Antagonizing leukotriene B4 receptors delays cardiac allograft rejection in mice.

BACKGROUND: Allograft rejection is a cellular immunological/inflammatory response that is, in part, directed by potent proinflammatory mediators. This study was designed to test the hypothesis that leukotriene B4 (LTB4) may have a role in graft rejection and that LTB4 receptor antagonists may be clinically useful in the treatment of allograft rejection. METHODS: We evaluated the potent and selective LTB4 receptor antagonist CP-105696 in a murine heterotopic cardiac allograft model with oral dosing daily for 28 days or in an induction protocol (day -1 to day 3). RESULTS: At a dose of 50 mg/kg/day (28 days), B10.BR (H2k) allografts transplanted into C57Bl/6 (H2b) recipients were significantly protected, as reflected by the mean survival time versus control grafts (27+/-20 days [n=10] vs. 12+/-6 days [n=14]; P=0.0146). Using an induction protocol (day -1 to day 3), CP-105696 at 100 mg/kg/day significantly prolonged allograft survival (33+/-23 days [n=9]; P=0.0026), but CP-105696 at 10 mg/kg/day did not (18+/-16 days [n=8]; P=0.1433). Syngeneic grafts survived indefinitely (n=11). Immunohistological evaluation of allografts at rejection revealed a mononuclear cell infiltrate composed primarily of CD3+ and CD11b+ (Mac-1+) cells, which were infrequent in syngeneic grafts. Allografts from mice treated with CP-105696 at 50 or 100 mg/kg/day demonstrated a selective reduction in beta2-integrin (Mac-1) expression on monocytes/macrophages, as demonstrated by CD11b staining density compared with allograft controls. CONCLUSIONS: The results suggest that LTB4 or other potential ligands for LTB4 receptors may be important mediators of allograft rejection and support the clinical evaluation of LTB4 receptor antagonists in human organ transplantation.

Early eosinophil infiltration into the optic nerve of mice with experimental allergic encephalomyelitis.

Experimental allergic encephalomyelitis is a murine model of preclinical autoimmune disease that has pathologic similarities to multiple sclerosis (MS). Although CD4+ T cells have been shown to play a crucial role in the development of disease, we recently demonstrated a link between the development of paralysis and eosinophil infiltration into the spinal cord. As such, CD4+ cells may initiate disease, but eosinophils may be the actual effector cells responsible for causing damage to myelin and causing paralysis. Because MS patients sometimes experience early visual problems, ie, optic neuritis, we explored whether an early eosinophil infiltrate was also observed in the optic nerves of SJL mice after the passive transfer of encephalitogenic T cells. Seven days after the passive transfer of myelin basic protein (MBP)-reactive T cell blasts, we observed a significant infiltration of eosinophils into the optic nerves of the mice. This infiltration persisted during the early phases of paralysis, then declined to baseline values by the peak of limb paralysis on Day 10, and remained at baseline during the remission phase of the disease. Remyelination of optic nerves was observed at this time. These results suggest that eosinophil infiltration into the optic nerve is one of the earliest events occurring after the passive transfer of encephalitogenic T cells in murine experimental allergic encephalomyelitis.

The preclinical pharmacological profile of the potent and selective leukotriene B4 antagonist CP-195543.

