NTPDase1 controls IL-8 production by human neutrophils

The ectonucleotidase NTPDase1 (CD39) terminates P2 receptor activation by the hydrolysis of extracellular nucleotides (i.e., the P2 receptor ligands). In agreement with that role, exacerbated inflammation has been observed in NTPDase1-deficient mice. In this study, we extend these observations by showing that inhibition of NTPDase1 markedly increases IL-8 production by TLR-stimulated human neutrophils. First, immunolabeling of human blood neutrophils and neutrophil-like HL60 cells displayed the expression of NTPDase1 protein, which correlated with the hydrolysis of ATP at their surface. NTPDase1 inhibitors (e.g., NF279 and ARL 67156) as well as NTPDase1-specific small interfering RNAs markedly increased IL-8 production in neutrophils stimulated with LPS and Pam(3)CSK(4) (agonists of TLR4 and TLR1/2, respectively) but not with flagellin (TLR5) and gardiquimod (TLR7 and 8). This increase in IL-8 release was due to the synergy between TLRs and P2 receptors. Indeed, ATP was released from neutrophils constitutively and accumulated in the medium upon NTPDase1 inhibition by NF279. Likewise, both human blood neutrophils and neutrophil-like HL60 cells produced IL-8 in response to exogenous nucleotides, ATP being the most potent inducer. In agreement, P2Y(2) receptor knockdown in neutrophil-like HL60 cells markedly decreased LPS- and Pam(3)CSK(4)-induced IL-8 production. In line with these in vitro results, injection of LPS in the air pouches of NTPDase1-deficient mice triggered an increased production of the chemokines MIP-2 and keratinocyte-derived chemokine (i.e., the rodent counterparts of human IL-8) compared with that in wild-type mice. In summary, NTPDase1 controls IL-8 production by human neutrophils via the regulation of P2Y(2) activation.     

alpha-Tocopherol Inhibits IL-8 synthesis induced by thrombin and high glucose in endothelial cells.

It has been reported that alpha-tocopherol, an antioxidant agent, may play a role in preventing diabetic angiopathy. However, there is little evidence to show the effect of alpha-tocopherol on the production of pro-inflammatory cytokines in endothelial cells. Therefore, we examined the effect of alpha-tocopherol on the regulation of IL-8 synthesis induced by high glucose and/or thrombin in endothelial cells. Thrombin alone markedly increased the IL-8 release. Furthermore, high glucose levels and thrombin combined had additive effects on IL-8 synthesis, and alpha-tocopherol diminished their effect; alpha-tocopherol also inhibited the phosphorylation of IkappaB-alpha induced by high glucose levels and/or thrombin. Our results suggest that the administration of alpha-tocopherol to diabetic patients may have a beneficial effect for the prevention of diabetic vascular complications by the inhibition of IL-8 synthesis from endothelial cells.     

Cleavage of CD14 on human gingival fibroblasts cocultured with activated neutrophils is mediated by human leukocyte elastase resulting in down-regulation of lipopolysaccharide-induced IL-8 production

Activated polymorphonuclear leukocytes (PMNs) release various types of proteases and express them on the cell surface. The proteases play important roles in PMN-mediated events. In the present study, flow cytometric analysis revealed that CD14 expression on human gingival fibroblasts (HGF) was markedly reduced by PMA-activated PMNs in a coculture system. We found that this reduction was caused by both secreted and cell surface proteases produced by activated PMNs. A protease responsible for the reduction was found to be human leukocyte elastase (HLE) secreted from the activated PMNs by use of various protease inhibitors, although HLE was only partially involved in CD14 reduction caused by cell-bound molecule(s) on fixed PMNs. Analysis with purified HLE revealed a time- and dose-dependent reduction of CD14 on HGF, and complete reduction was observed by 20 microg/ml HLE treatment for 30-60 min, but the other molecules such as CD26, CD59, CD157, and MHC class I on HGF were only slightly reduced. This reduction of CD14 resulted from direct proteolysis by HLE on the cell surface, because HLE reduced CD14 on fixed HGF and also on purified cell membranes. As a result of CD14 proteolysis, IL-8 production by HGF was suppressed when triggered by 10 ng/ml LPS, but not by IL-1alpha, indicating that HLE inhibited a CD14-dependent cell activation. These findings suggested that activated PMNs have a potential negative feedback mechanism for HGF function at the inflammatory site, particularly in periodontal tissues.     

