The Effects of Bruton Tyrosine Kinase Inhibition on Chemotaxis and Superoxide Generation in Human Neutrophils

Purpose

The role of the Bruton tyrosine kinase (Btk) protein in neutrophil function has been evaluated using neutrophils from healthy volunteers after incubation with a Btk inhibitor, leflunomide metabolite analog (LFM-A13), suggesting an important role for Btk in neutrophil function. We sought to determine the role of Btk protein on neutrophil superoxide generation and chemotaxis stimulated by N-formyl-methionine-leucine-phenylalanine (fMLP).

Methods

Chemotaxis was assayed on agarose gel and superoxide generation by cytochrome C reduction. The affects of LFM-A13 on chemotaxis and superoxide generation in unstimulated and fMLP stimulated neutrophils were studied in Btk deficient neutrophils from XLA patients compared with matched controls analyzed simultaneously.

Results

Chemotaxis and stimulated superoxide production were similar in the normal and Btk deficient neutrophils and were similarly inhibited by LFM-A13. In one patient, LFMA13 had no effect on superoxide generation in Btk deficient neutrophils up to a concentration of 25 microM, while inhibited superoxide production by control neutrophils.

Conclusions

Our results suggest that Btk does not have a specific role in neutrophil fMLP-stimulated superoxide generation and chemotaxis since these activities were similarly inhibited by LFM-A13 in Btk deficient and normal neutrophils. The lack of superoxide generation following Btk inhibition by LFM-A13 in Btk deficient neutrophils from one patient may suggest some heterogeneity in the role of Btk in fMLP induced neutrophil superoxide generation.     

Interaction Between Interleukin 10 and Interleukin 6 in Human B-Cell Differentiation

Contrary to their opposing action on human T-lymphocytes and monocytes, both Interleukin (IL-)10 and IL-6 are potent differentiation factors of human B-cells. Both are known to induce immunoglobulin (Ig) production. The precise mechanism of this converging effect of IL-6 and IL-10 remains elusive, however. Here we investigated the role of IL-6 in the IL-10 dependent B-cell differentiation into Ig secreting cells. We found that co-stimulation of SAC-stimulated human peripheral B-lymphocytes with IL-10 and IL-6 exhibited no additive effect on Ig production over stimulation with IL-10 alone, and that IL-6 receptor blockade only mildly inhibited IL-10 induced Ig synthesis. In fact, we could show that stimulation of B-cells with IL-10 somewhat suppressed SAC induced autocrine IL-6 production. Despite this suppression IL-6 levels remained sufficiently high to stimulate its receptor, and IL-6 binding to the B-cell surface was not affected. The failure of IL-6 to exert an additional effect on SAC+IL-10 induced Ig production suggests that IL-10 may recruit components of the IL-6 intracellular pathway for Ig induction. In conclusion we could demonstrate that IL-10 acts on B-cell differentiation independently of autocrine IL-6 and that it had a considerably mild effect on B lymphocytic autocrine IL-6 secretion. This still allows an IL-6 effect in the presence of IL-10 which appears adaptive with a view to the converging effects of these two cytokines on human B lymphocytes. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Interaction Between Interleukin 10 and Interleukin 6 in Human B-Cell Differentiation