CP-195543 [(+)-2-(3-benzyl-4-hydroxy-chroman-7-yl)-4-trifluoromethyl-benzoic acid] is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro CP-195543 inhibited [3H]LTB4 binding to high-affinity LTB4 receptors on human neutrophils (HN) and murine spleen membranes with IC50 values of 6.8 nM (Ki = 4.9 nM) and 37.0 nM (Ki = 26.9 nM), respectively. CP-195543 inhibited human and mouse neutrophil chemotaxis mediated by LTB4 with IC50 values of 2.4 nM and 7.5 nM, respectively. Evidence of noncompetitive antagonist effects on the HN high-affinity LTB4 receptor was obtained by Scatchard analysis of [3H]LTB4 binding to and chemotaxis of HN to LTB4. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on HN indicated that CP-195543 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b up-regulation on HN was inhibited competitively by CP-195543 (pA2 = 7.66). In whole blood, CP-195543 also blocked CD11b up-regulation on HN (pA2 = 7.12) and murine neutrophils (pA2 = 7.06) with a similar potency. LTB4-mediated CD11b up-regulation on human monocytes and eosinophils in whole blood were inhibited by CP-195543 with IC50 values of 270 nM and 420 nM, respectively. CP-195543 at 10 microM failed to inhibit HN chemotaxis and CD11b up-regulation mediated through alternative (i.e., complement fragment 5a, interleukin-8, platelet-activating factor) G-protein-coupled chemotactic factor receptors. In vivo, after oral administration, CP-195543 blocked LTB4-mediated neutrophil infiltration in guinea pig and murine skin with ED50 values of 0.1 mg/kg and 2.8 mg/kg p.o., respectively. When administered in osmotic pumps, CP-195543 reduced the clinical symptoms and attendant weight loss in an IL-1-exacerbated murine model of collagen-induced arthritis with half-maximal effects associated with plasma drug levels of 0.4 to 0.5 microg/ml. Collectively these data provide evidence of the in vitro potency and in vivo efficacy of a novel LTB4 antagonist and support its clinical evaluation in a variety of inflammatory diseases in man.

Pharmacokinetics and pharmacodynamics of the leukotriene B4 receptor antagonist CP-105,696 in man following single oral administration.

AIMS: CP-105,696, (+)-1-(3S,4R)-[3-(4-phenylbenzyl)-4-hydroxy-chroman-7-yl] cyclopropane carboxylic acid is a potent, novel LTB4 receptor antagonist advanced to clinical trials to determine its efficacy in inflammatory diseases. The pharmacokinetics and pharmacodynamics of CP-105,696 were investigated in healthy male volunteers following oral administration of single doses of 5 to 640 mg. METHODS: Forty-eight subjects participated in a randomized, double-blind, parallel group study. Plasma and urine concentrations of CP-105,696 were determined at intervals after drug administration. As an indication of LTB4 receptor antagonism following oral administration of CP-105,696, the inhibiton of LTB4-induced upregulation of the neutrophil cell surface complement receptor (CR3), CD11b/CD18, was monitored at 4 h following drug administration using an ex vivo whole blood flow cytometry assay. RESULTS: Cmax and AUC(0, infinity) increased in a dose-related manner. Respective mean Cmax values were 0.54 to 30.41 microg ml(-1) following doses of 5 to 640 mg. Respective mean AUC(0, infinity) values were 1337 to 16819 microg ml(-1) h for the 40 to 640 mg dose groups. Plasma concentrations declined in a monoexponential manner, with terminal elimination half-lives ranging from 289 to 495 h. Group mean terminal elimination half-lives were dose-independent. Urinary excretion of unchanged drug accounted for < 1% of the administered dose. A linear relationship was observed between CP-105,696 plasma concentrations and inhibition of LTB4-mediated CD11b upregulation on human neutrophils in whole blood. CP-105,696 plasma concentrations of 5-6 microg ml(-1) were necessary to elicit a two-fold shift to the right of the LTB4 concentration response curve for CD11b upregulation. CONCLUSIONS: These studies demonstrate pharmacologically significant LTB4-receptor antagonism following a single dose of CP-105,696 and pharmacokinetics consistent with once-daily dosing.

trans-3-Benzyl-4-hydroxy-7-chromanylbenzoic acid derivatives as antagonists of the leukotriene B4 (LTB4) receptor.

The SAR of a series of 2-(7-chromanyl)benzoic acids has been investigated with the aim of identifying potent and selective LTB4 receptor antagonists that maintain potency in complex biological fluids. We found optimal activity in derivatives with electron-withdrawing groups in the benzoic acid ring and with an unsubstituted C-3 benzyl group on the chromanol nucleus. While compounds containing a 3-(4-phenyl)benzyl chromanol substituent were potent LTB4 receptor antagonists, the increased lipophilicity imparted by the additional phenyl substituent led to decreased potency in the presence of plasma proteins. From among the potent compounds identified, CP-195543, the 5'-trifluoromethyl 3-benzyl chromanol, was selected for development.