Platelet responses to compound interactions with thrombin.

Catalytic and noncatalytic interactions of thrombin with platelets are investigated with use of thrombin variants with altered specificities and with ligands of thrombin receptors on platelets. Both alpha-thrombin and weakly coagulant meizothrombin-des-fragment-1 (mu-thrombin) hydrolyze proteolytically activated receptor 1 for thrombin (rPAR1(T), recombinant) with catalytic efficiencies of >10(7) M(-)(1) s(-)(1), whereas rPAR1(T) is not a substrate for weakly coagulant beta-thrombin. In contrast, both mu-thrombin and beta-thrombin are weak agonists of platelet dense body (ATP) secretion. Antibodies that block rPAR1(T) cleavage strongly inhibit the secretory reaction to alpha- and mu-thrombins but not to beta-thrombin or to thrombin receptor activating peptide (TRAP). However, catalytically inactive FPR-thrombin, which binds glycoprotein Ib but does not inhibit rPAR1(T) cleavage, inhibits responses to TRAP as well as those to alpha- and mu-thrombins, which indicates that binding of the inactive enzyme to platelets influences the function of PAR1(T). An antibody that inhibits binding of thrombin to platelet glycoprotein Ib inhibits secretory responses to thrombin but not to TRAP, so occupancy of glycoprotein Ib per se accounts for only part of the attenuation. All three thrombins stimulate a rise in cytosolic Ca(II), and the dose response to beta-thrombin is congruent with that for ATP secretion. However, the response of cytosolic Ca(II) is 10-100 times more sensitive to mu-thrombin and alpha-thrombin than ATP secretion is, and is inhibited by neither anti-PAR1(T) Ig nor FPR-thrombin. Thus, alpha-thrombin appears to have an activity not shared by either mu- or beta-thrombins. This activity is owed to more than coupling of independent signals from cleavage of two proteolytically activated receptors, as there is no synergism when mu-thrombin and beta-thrombin costimulate secretion. It is concluded either that alpha-thrombin has a third interaction site on platelets with which neither mu-thrombin nor beta-thrombin interacts or that dual receptors are coordinately cleaved. In either case, the strong secretory response to thrombin appears to be moderated, independently of cytosolic Ca(II), by occupancy of a noncatalytic interaction site such as glycoprotein Ib.     

Structure-function relationships of hirulog peptide interactions with thrombin.

Using hirudin as a model, a novel class of bivalent thrombin inhibitors has been designed and characterized (Maraganore et al. (1990) Biochemistry 29, 7095-7101). These peptides, designated 'hirulogs', interact with both thrombin's catalytic center and its anion-binding exosite for fibrinogen recognition. In order to investigate structure-activity relationships in hirulog peptides, a number of peptide and peptidomimetic derivatives with alterations in catalytic-site binding and anion-binding exosite binding moieties were prepared. Replacements or modifications in the catalytic site and exosite binding moieties were achieved with the consequences of maintaining or improving antithrombin activity. In addition to showing improved affinity for thrombin, some derivatives with Ki's in the sub-nanomolar range showed increased anticoagulant activities. These findings highlight the versatility of hirulog peptides in their bivalent interactions with thrombin.     

Intravascular IL-8. Inhibitor of polymorphonuclear leukocyte accumulation at sites of acute inflammation.