Contrary to their opposing action on human T-lymphocytes and monocytes, both Interleukin (IL-)10 and IL-6 are potent differentiation factors of human B-cells. Both are known to induce immunoglobulin (Ig) production. The precise mechanism of this converging effect of IL-6 and IL-10 remains elusive, however. Here we investigated the role of IL-6 in the IL-10 dependent B-cell differentiation into Ig secreting cells. We found that co-stimulation of SAC-stimulated human peripheral B-lymphocytes with IL-10 and IL-6 exhibited no additive effect on Ig production over stimulation with IL-10 alone, and that IL-6 receptor blockade only mildly inhibited IL-10 induced Ig synthesis. In fact, we could show that stimulation of B-cells with IL-10 somewhat suppressed SAC induced autocrine IL-6 production. Despite this suppression IL-6 levels remained sufficiently high to stimulate its receptor, and IL-6 binding to the B-cell surface was not affected. The failure of IL-6 to exert an additional effect on SAC+IL-10 induced Ig production suggests that IL-10 may recruit components of the IL-6 intracellular pathway for Ig induction. In conclusion we could demonstrate that IL-10 acts on B-cell differentiation independently of autocrine IL-6 and that it had a considerably mild effect on B lymphocytic autocrine IL-6 secretion. This still allows an IL-6 effect in the presence of IL-10 which appears adaptive with a view to the converging effects of these two cytokines on human B lymphocytes. Our study thus adds to the appreciation of the complex cytokine regulation of the immune system.     

Tissue Plasminogen Activator (tPA) Inhibits Human Neutrophil Superoxide Anion Production in Vitro

Because neutrophils contribute to reperfusion injury associated with acute myocardial infarction (MI), and because tissue plasminogen activator (tPA) is often used in the management of MI, we evaluated the effect of tPA on superoxide ( ) production by human neutrophils in vitro. We found that adding increasing amounts of tPA significantly (r = 0.89, P < 0.025) and progressively reduced generation by neutrophils treated with phorbol myristate acetate (PMA) in vitro. Furthermore, adding tPA that had been previously treated with the protease inhibitor, D-Phe-Pro-Arg-chloromethyl ketone HCl (PPACK), also decreased neutrophil generation in vitro (P < 0.05). In contrast, adding L-arginine, a component of the tPA preparation and a precursor of nitric oxide (NO), did not inhibit PMA-induced neutrophil production. Also, adding increasing concentrations of tPA did not reduce (P > 0.05) the concentrations of produced by xanthine oxidase (XO) in vitro. Our findings suggest that tPA reduces neutrophil generation by a mechanism that is not related to L-arginine, is not dependent on tPA proteolytic activity, and is not a function of direct scavenging. This property may account for some of the effectiveness of tPA in the treatment of MI and/or make tPA valuable for treating acute respiratory distress syndrome (ARDS) or other inflammatory disorders involving neutrophil production.     

Interaction of 2-(9H-purin-6-ylamino)-4-(methylthio) Butanoic Acid with Human Serum Albumin: Fluorescence and Modeling Studies

This study was designed to investigate the interaction between (S)-2-(9H-purin-6-ylamino)-4-(methylthio) butanoic acid (PYMBA) and human serum albumin (HSA) using fluorescence spectroscopy. The combination of UV absorption and molecular docking under simulative physiological conditions was also applied to comprehensively understand the binding mechanism of PYMBA to HSA. Fluorescence data indicated that PYMBA has a strong ability to quench the intrinsic fluorescence of HSA. The binding constants (K) at different temperatures, thermodynamic parameters including enthalpy change (ΔH) and entropy change (ΔS) of PYMBA–HSA were correlated to the relevant fluorescence data, which suggested that the hydrophobic force played a very important role for the PYMBA binding to the HSA. The experimental results were in agreement with the results obtained via a molecular docking study. The effects of other ions on the binding constants were also studied.     

纳洛酮对缺血/再灌注损伤家兔心肌前列腺素代谢产物及再灌注血流的影响

目的 观察纳洛酮对缺氧心肌组织再灌注血流及前列腺素代谢产物生成的影响。方法 30只新西兰大白兔随机分为：再灌注组10只，结扎冠状动脉左前降支1h再开放4．5h；纳洛酮组10只，手术过程同前，同时静注纳洛酮；假手术组10只，手术过程同前，不结扎前降支动脉。用激光多谱勒血流测定仪测定再灌注组及纳洛酮组结扎前、后及稃灌注4．5h后缺血区心肌再灌注血流量：放射免疫法测定各组再灌注4．5h后的血浆6-酮-前列腺素F1α血栓素B，含量。结果 （1）结扎前，再灌注组及纳洛酮组局部心肌组织血流无差别；再灌注后，两组血流均较前下降，再灌注组的缺血区血流量明显低于纳洛酮组（P〈0．01）。（2）再灌注后，再灌注组、纳洛酮组血浆血栓素B1含量及血栓素B2／6-酮-前列腺素F1α比值显著高于假手术组，再灌注组又高于纳洛酮组，各组间血浆6．酮．前列腺素F1α含量无显著性差异。结论 纳洛酮可以抑制缺血／再灌注后家兔心肌血栓素B2的生成。改善再灌注部位血流。     