The phosphodiesterase type 4 (PDE4) inhibitor CP-80,633 elevates plasma cyclic AMP levels and decreases tumor necrosis factor-alpha (TNFalpha) production in mice: effect of adrenalectomy.

Rolipram was previously reported to elevate plasma cyclic adenosine 3',5'-monophosphate (cAMP) and inhibit serum tumor necrosis factor-alpha (TNF-alpha) production in mice. CP-80,633, a new cyclic nucleotide phosphodiesterase (PDE4) inhibitor, has been shown to augment intracellular cAMP levels and to inhibit TNFalpha release from human monocytes in vitro. This study was undertaken to determine the effect of p.o. CP-80,633 on plasma cAMP levels and lipopolysaccharide-induced TNFalpha production in mice with and without adrenal glands. CP-80,633 dose-dependently (3-32 mg/kg p.o.) elevated plasma cAMP levels and decreased systemic TNFalpha production in response to i.p. injection of lipopolysaccharide. Elevated plasma cAMP levels can be detected for up to 4 hr. CP-80,633 (10 mg/kg p.o.) caused a 6-fold increase in the plasma cAMP level, a 2-fold increase in the plasma epinephrine level and a greater than 95% reduction in TNFalpha production. Unlike CP-80,633, neither vinpocetine, dipyridamole, SKB-94,120 nor zaprinast, at 100 mg/kg p.o., modified the cAMP response, which suggests that this response is mediated by inhibition of PDE4. Adrenalectomy reduced the cAMP response and completely blocked the epinephrine response; however, the levels of plasma cAMP in the CP-80,633-treated mice (10 mg/kg p.o.) remained elevated (vehicle: 47.3 +/- 6.8 vs. CP-80,633: 98.4 +/- 10.3 pmol/ml, n = 7, P < .05). This effect is mimicked by treatment of control mice with propranolol, which demonstrates that beta adrenoreceptors contribute to the cAMP response. Removal of adrenal glands significantly increased the LPS-induced elevation of serum TNFalpha. The ability of CP-80,633 to block the TNFalpha response was only slightly affected by adrenalectomy (ED50 = 1.2 mg/kg in controls vs. 3.9 mg/kg in adrenalectomized mice). Taken together, these results show that CP-80,633, when given p.o. to mice, is capable of elevating plasma cAMP and inhibiting TNFalpha production and that adrenal catecholamines contribute significantly to the effect of CP-80,633 on the cAMP response but only slightly to its effect on the systemic TNFalpha response.

Biological activity C-X-C and C-C chemokines on leukocyte subpopulations in human whole blood.

We have described an assay that monitors the activating effects of a variety of chemokines on leukocyte subsets in human whole blood. This procedure has the following advantages: (1) minimal manipulation of the cells, (2) maintenance of more physiological conditions, and (3) simultaneous monitoring of the responses of monocytes, neutrophils, and eosinophils.

Chemokine-dependent upregulation of CD11b on specific leukocyte subpopulations in human whole blood: effect of anticoagulant on rantes and MIP-1 beta stimulation.

The effect of anticoagulant (heparin vs EDTA) on chemokine induced CD11b upregulation on neutrophils, eosinophils, and monocytes in human whole blood was determined. For most of the chemokines (IL-8, GRO-alpha, MCP-1, MIP-1 alpha) the difference in the response of leukocytes in EDTA anticoagulated blood vs those in heparinized blood was the degree of their maximal response, with a slightly higher maximal increase in CD11b expression usually seen in cells from EDTA anticoagulated blood. Two chemokines were exceptions to this: RANTES and MIP-1 beta. RANTES is considered to be a stimulator of monocytes and eosinophils and not of neutrophils. As expected, neutrophils in heparinized whole blood did not respond to RANTES; however, neutrophils in EDTA anticoagulated blood had a significant increase in CD11b when exposed to high concentrations (1 microM) of RANTES. RANTES-induced CD11b expression on monocytes and eosinophils in these samples were the same in either heparin or EDTA. In EDTA anticoagulated blood, MIP-1 beta did not elicit a response in either monocytes, eosinophils or neutrophils; however, in heparinized blood, all three cell types increased CD11b expression upon exposure to 1 microM MIP-1 beta.