IL-8 has been characterized primarily as a polymorphonuclear leukocyte (PMN) chemoattractant and proinflammatory mediator. Recently, we have reported that [Ala-IL-8]77 is secreted by activated cultured human endothelial cells and can function as a potent inhibitor of PMN adhesion to these monolayers. The pathophysiologic relevance of this in vitro observation was examined by determining the effects of intravascular or extravascular administration of IL-8 on PMN emigration at sites of acute inflammation in the skin of NZW rabbits. An i.v. bolus of [Ala-IL-8]77 (12 micrograms/kg) produced a marked and selective reduction of circulating PMN within 3 min, which returned toward preinjection levels within 30 min, and subsequently exceeded this level. A similar response was observed for circulating radiolabeled PMN, and gamma-scintigraphy determined that the lungs were the primary site of leukosequestration. During the 30- to 150-min interval after i.v. infusion of [Ala-IL-8]77, PMN emigration into acute inflammatory sites, elicited by various chemoattractants or cytokines, was significantly reduced, as judged histologically and quantitated with 51Cr-labeled PMN and myeloperoxidase measurements. Intravenous administration of [Ser-IL-8]72 yielded similar results. This inhibitory effect of i.v. IL-8 was transient and reinducible and did not reflect a suppression of the responsiveness of circulating PMN to chemoattractants. Intradermal injections of [Ala-IL-8]77 or [Ser-IL-8]72 induced dose-dependent PMN accumulation, which also was significantly reduced by i.v. administration of either form of IL-8. These results indicate that i.v. IL-8 can function as a PMN-directed leukocyte adhesion inhibitor and suggest that local secretion of IL-8 by activated endothelium may differentially modulate leukocyte-endothelial interactions at sites of acute inflammation.     

Anti-IL-8 autoantibody:IL-8 immune complexes suppress spontaneous apoptosis of neutrophils

Our previous studies demonstrated that a significant fraction of interleukin-8 (IL-8) in lung fluids from patients with acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS) is associated with anti-IL-8 autoantibodies (anti-IL-8:IL-8 immune complexes). Neutrophils have been implicated in the pathogenesis of ALI/ARDS, and moreover, it is well-established that apoptosis of neutrophils is delayed in patients with ALI/ARDS. The aim of this study was, therefore, to examine the role of anti-IL-8:IL-8 immune complexes in modulating spontaneous apoptosis of normal human neutrophils. Apoptosis was assessed by evaluating morphological changes, measuring enzymatic activity of caspase-3, and determining the extent of DNA degradation. We found that samples containing anti-IL-8:IL-8 immune complexes but not samples from which these complexes were removed inhibited neutrophil apoptosis. Furthermore, the former samples or effectively anti-IL-8:IL-8 complexes induced an increase in the level of antiapoptotic protein, Bcl-X(L). In contrast, levels of proapoptotic proteins Bax and Bak were decreased in the same conditions. Activity of both caspase-3 and caspase-9 was also suppressed by anti-IL-8:IL-8 complex-containing samples. Finally, we established that IgG receptor, FcgammaRIIa, mediates antiapoptotic activity of anti-IL-8:IL-8 complexes and that the key components of the FcgammaRIIa signaling pathway, Src, Syk, PI3 kinase, and ERK, may be involved in regulation of neutrophil apoptosis by the complexes. These studies demonstrate for the first time that anti-IL-8:IL-8 immune complexes have the ability to prolong neutrophil life.     

Functional expression of IL-9 receptor by human neutrophils from asthmatic donors: role in IL-8 release

Human polymorphonuclear neutrophils (PMNs) express surface receptors for various inflammatory mediators, including IgE and IL-4. Recently, the IL-9R locus has been genetically linked to asthma and bronchial hyperresponsiveness in humans. In this study, we evaluated expression of the IL-9R and the effect of IL-9 on human PMNs. RT-PCR analysis showed the presence of IL-9Ralpha-chain mRNA in PMN RNA preparations from asthmatic patients. Using FACS analysis, surface expression of IL-9Ralpha was detected on PMNs freshly isolated from asthmatics, and to a lesser extent on normal controls. In addition, protein expression of IL-9Ralpha was also detected in peripheral blood and bronchoalveolar lavage PMNs. Furthermore, functional studies showed that IL-9 stimulation of PMNs results in the release of IL-8 in a concentration-dependent manner. The anti-IL-9 neutralizing Ab suppressed this effect, but had no effect on GM-CSF-induced IL-8 release from PMNs. Taken together, these findings suggest a novel role for PMNs in allergic disease through the expression and activation of the IL-9R.     