The Effect of Monoclonal Antibodies against Escherichia coli Type 1 Pili and Capsular Polysaccharides on the Interaction between Bacteria and Human Granulocytes

Monoclonal antibodies against Escherichia coli type 1 pili and the K13 capsular polysaccharide strongly influenced the interaction between human polymorphonuclear leucocytes (PMNL) and E. coli 06:K13:H1. Bacteria with type 1 pili, associated with the neutrophils, caused a metabolic activation but were not ingested. Addition of monoclonal antibodies to type 1 pili resulted in increased association, but also phagocytosis and metabolic activation of the granulocytes. Monoclonal antibodies against the K13 polysaccharide strongly stimulated phagocytosis, especially of the type 1 piliated bacteria, suggesting a synergistic effect between binding of type 1 pili and the Fc part of the capsular antibodies to the PMNL. Addition of D-mannose inhibited the opsonization of type 1 piliated E. coli 06:K13:H1 in the presence of both type 1 pilus antibodies and capsular antibodies. Monoclonal anti-idiotypic (anti-anti-capsular) antibodies reduced the association with the PMNL of the bacteria preopsonized with anti-capsular antibodies. The bacterial ingestion and the metabolic activation of the PMNL were also reduced, suggesting a role for anti-idiotypic antibodies in specific modulation of inflammation.     

人类ZAKβ与YWHAZ蛋白相互作用的初步研究

目的研究ZAKβ与YWHAZ蛋白之间的相互作用。方法以ZAKβ为诱饵蛋白,利用酵母双杂交筛选人类肝脏cDNA文库,经GST Pull-down、免疫共沉淀和细胞共定位实验分别验证ZAKβ与猎物蛋白在体外和细胞内的相互作用,构建ZAKβ的截短体以确定相互作用的区域,用磷酸化抗体检测ZAKβ在相互作用中的磷酸化作用,并通过流式细胞仪检测这对蛋白相互作用对细胞周期的影响。结果利用酵母双杂交筛选到一个能与ZAKβ相互作用的蛋白YWHAZ,并在体外和细胞内都验证了这二者的相互作用,发现这两个蛋白都能共定位于293T细胞的胞质中,确定了ZAKβ的N端1~250氨基酸为与YWHAZ蛋白相互作用的区域。体外实验发现了这二者的相互作用涉及到磷酸化反应,发现这对蛋白的相互作用能提高293T细胞的G2期水平。结论首次发现并证实了ZAKβ和YWHAZ之间的相互作用,发现该相互作用涉及到磷酸化过程并能够对细胞周期产生影响。     

Superoxide anion (O2-) production and bactericidal capacity of polymorphonuclear neutrophils obtained from patients subjected to glucagon test

Superoxide anion (O₂^-) production and bactericidal capacity of morphologically mature bone marrow polymorphonuclear neutrophils (PMN) were evaluated in 30 haematologically normal individuals. These same parameters of peripheral PMNs were estimated in 15 healthy volunteers before and after glucagon-induced marrow granulocyte reserve mobilization. Bone marrow PMN in comparison with cells obtained from peripheral blood manifested impaired superoxide anion production and diminished bactericidal capacity. The admixture of bone marrow PMN released into the circulation by the use of glucagon administration significantly lowered both estimated PMN functions.     