The in vivo pharmacology of CP-80, 633, a selective inhibitor of phosphodiesterase 4.

The following studies were conducted to characterize the bron-chodilatory and antiinflammatory activity of the novel, selective phosphodiesterase-IV inhibitor, CP-80,633 (2'S)5-[3-(2'-exobicyclo[2.2.1]heptyloxy-4-methoxy-phenyl]te trahydro- 2(1H)-pyrimidone, a compound in clinical development for atopic disease. In IgG1 passively sensitized guinea pigs, aerosolized ovalbumin challenge increases both pulmonary eosinophil peroxidase levels and airway obstruction. CP-80,633, administered before ovalbumin challenge, significantly attenuated both the increase in tissue eosinophil peroxidase levels (ED50 = 1.4 mg/kg, p.o.) and airway obstruction (ED50 = 0.93 +/- 0.14 mg/kg,p.o.) 10 to 30 times more potently than theophyl-line. Intraarterially administered CP-80,633 also reversed an established bronchoconstriction initiated by continuous infusion of histamine to guinea pigs (ED50 of 8.2 micrograms/kg vs. 5.6 mg/kg for theophylline). The antiinflammatory effect of CP-80,633 was also examined in atopic monkeys challenged with Ascaris suum (Ag) aerosol. CP-80,633 (1 mg/kg, qid, s.c., 1 hr before antigen challenge) significantly reduced antigen-induced increases in bronchoalveolar lavage neutrophils (72.8 +/- 15.8% inhibition) and eosinophils (61.1 +/- 5.7% inhibition) 4 hr postchallenge, but did not reduce leukocytes 24 hr postchallenge. CP-80,633 did not inhibit antigen-induced increases in BAL levels of interleukin-1 beta, -6 or -8 as measured by enzyme-linked immunosorbant assay. These results indicate that CP-80,633 possesses bronchodilatory activity in guinea pigs and some antiinflammatory effects in both guinea pigs and monkeys.

In vitro pharmacology of the novel phosphodiesterase type 4 inhibitor, CP-80633.

We present the in vitro pharmacology of a novel adenosine 3'-5' -cyclic monophosphate-specific phosphodiesterase (PDE) type 4 inhibitor, CP-80633 ((2'S)5-[3-(2' -exobicyclo[2.2.1]-heptyloxy)4-methoxyphenyl] tetrahydro-2(1H)-primidone), which has shown efficacy in phase II clinical trials for atopic dermatitis. CP-80633 inhibits PDE4 isozymes (human lung IC50 = 1.27 microM) in the absence of effects on PDE1, PDE2, PDE3 and PDE5 isozymes (IC50 > 100 microM). It exhibits no significant selectivity for any single cloned PDE4A, B, C or D isoform. CP-80633 inhibits adenosine 3'-5'-cyclic monophosphate hydrolysis in partially purified human peripheral blood monocyte cytosol (IC50 = 3.52 microM), eosinophil membrane (IC50 = 1.10 microM) and T cell membrane (IC50 = 2.28 microM) preparations. Inhibition of eosinophil PDE4 adenosine 3'-5'-cyclic mono-phosphate hydrolysis by CP-80,633 occurs in a noncompetitive manner. Unlike theophylline, CP-80,633 is inactive against ratrain adenosine (A1,A2) receptors. Consistent with its action as a PDE4 inhibitor in whole cells, CP-80633 potentiates PGE1 dependent increases in adenosine 3'-5'-cyclic monophosphate levels in human U937 cells, and in human eosinophils, monocytes and T cells (EC200 approximately 1.0 microM). Consequently, CP-80633 inhibits many inflammatory cell functions including 1) human eosinophil superoxide anion production (IC50 < 0.6 microM), 2 C5a-(IC50 = 0.40 microM) and LTB4-(IC50 = 0.20 microM) mediated guinea pig peritoneal eosinophil chemotaxis and 3) lipopolysac-charide-induced tumor necrosis factor-alpha release from human monocytes (IC50 = 0.219 microM). These data clearly demonstrate that CP-80633 is a selective inhibitor of PDE4 isozymes, and support its potential use as a therapeutic agent for a number of inflammatory and immune disorders.