Synthesis of platelet-activating factor by polymorphonuclear neutrophils stimulated with interleukin-8.

Human interleukin-8 (IL-8) was evaluated for its capability to induce the synthesis and release of platelet-activating factor (PAF) from human polymorphonuclear neutrophils (PMN). IL-8 promotes in a dose-dependent fashion (1-100 ng/ml) a rapid synthesis of PAF, which is only partially released. The synthesis of PAF is preceded by the activation of acetyl-CoA: 1-alkyl-2-lyso-sn-glycero-3-phosphocholine acetyl-transferase, suggesting that IL-8 activates the "remodeling pathway" of PAF synthesis. By thin layer chromatography and reverse-phase high pressure liquid chromatography, we demonstrated that PAF synthesized by human PMN stimulated with IL-8 is heterogeneous: the 2-acetylated phospholipids having the biological and physicochemical characteristics of PAF include the 1-O-alkyl form, which is produced in large extent (51%), and the 1-acyl form (20%). The analysis of the individual molecular species of radyl chain indicated nine peaks, 16:0 and 18:0 being the predominant forms. These results identify PAF as a direct product of IL-8 stimulation in PMN.     

Analysing lymphokine-receptor interactions of IL-1 and IL-2 by recombinant-DNA technology.

The development of lymphokines as pharmaceutical agents is at the forefront of current biotechnological research. Lymphokines exert their regulatory action on cellular function through interaction with cell-surface receptors. An understanding of the biochemical nature of these molecular interactions should facilitate the design of small peptide analog pharmaceuticals which mimic lymphokines or their receptors.     

Ligand binding specificity of the leukocyte response integrin expressed by human neutrophils.

The ligand binding specificity of the leukocyte response integrin (LRI) expressed by polymorphonuclear leukocyte (PMN) was investigated by examining its interaction with two adhesion motifs within fibrinogen: the alpha chain sequence RGD and the gamma chain sequence KQAGDV. The effect of the hexapeptides KQAGDV, KQRGDV, and KGAGDV on fibrinogen-stimulated phagocytosis, a LRI-dependent function, was examined. Surprisingly, the sequence KGAGDV was most potent for inhibition of fibrinogen-stimulated ingestion; the order of potency of these peptides was KGAGDV greater than KQAGDV greater KQRGDV = GRGDSPA. Latex spheres coated with multivalent KGAGDV bound specifically to PMN and antibodies that recognized either the LRI beta chain (7G2) or an associated protein (IAP)-abrogated bead binding. Various control and anti-beta 1 and anti-beta 2 antibodies did not affect bead binding. Monovalent peptides KGAGDV and KQRGDV were equipotent for inhibition of bead binding to unstimulated PMN (ID50 = 19 microM). In contrast, KGAGDV was more potent than KQRGDV for inhibition of bead binding to N-formylmethionylleucylphenylalanine-stimulated PMN (ID50 = 2.5 microM versus ID50 = 60 microM). A control peptide, KGALEVA, did not inhibit LRI ligand binding or function. These data suggest that the unique amino acid sequence KGAGDV may represent a specific ligand for LRI and that LRI ligand binding specificity may be regulated by the activation state of the cell.     