The Interaction Between the Indirect Anticoagulant Coumatetralyl and Calciferol (Vitamin D3) in Warfarin-resistant Rats (Rattus norvegicus)

Resistance to 4-hydroxycoumarin anticoagulant rodenticides has been a problem in some local foci for a number of years. One method of managing resistance could be to use synergists in conjunction with anticoagulants. We investigated the effect of administering cholecalciferol and coumatetralyl alone and in a synergistic mixture to anticoagulant-resistant rats by monitoring plasma factor X concentrations. The study showed that the efficacy of the compounds was significantly improved when they were used together. This synergistic effect led to an increased mortality in female rats that had been treated with both compounds compared to both a control group and groups of rats that had received the compounds singly. An unexpected result from this work was that cholecalciferol when given on its own to rats caused a decrease in plasma factor X concentration. We hypothesise that this effect was due to a vitamin D-induced increase in production of vitamin K-dependent proteins leading to a saturation of the carboxylation process and hence to a significant number of factor X molecules being under-carboxylated and therefore dysfunctional. It is suggested that the reduction in factor X levels is a major component of the increased efficacy of the anticoagulant/calciferol mixture and also that effects on other vitamin K-dependent proteins, e.g. matrix GLA protein, may play a role in the increased mortality seen in female rats.  This work illustrates that synergists could be utilised in managing anticoagulant resistance. There are also possible implications for human patients on anticoagulant therapy who may be receiving increased amounts of vitamin D analogues through, for example, dietary supplements.     

Granulocytes (the red,white, and blue) in hypersensitivity reactions: A review

This review is an attempt to summarize studies which demonstrate the participation of all three kinds of granulocytes in reactions of both immediate and delayed hypersensitivity. The focus is on the biologic control of these cell types by various mediator substances, in particular the lymphokines and the complement-derived factors. These factors collectively mobilize, attract, activate, immobilize, and otherwise affect granulocyte function in vitro and in vivo. These mechanisms which lead to control of the distribution and properties of the granulocytes are of prime importance in the production of an inflammatory reaction following an underlying immunologic event. This interface between inflammation and immunity underlies all of clinical and experimental hypersensitivity.Supported by NIH grants AI-12225 and AI-09529.     

Superoxide anion (O2-) production by neutrophils in refractory anemia with excess of blasts

The O2- production by neutrophils was examined in 4 cases of refractory anemia with excess of blasts (RAEB) in order to evaluate the possible causes of enhanced susceptibility to infection and to gain some informations on the differentiation of neutrophils in this hematological disorder. In three of the four RAEB cases there was little O2- production by neutrophils, in addition to there being morphological anomalies of the neutrophils such as a Pelger-Hüet-like anomaly, granular deficiency and binucleated cells. These results suggest that the impairment of O2- production by neutrophils in RAEB is one of the possible causes of susceptibility to infection and also suggest that the differentiation of neutrophils in this hematological disorder is faulty. The estimation of O2- production by neutrophils may be a useful diagnostic method for preleukemia.     

Generation and function of bone marrow-derived dendritic cells from CD4/CD8(-/-) double-knockout mice.

We present a novel, simple and straightforward method to obtain mouse bone marrow-derived dendritic cells (DC) from C57Bl/6 CD4/CD8(-/-) double knock-out mice. This new method, involving culture of bone marrow cells in medium supplemented with Interleukin 4 and Granulocyte-Macrophage Colony-Stimulating Factor, does not involve negative immunodepletion of CD4+ and CD8+ populations, or extensive prior manipulations of the starting population. The resulting, loosely adherent cell population, exhibited the morphological characteristics and typical surface markers of DCs, and were endowed with the functional activities characteristic of bone marrow-derived DCs of wild-type mice. Interestingly, LCMV GP33-41 peptide-loaded CD4/CD8(-/-) DCs were efficiently lysed by peptide-specific activated CTLs in vitro. Furthermore, these peptide-loaded CD4/CD8(-/-) DCs induced a peptide-specific CTL response upon immunization of wild-type C57Bl/6 mice.     