Production and characterization of guinea pig IL-5 in baculovirus-infected insect cells.

To study the role interleukin (IL)-5 may play in altering airway function in asthma, we have produced recombinant protein for exogenous administration to guinea pigs. The guinea pig IL-5 (gpIL-5) cDNA was cloned by polymerase chain reaction (PCR) amplification of guinea pig spleen RNA and expressed as a secretion product from recombinant baculovirus-infected Sf9 insect cell cultures. The protein was purified to homogeneity by a four-step procedure that included immunoaffinity chromatography using polyclonal antipeptide antibodies against a region of the mature secreted cytokine. The cytokine was properly processed after the signal sequence by the Sf9 cells, was glycosylated with terminal mannose-containing oligosaccharide, and had proper disulfide-linked dimer structure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified preparation was active in vitro and in vivo as determined by its ability to prime human basophils to release leukotriene C4 in the presence of C5a and to induce airway eosinophilia in naive guinea pigs.

Inhibition of leukotriene B4-receptor interaction suppresses eosinophil infiltration and disease pathology in a murine model of experimental allergic encephalomyelitis.

Leukotriene B4 (LTB4) is a chemotactic and cell-activating factor present at inflammatory sites in a variety of autoimmune diseases including multiple sclerosis (MS). In this study, we used a murine model of MS, experimental allergic encephalomyelitis (EAE), to assess the potential role of LTB4 on cell infiltration and paralysis. Injection of encephalogenic T cells into naive animals induced paralysis and weight loss that was completely inhibited by treatment with the selective LTB4 receptor antagonist CP-105,696 (ED50= 8.6 mg/kg orally). Although migration of lymphocytes into the central nervous system was unaffected, the efficacious effects of CP-105,696 correlated with up to a 97% decrease in eosinophil infiltration into the lower spinal cord as determined by light and electron microscopy and quantitated by levels of the specific enzyme marker eosinophil peroxidase. These results demonstrate that eosinophil recruitment in EAE is dependent on LTB4 receptor ligation and further reveal a previously unrecognized role for eosinophils in the pathogenesis of this disease.

Characterization of the pharmacological profile of the potent LTB4 antagonist CP-105,696 on murine LTB4 receptors in vitro.

1. Binding of [3H]-leukotriene B4 ([3H]-LTB4) to murine spleen membranes (MSM) was determined. 2. Scatchard analyses of [3H]-LTB4 binding indicated the presence of high (KD1 = 1.7 nM) and low (KD2 = 7.5 nM) affinity receptors on MSM with Bmax values of 151 fmol mg-1 protein (Bmax1) and 354 fmol mg-1 protein (Bmax2), respectively. 3. CP-105,696, a potent LTB4 antagonist, inhibited [3H]-LTB4 (0.67 nM) binding to the high affinity receptor on MSM, IC50 = 30.2 nM, Ki = 17.7 nM with a Hill coefficient of 0.93. 4. Scatchard analyses of [3H]-LTB4 binding to MSM in the presence of CP-105,696 indicated that the high-affinity receptor was inhibited in a non-competitive manner and the low-affinity receptor in a competitive manner. 5. Isolated peripheral blood murine neutrophils (MN) responded chemotactically to LTB4, EC50 = 2.5 nM. CP-105,696 blocked this response, IC50 = 2.3 nM. When examined over a full concentration-response range of LTB4, CP-105,696 inhibited chemotaxis in a non-competitive manner. 6. Murine neutrophils in anticoagulated whole blood upregulated the integrin, complement receptor type 3 (CD11b/CD18, Mac-1) in response to LTB4, EC50 = 20 nM and this was inhibited by CP-105,696 in a competitive manner. 7. These results provide evidence that MSM have specific binding sites for LTB4, and as exemplified by CP-105,696, that these receptors may be useful for determining the potency and nature of antagonism of novel LTB4 receptor antagonists.

