF2 alpha levels are evaluated before and after nasal challenge in sodium cromoglycate treated and untreated subjects. This study provides "in vivo" evidence that the pathophysiological responses to nasal antigen challenge could be related to the release of 15-HETE as well as other arachidonic acid metabolites, mainly arising from the lipoxygenase pathway.     

Feedback control of the arachidonate cascade in rheumatoid synoviocytes by 15-deoxy-Delta(12,14)-prostaglandin J2

Rheumatoid arthritis (RA) is a chronic polyarticular joint disease associated with massive synovial proliferation, inflammation, and angiogenesis. PPAR-gamma ligands, both 15-deoxy-Delta(12,14)-prostaglandin J2 (15d- PGJ2) and troglitazone (TRO), can inhibit the growth of RA synoviocytes in vitro, and suppress the chronic inflammation of adjuvant-induced arthritis in rats, but the potency of 15d-PGJ2 is higher than TRO. Prostaglandin (PG) E2 plays important roles in joint erosion and synovial inflammation. In the present study, 15d-PGJ2, but not TRO and other prostanoids, suppressed interleukin (IL)-1beta-induced PGE2 synthesis in rheumatoid synovial fibroblasts (RSFs) through the inhibition of cyclooxygenase (COX-2) and cytosolic phospholipase A2 (cPLA2) expression. Furthermore, the inhibition was not affected by pretreatment with anti-PPAR-gamma antibody. It means that this anti-inflammatory effect of 15d-PGJ2 for PG synthesis may be independent of PPAR-gamma and 15d-PGJ2 is a key regulator of negative feedback of the arachidonate cascade on the COX pathway. These findings provide new insight into the feedback mechanism of the arachidonate cascade.     

15(S)-HETE modulates LTB(4) production and neutrophil chemotaxis in chronic bronchitis

We evaluated the levels of15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE] and the expression of15-lipoxygenase (15-LO) mRNA in induced sputum obtained from 10 controland 15 chronic bronchitis subjects. 15(S)-HETE was evaluated by reversephase high-performance liquid chromatography separationfollowed by specific RIA. 15-LO mRNA expression was determined byprimed in situ labeling. The levels of both soluble and cell-associated 15(S)-HETE resulted significantly higher in chronic bronchitis than incontrol subjects. The percentage of cells expressing 15-LO mRNA wassignificantly higher in chronic bronchitis than in control subjects(P < 0.01). Double staining for specific cell typemarkers and 15-LO mRNA showed macrophages and neutrophils positive for 15-LO, whereas similar staining of peripheral blood neutrophils did notshow evidence for 15-LO expression, suggesting that expression of 15-LOin neutrophils takes place on migration into the airways. Because15(S)-HETE inversely correlated with the percentage of neutrophils insputum of chronic bronchitis subjects, we studied the effect of15(S)-HETE on leukotriene B₄ (LTB₄) productionin vitro and evaluated the concentration of LTB₄ in inducedsputum and the contribution of LTB₄ to the chemotacticactivity of induced sputum samples ex vivo. The results obtainedindicate that macrophages and neutrophils present within the airways ofchronic bronchitis subjects express 15-LO mRNA; increased basal levelsof 15(S)-HETE may contribute to modulate, through the inhibition of5-lipoxygenase metabolites production, neutrophil infiltration andairway inflammation associated with chronic bronchitis.

     

Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.

Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis.     

Endogenous monocyte chemoattractant protein-1 (MCP-1) protects mice in a model of acute septic peritonitis: cross-talk between MCP-1 and leukotriene B4

We investigated the involvement of monocyte chemoattractant protein (MCP)-1 in a murine model of septic peritonitis induced by cecal ligation and puncture (CLP). Initial studies demonstrated that CLP induced a dramatic increase in MCP-1 production in the peritoneum, followed by an increase in the recruitment of leukocytes. MCP-1 blockade with anti-MCP-1 antiserum significantly decreased the survival rate following CLP, which was accompanied by an enhanced recovery of viable bacteria from the peritoneum. This was likely due to the reduction in the recruitment and activation of both macrophages and neutrophils. To understand the mechanisms whereby MCP-1 may influence neutrophil infiltration, levels of chemokines known to attract neutrophils were monitored, which showed that peritoneal levels of macrophage-inflammatory protein (MIP)-2, KC, and MIP-1alpha were not altered with anti-MCP-1 Abs. However, anti-MCP-1 Abs reduced the peritoneal levels of leukotriene B4 (LTB4) by 59%. The i.p. injection of MCP-1 into normal mice resulted in elevated levels of LTB4 in the peritoneum. In vitro, MCP-1 stimulated the production of LTB4 from peritoneal macrophages, in a dose-dependent manner. A specific LTB4 receptor antagonist (CP-105, 696) inhibited CLP-induced recruitment of both neutrophils and macrophages, which was accompanied by a reduced level of MCP-1 in the peritoneum. Finally, administration of CP-105,696 was extremely detrimental to the survival of mice following CLP. These experiments demonstrate that endogenous MCP-1 serves as an indirect mediator to attract neutrophils via the production of LTB4, and suggest the cross-talk can occur between MCP-1 and the lipid mediator LTB4 during septic peritonitis.     

The combined use of isolated strips of guinea-pig lung parenchyma and ileum as a sensitive and selective bioassay for leukotriene B4

The biological effects of leukotriene (LT)B4 were compared, on a molar basis, with those of LTC4, LTD4, LTE4, 5-hydroxyeicosatetraenoic acid (5-HETE), PGD2, PGE1, PGE2, PGF2 alpha, PGI2, 6-oxo-PGF1 alpha, bradykinin (BK) and angiotensin II (Ang II) on isolated strips of guinea-pig lung parenchyma (GPP) and ileum smooth muscle (GPISM) superfused in series. LTB4 was similar to LTC4 and LTD4 on GPP, in relation to potency and contractions induced, but differed from LTE4 in being ten times more active and causing contractions of a much shorter duration of action on this tissue. However, unlike the other LTs, LTB4 produced contractions which were resistant to FPL 55712 (1.9 microM) and, when given repeatedly, caused tachyphylaxis in GPP. LTB4 was considerably more active on GPP than the other substances investigated. Further, PGD2, PGF2 alpha and PGI2 contracted GPP, the order of potency being PGD2 greater than PGF2 alpha approximately equal to PGI2, whereas PGE1 and PGE2 relaxed this tissue. In contrast to all other agonists tested which contracted GPISM, LTD4 displaying the highest activity, LTB4 was inactive on this tissue. 5-HETE and 6-oxo-PGF1 alpha were inactive on both GPP and GPISM. On the basis of differential effects of LTB4 on GPP and GPISM, this assay represents a simple and selective means to distinguish LTB4-like materials from other naturally-occurring substances likely to be generated in inflammatory fluids.