Superoxide dismutase and pulmonary oxygen toxicity: lessons from transgenic and knockout mice (Review)

Superoxide (O2-) has been implicated in the pathogenesis of pulmonary O2 toxicity. The studies using transgenic and knockout mice of each of the three isoforms of superoxide dismutase (SOD) e.g. , CuZnSOD, MnSOD and extracellular SOD (EC-SOD), have demonstrated that O2- produced in the mitochondria from its electron transport system and extracellular O2- generated by infiltrating neutrophils, and possibly its derivatives e.g., hydroxyl radical and peroxynitrite, are important mediators of hyperoxia-induced pulmonary injury, while cytoplasmic O2- plays a limited, if any, role in the pathogenesis of pulmonary O2 toxicity. Distal airway epithelial cells including type II alveolar and non-ciliated bronchiolar epithelial cells, are important targets for O2 radicals under the hyperoxic condition. The accessibility of these distal airway epithelial cells to in vivo gene transfer through the tracheal route of administration, suggests the potential for in vivo transfer of MnSOD and EC-SOD genes as a future approach in the prevention of pulmonary O2 toxicity.     

Host Defense Functions of Proteolytically Processed and Parent (Unprocessed) Cathelicidins of Rabbit Granulocytes

Members of the cathelicidin family are present in all mammals studied. Generally, these proteins contain a conserved N-terminal domain and a structurally and functionally divergent C-terminal region that expresses antibacterial or other activities when proteolytically released. Rabbit granulocytes produce CAP18, a cathelicidin that conforms to this structural and functional organization, and also 15-kDa protein isoforms (p15s) that share several key structural features with other cathelicidins but apparently do not undergo processing with release of an active peptide. To further define the importance of proteolysis in the antibacterial activities of these proteins, we have purified from granulocytes proCAP18, its C-terminal peptide (CAP18p), and two p15 isoforms to apparent homogeneity. Of these four polypeptides, only CAP18p was independently cytotoxic to encapsulated Escherichia coli (90% inhibitory concentration, approximately 600 nM) but it was approximately 50-fold less potent on a molar basis than the bactericidal/permeability-increasing protein (BPI). However, all four cathelicidin species, notably including proCAP18, exhibited antibacterial synergy with BPI, and the p15s also displayed synergy with CAP18p in the absence of BPI. Subnanomolar concentrations of proCAP18 blocked lipopolysaccharide-induced chemiluminescence of human leukocytes, showing a molar potency more than 100-fold greater than that of CAP18p ( approximately 20 nM) or BPI ( approximately 50 nM). Thus, while independent bactericidal activity of cathelicidins requires processing, other host-defense functions do not and are more potently expressed by the unprocessed protein than by the C-terminal peptide.     

Expression of CD15 (Lewisx) Antigen on Human Sperm and its Role in Sperm-Egg Interaction