In vitro and in vivo effects of leukotriene B4 antagonism in a primate model of asthma.

To test the hypothesis that leukotriene (LT) B4 antagonists may be clinically useful in the treatment of asthma, CP-105,696 was evaluated in vitro, using chemotaxis and flow cytometry assays, and in vivo, using a primate asthma model. CP-105,696 inhibited LTB4-mediated monkey neutrophil chemotaxis (isolated cells, LTB4 = 5 nM) and CD11b upregulation (whole blood, LTB4 = 100 nM) with IC50 values of 20 nM and 16.5 microM, respectively. Using a modification of a previously described in vivo protocol (Turner et al. Am. J. Respir. Crit. Care Med. 1994. 149: 1153-1159), we observed that treatment with CP-105,696 inhibited the acute increase in bronchoalveolar lavage (BAL) levels of IL-6 and IL-8 by 56.9 +/- 13.2% and 46.9 +/- 14.5%, respectively, 4 h after challenge with Ascaris suum antigen (Ag). CP-105,696 tended to reduce the increase in BAL protein levels 0.5 h after Ag challenge by 47.5 +/- 18.3%, but this was not statistically significant. In addition, CP-105,696 prevented the significant 11-fold increase in airway responsiveness to methacholine after multiple Ag challenge. These results suggest that LTB4 partially mediates acute and chronic responses to antigen in an experimental primate asthma model and support the clinical evaluation of LTB4 antagonists in human asthma.

Expression and biologic characterization of the murine chemokine KC.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was expressed in Escherichia coli by using a maltose binding protein vector and biochemically characterized as a ligand for both murine and human polymorphonuclear neutrophils (PMN). On murine PMN, KC is both a potent chemoattractant and up-regulator of Mac-1 cell surface expression. On human PMN, in contrast, KC exhibits dissociation of its chemoattractant and Mac-1 up-regulatory activities. Although KC strongly increases Mac-1 expression on human PMN, it does not induce chemotaxis in vitro. 125I-KC-Tyr binds to both mouse and human PMN with two classes of binding sites, including high affinity sites of 0.8 and 2 nM, with approximately 9,000 and 10,000 sites per cell, respectively. On mouse PMN, human macrophage inflammatory protein (MIP)-2 alpha and MIP-2 beta compete for 125I-KC-Tyr binding with high affinity, whereas the murine beta-chemokine TCA-3 does not compete. KC binds to human PMN by the IL-8 type B receptor and to murine PMN by a murine IL-8 type B receptor homologue. 125I-KC-Tyr also binds to human RBC with a single class of high affinity sites. KC mRNA is constitutively expressed in multiple murine tissues. With human IL-8 and KC cDNA as probes, a mouse neutrophil exudate library was screened: KC and MIP-2 were the dominant chemokine species found. Thus, KC appears to be intimately involved in murine inflammation and its constitutive expression may have a role in the basal trafficking of neutrophils.

The Duffy antigen/receptor for chemokines (DARC) is expressed in endothelial cells of Duffy negative individuals who lack the erythrocyte receptor.

The Duffy antigen/receptor for chemokines (DARC), first identified on erythrocytes, functions not only as a promiscuous chemokine receptor but also as a receptor for the malarial parasite, Plasmodium vivax. The recent finding that DARC is ubiquitously expressed by endothelial cells lining postcapillary venules provides a possible insight into the function of this receptor because this anatomic site is an active interface for leukocyte trafficking. However, the biological significance of DARC is questionable since it has not yet been determined whether individuals lacking the expression of this protein on their erythrocytes (Duffy negative individuals), who are apparently immunologically normal, express the receptor on endothelial cells. However, we report here that DARC is indeed expressed in endothelial cells lining postcapillary venules and splenic sinusoids in individuals who lack the erythrocyte receptor. These findings are based on immunohistochemical, biochemical, and molecular biological analysis of tissues from Duffy negative individuals. We also present data showing that, in contrast to erythrocyte DARC, cells transfected with DARC internalize radiolabeled ligand. We conclude that the DARC may play a critical role in mediating the effects of proinflammatory chemokines on the interactions between leukocyte and endothelial cells since the molecular pathology of the Duffy negative genotype maintains expression on the latter cell type.