晚期糖基化终产物通过其受体促进内皮细胞分泌IL-8的研究

探讨晚期糖基化终产物(AGE)修饰蛋白对内皮细胞生成白介素8(IL-8)的作用,及晚期糖基化终产物受体(RAGE)在此病理过程中的作用.内皮细胞来自培养的人脐静脉内皮细胞(HUVEC).将内皮细胞与不同浓度的AGE修饰人血清白蛋白(AGE-HSA)在体外共同培养,或以可溶性晚期糖基化终产物受体(sRAGE)对AGE-HSA进行预处理后再与HUVEC共同培养.用蛋白质液相芯片法检测HUVEC培养上清中IL-8水平,并提取细胞RNA,进行RT-PCR反应,检测细胞中IL-8 mRNA的表达水平.结果表明,AGE-HSA以时间和剂量依赖的方式刺激HUVEC生成IL-8,未经修饰的HSA无此作用.AGE-HSA用sRAGE预处理后,刺激HUVEC生成IL-8的作用被抑制,并且此抑制作用呈剂量依赖的方式.AGE-HSA刺激HUVEC使IL-8 mRNA表达增高,未经修饰的HSA无此作用.sRAGE能够阻断AGE-HSA诱导HUVEC表达IL-8mRNA的作用.整个变化趋势与蛋白质水平一致.研究首次证实,AGE-HSA与细胞表面受体RAGE相互作用可刺激内皮细胞分泌IL-8,并上调IL-8 mRNA的表达.这为研究加速型血管病变的发病机制提供了新视角,也为治疗由AGE增多和潴留所引起的病理损害提供了新靶点.     

Differential effects of neutrophil-activating peptide 1/IL-8 and its homologues on leukocyte adhesion and phagocytosis.

Several structural homologues of the chemotactic peptide neutrophil-activating peptide 1/IL-8 (NAP-1/IL-8) were tested for their ability to influence the expression and function of adhesion-promoting receptors on human polymorphonuclear leukocytes (PMN). NAP-2, melanoma growth stimulatory activity, and two forms of NAP-1/IL-8 (ser-NAP-1/IL-8 and ala-NAP-1/IL-8, consisting of 72 and 77 amino acids, respectively), each caused an increase in the expression of CD11b/CD18 (CR3) and CR1, which was accompanied by a decrease in the expression of leukocyte adhesion molecule-1 (LAM-1, LECAM-1). The binding activity of CD11b/CD18 was also enhanced 3- to 10-fold by these peptides, but enhanced function was transient: binding of erythrocytes coated with C3bi reached a maximum by 30 min and declined thereafter. Ser-NAP-1/IL-8, ala-NAP-1/IL-8, NAP-2, and melanoma growth stimulatory activity also caused a two- to threefold enhancement of the phagocytosis of IgG-coated erythrocytes (EIgG) by PMN without causing a large increase in the expression of Fc gamma receptors. Enhanced phagocytosis of EIgG appeared to be mediated through CD11b/CD18, because F(ab')2 fragments of an antibody directed against CD18 inhibited NAP-1/IL-8-stimulated ingestion of EIgG. The four active peptides caused a rapid, transient increase in the amount of F-actin within PMN, indicating that they are capable of influencing the structure of the microfilamentous cytoskeleton, which participates in phagocytosis. Two other NAP-1/IL-8-related peptides, platelet factor 4 and connective tissue-activating peptide III, were without effect on expression of CD11b/CD18, CR1, and LAM-1, binding activity of CD11b/CD18, or Fc-mediated phagocytosis, and increased actin polymerization only slightly. Our observations indicate that several members of the NAP-1/IL-8 family of peptides were capable of promoting integrin-mediated adhesion and Fc-mediated phagocytosis, processes important in the recruitment of PMN to sites of inflammation and antimicrobial responses of PMN.     

In vitro inhibitory effect of IL-8 and other chemoattractants on neutrophil-endothelial adhesive interactions.