PROBLEM: The carbohydrate epitope 3-fucosyl-N-acetyllactosamine (CD15) is a constituent of cell surface glycoconjugates that has been implicated in cell-cell adhesion mediated by carbohydrate-specific ligands. The present study was designed to investigate whether CD15 is present on human sperm and whether it plays a role in human sperm-egg interaction. METHODS: Fluorescent flow cytometry was used to quantitate the binding of monoclonal antibodies (mAb) to sperm-bound CD15 and CD46 antigens on acrosome-intact (AI) and acrosome-reacted (AR) sperm. The location of the binding site of these mAbs was assessed by fluorescence microscopy. The effects of anti-CD15 and anti-CD46 mAbs on gamete interaction were tested utilizing both homologous (human zona binding and penetration) and heterologous (zona-free hamster egg binding and penetration) systems. RESULTS: The mean percentage of capacitated sperm which bound anti-CD15 or anti-CD46 mAbs was low (4.8% and 5.1%, respectively). Exposure to calcium ionophore A23187 (CaI) resulted in an increase in anti-CD15 (38.6 ± 4%) and anti-CD46 (83.4 ± 2%) binding to sperm. Both anti-CD 15 and anti-CD46 binding sites were localized by fluorescence microscopy on the sperm acrosomal region. In four experiments, the percent of zona-free hamster eggs penetrated by human sperm were medium control 93% (62/66), irrelevant mAb 74% (70/94), anti-CD46 0% (0/107), and anti-CD15 10% (9/90). One hundred percent (6/6) of human zona were penetrated by human sperm exposed to medium control, 88% (8/9) following exposure to irrelevant mAb, 0% (0/11) following exposure to anti-CD46, and 50% (5/10) following exposure to anti-CD 15. The mean (± SD of tightly bound sperm to hamster eggs were medium control 57 ± 18%, irrelevant mAb 64 ± 16%, anti-CD46 37 ± 13%, and anti-CD15 19 ± 10%. The corresponding values for human zona were: medium control 118 ± 14%, irrelevant mAb 61 ± 11%, anti-CD46 39 ± 18%, and anti-CD15 99 ± 19%. CONCLUSION: CD15 antigen is expressed on human sperm that have undergone acrosomal loss. mAb to CD15 was shown to inhibit significantly sperm binding and penetration of zona-free hamster eggs and penetration of human zona pellucida. These findings suggested that sperm-egg interaction may be mediated in part by the CD15 antigen. Capsule: Acrosome-reacted human sperm bind monoclonal antibodies specific for CD15 (Lewis^x) epitope. The binding sites were located on the sperm head. Anti-CD15 antibody impaired both the binding and penetration of zona-free hamster eggs and the penetration of human zona by human sperm.     

Chemotherapeutic Interaction Between Khaya Grandifoliola (WELW) CDC Stem Bark Extract and Two Anti-Malarial Drugs in Mice

In malarial endemic countries especially in the tropics, conventional antimalarial drugs are used with herbal remedies either concurrently or successively. Khaya grandifoliola is one of such popular herbs used in the treatment of malaria.Various doses of ethanol extract of K. grandifoliola stem bark (50–400 mg/kg/day) were administered orally to Swiss albino mice infected with Plasmodium yoelii nigerense. A dose of 100 mg/kg/day of the extract was also combined with 2.5 mg/kg/day of chloroquine or 6.25 mg/kg/day of halofantrine in both early and established malaria infection test models. The results showed that in the early malaria infection test, K. grandifoliola in combination with chloroquine or halofantrine elicited enhanced antiplasmodial effect in the established infection, there was significantly greater parasite clearance following administration of the combination when compared to the effects of K. grandifoliola or the conventional drugs alone. The mean survival period of parasitized animals was also enhanced by the extract/halofantrine combination. Lower therapeutic doses of halofantrine may be required to potentiate parasite clearance when used in combination with K. grandifoliola. This may constitute great advantage to halofantrine which is associated with cardiotoxicity at high doses.Key words: Khaya grandifoliola, Antimalarial, Chemotherapeutic interaction, Chloroquine, Halofantrine     

The IgG FcRII and the PI-linked IgG FcRIII Trigger Cytoplasmic Calcium Fluxes Independently in Human Granulocytes

The aim of this study was to investigate signal transduction through the two Fc receptors for IgG on human granulocytes. Using flow cytometry and the calcium indicator Fluo-3, we measured changes in leucocyte cytoplasmic calcium concentrations following cross-linking of cellular Fc receptors with specific antibodies. Two different approaches were used in order to study the two Fc receptors independently of each other. One was to avoid the presence of IgG Fc fragments, capable of binding to both types of receptors. The other was to use leucocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) deficient in granulocyte FcRIII. In contrast to earlier reports, both approaches showed that the two types of IgG Fc receptors on granulocytes are capable of increasing cytoplasmic free calcium concentrations independently of each other. The results suggest that free cytoplasmic calcium ions are involved in the signal transduction pathway of both types of IgG Fc receptors on human granulocytes.