The in vitro and in vivo pharmacologic activity of the potent and selective leukotriene B4 receptor antagonist CP-105696.

CP-105696, (+)-1-(3S,4R)-[3-(4-phenyl-benzyl)-4-hydroxy-chroman-7-yl] cyclopentane carboxylic acid, is a structurally novel, selective and potent leukotriene B4 (LTB4) receptor antagonist. In vitro, CP-105696 inhibited [3H]LTB4 (0.3 nM) binding to high-affinity LTB4 receptors on human neutrophils with an IC50 value of 8.42 +/- 0.26 nM. Scatchard analyses of [3H]LTB4 binding to these high-affinity receptors indicated that CP-105696 acted as a noncompetitive antagonist. CP-105696 inhibited human neutrophil chemotaxis mediated by LTB4 (5 nM) in a noncompetitive manner with an IC50 value of 5.0 +/- 2.0 nM. Scatchard analyses of [3H]LTB4 binding to low-affinity receptors on neutrophils indicated that CP-105696 acted as a competitive antagonist at this receptor, and inhibition of LTB4-mediated CD11b upregulation on human neutrophils was competitively inhibited by CP-105696 (pA2 = 8.03 +/- 0.19). CP-105696 at 10 microM did not inhibit either human neutrophil chemotaxis or CD11b upregulation mediated through alternate (i.e., C5a, IL-8, PAF) G-protein coupled chemotactic factor receptors. In isolated human monocytes, LTB4 (5 nM)-mediated Ca++ mobilization was inhibited by CP-105696 with an IC50 value of 940 +/- 70 nM. In vivo, after oral administration, CP-105696 blocked neutrophil and eosinophil infiltration in cavine dermis mediated by either LTB4 or arachidonic acid with ED50 values of 0.3 +/- 0.1 mg/kg. 12(R)-Hydroxyeicosatetraenoic acid-mediated neutrophil infiltration was blocked by 76.4 +/- 14.8% at 3 mg/kg.(ABSTRACT TRUNCATED AT 250 WORDS)

Synovial fluid from rheumatoid arthritis patients contains sufficient levels of IL-1 beta and IL-6 to promote production of serum amyloid A by Hep3B cells.

The presence of positive acute phase proteins within the circulation of rheumatoid arthritis patients suggests that elevated cytokine production associated with this chronic inflammatory disorder initiates the hepatic acute phase response. Cytokines produced at inflammatory lesions are believed to travel via the circulation to the liver where they induce acute phase protein production by hepatocytes. To test whether serum from rheumatoid arthritis patients contained sufficient levels of cytokines to promote an acute phase response in vitro, a bioassay was developed that employed the human hepatoma cell line Hep3B. These cells produced the acute phase protein serum amyloid A (SAA) in response to a combination of recombinant IL-1 beta and IL-6 or to monocyte conditioned medium. Serum (or plasma) from normal individuals or from rheumatoid arthritis patients did not induce SAA production by Hep3B cells. Moreover, these serum samples did not prevent SAA production induced by monocyte conditioned medium, indicating that they did not contain inhibitors of cytokine activity. Despite the inactivity of serum samples, synovial fluid samples obtained from rheumatoid arthritis patients were active in the hepatocyte bioassay and promoted SAA synthesis. One synovial fluid sample was analysed in detail to identify cytokines responsible for the SAA-inducing activity. Neutralizing antisera against IL-6 and IL-1 beta blocked this activity by > 90% whereas anti-IL1 alpha and anti-TNF-alpha sera were without effect. Absolute cytokine levels within the synovial fluid sample were determined by ELISA; IL-6, IL-beta and TNF-alpha, but not IL-1 alpha, were confirmed to be present. Moreover, the synovial fluid sample contained a large amount of the IL-1 receptor antagonist. These data indicate, therefore, that synovial fluid recovered from an inflamed joint contains all the necessary cytokines in balance with inhibitors to promote SAA production by Hep3B cells. The steady state levels of these factors within the plasma compartment, however, were insufficient to induce the acute phase response by cultured Hep3B cells, suggesting that this system does not mimic the relationship between the circulation and the liver that likely exists in rheumatoid arthritis patients.