We have previously reported that cytokine- or LPS-activated human umbilical vein endothelial cell (HUVEC) monolayers secrete IL-8 that can act as a neutrophil-selective adhesion inhibitor. In our study we investigated the mechanisms involved in the leukocyte adhesion inhibitory action of IL-8. The leukocyte adhesion inhibitory effect appears to be mediated by the action of IL-8 on the neutrophil, does not involve down-regulation of relevant endothelial adhesion molecules such as endothelial-leukocyte adhesion molecule-1 or intercellular adhesion molecule-1, and is quantitatively similar in different endothelial activation states that are predominantly endothelial-leukocyte adhesion molecule-1 dependent or intercellular adhesion molecule-1 dependent. In addition to inhibiting the attachment of freshly isolated peripheral blood neutrophils to cytokine-activated HUVEC monolayers, IL-8 also promoted a rapid detachment of tightly adherent neutrophils from activated HUVEC, and abolished neutrophil transendothelial migration. Certain other chemoattractants, including FMLP and C5a, had similar inhibitory actions, indicating IL-8 was not unique in its ability to inhibit various neutrophil-endothelial interactions. In contrast, two other neutrophil agonists 1-0-alkyl-2-acetyl sn-glycero-3-phosphocholine and granulocyte-macrophage-CSF, which, like IL-8, are produced by activated HUVEC, as well as the leukocyte-derived chemoattractant leukotriene B4, exerted minimal inhibitory effects on adhesion. Regardless of their ability to modulate neutrophil-endothelial cell adhesion, all these agents induced altered leukocyte surface expression of functionally important adhesion molecules, including loss of L-selectin (leukocyte adhesion molecule-1, LECAM-1) and increase in CD11b/CD18. Thus, although the above agonists have been characterized primarily as chemoattractants, our findings demonstrate that these agents can exert a wide range of modulatory effects on neutrophil-endothelial adhesive interactions.     

Induction of lactoferrin and IL-8 release from human neutrophils by tryptic enzymes via proteinase activated receptor-2

Tryptic enzymes such as tryptase, trypsin and thrombin are reportedly able to alter neutrophil behavior. However, little is known of the influence of these proteinases on lactoferrin or IL-8 release from neutrophils. In the present study, we investigated the effects of tryptase, trypsin, thrombin and elastase, and agonist peptides of PAR-1 SFLLR-NH(2) and PAR-2 SLIGKV-NH(2) and tc-LIGRLO-NH(2) on lactoferrin and IL-8 release from highly purified human neutrophils. Flow cytometry shows CD16(+) neutrophils express PAR-1 and PAR-2, but not PAR-3 and PAR-4 proteins. RT-PCR analysis reveals that neutrophils express only PAR-2 genes. Tryptase and trypsin, but not thrombin and elastase, induced significant lactoferrin and IL-8 secretion from neutrophils. SLIGKV-NH(2) and tc-LIGRLO-NH(2), but not SFLLR-NH(2), also stimulated lactoferrin and IL-8 secretion from neutrophils. In conclusion, only a proportion of neutrophils express PAR-1 and/or PAR-2. Tryptase and trypsin-induced lactoferrin and IL-8 secretion from neutrophils most likely occur through activation of PAR-2.     

Preparation and partial characterization of two forms of bovine thrombin.

A chromatographic procedure has been developed for the separation and purification of bovine α- and β-thrombin. α-Thrombin has a specific activity of 2400–3000 NIH U/mg while β-thrombin has only approximately 100 NIH U/mg. When assayed with an ester substrate, the two forms have equivalent activity while β-thrombin has only 30% of the activity of α-thrombin toward an anilide substrate. Previous studies have suggested that the degradation of α-thrombin produces a species in which a peptide fragment containing a disulfide bridge is lost. The amino acid composition determined in the present study indicates that the content of cysteine is identical in the two forms of the enzyme thus permitting the proposal of a structure for β-thrombin which differs from that currently in the literature. It is suggested that changes in the environment of the active site histidine residue in the two species is largely responsible for the observed changes in the catalytic activity.     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.