Prolonged microvascular vasodilation induced by leukotriene B4 in human skin is cyclooxygenase independent.

When the potent chemoattractant leukotriene B4 (LTB4) is applied topically to human skin it causes delayed onset, long-lasting leukocyte accumulation and erythema. We investigated the role of prostaglandins in the increase in local blood flow by applying LTB4 topically to the forearm skin of 22 healthy male volunteers and measuring the effect of the anti-inflammatory compounds tenidap, naproxen and indomethacin. Local microvascular blood flow responses were measured by laser Döppler flow probe and planimetry. LTB4 induced dose-dependent increases in blood flow which were maximal at 48 hr and lasted 4 days. Laser Döppler flow (% flux) at 48 hr was 2.7 +/- 0.1, 20.6 +/- 3.1, 28.7 +/- 2.4 and 30.2 +/- 2.3% in control and 3, 10, 30 ng/site LTB4, respectively (mean +/- S.E.M.). In eight subjects the intradermal injection of indomethacin, at a dose (3 x 10(-9) mol/site) that inhibited significantly the increased flow induced by intradermally injected arachidonic acid (1 x 10(-9) mol/site, n = 6), had no effect on the increased skin blood flow response induced by LTB4 (10 ng/site) at 48 hr. Blood flow in vehicle-injected LTB4 sites was 810 +/- 150% above basal and 819 +/- 149% in sites injected with indomethacin. In 20 subjects, the effect of the anti-inflammatory drugs naproxen and tenidap given orally on the skin blood flow responses to LTB4 were compared in a double-blind crossover design. The 1085 +/- 98% increase in local blood flow induced by 30 ng of LTB4 at 48 hr was unaltered at the end of the treatment periods with either naproxen or tenidap, where blood flow in the LTB4-treated sites was increased 1018 +/- 131% and 1034 +/- 130%, respectively. Because the vasodilator response to exogenous LTB4 was not altered by nonsteroidal anti-inflammatory drugs either injected locally or taken orally, we suggest that endogenous prostaglandins are not involved in this response.

The murine interleukin 8 type B receptor homologue and its ligands. Expression and biological characterization.

KC, the product of an immediate early gene induced in mouse fibroblasts by platelet-derived growth factor, was synthesized as a recombinant protein in Escherichia coli and binds with 0.8 nM affinity to mouse neutrophils. Human neutrophils also bind recombinant KC at a site competitive with human interleukin (IL8) and Gro-alpha/MGSA, consistent with binding at the IL8 type B receptor (IL8RB). The cDNA corresponding to human IL8RB hybridizes strongly with two restriction fragments in murine genomic DNA, representing candidate receptor genes for KC. Molecular cloning of both mouse genomic DNA and neutrophil exudate cell cDNA libraries yielded a receptor with approximately 68% sequence identity to both the human IL8 type A and B receptors. Transient expression of the murine receptor cDNA in COS cells conferred binding ability to KC and a related gene product, macrophage inflammatory protein-2 (MIP-2) with high affinity (approximately 5 nM). Human IL8 was a poor agonist for this expressed receptor (Kd = approximately 400 nM). The potent activity of human IL8 on mouse polymorphonuclear neutrophils is not consistent with binding on the cloned receptor and suggests that murine homologues of IL8 and an IL8 type A receptor remain to be identified. Our data indicate that KC is the murine homologue of human Gro-alpha, and the KC receptor is an IL8 type B receptor homologue capable of binding both KC and macrophage inflammatory protein-2 with high affinity.