Human Peripheral Blood Polymorphonuclear Leukocytes Research Articles

Naringenin suppresses K562 human leukemia cell proliferation and ameliorates Adriamycin-induced oxidative damage in polymorphonuclear leukocytes.

Treatments for leukemia remain unsatisfactory. Conventional chemotherapy agents that aim to kill tumor cells may also damage normal cells and thus result in severe side-effects. Naringenin, a natural polyphenolic compound with antioxidant effects, has been revealed to have significant antitumor effects with low toxicity in preliminary studies. Thus, it is considered as one of the most promising flavonoids in the treatment of leukemia. In the present study, the effects of naringenin on the K562 human leukemia cell line and the underlying mechanisms were explored in vitro. In addition, human peripheral blood polymorphonuclear leukocytes (PMNs) were used as a normal control in order to evaluate the effects of naringenin on normal granulocytes and in the mediation of Adriamycin (ADM)-induced oxidative damage. The results revealed that K562 proliferation was significantly inhibited by naringenin in a time- and concentration-dependent manner; however, minimal cytotoxic effects were observed in PMNs when naringenin was used at concentrations <400 μmol/l. Morphological changes indicative of apoptosis were observed in naringenin-treated K562 cells. Flow cytometric analysis indicated that the K562 cells were arrested in the G0/G1 phase of the cell cycle with a significantly upregulated rate of apoptosis. Furthermore, in the naringenin-treated K562 cells, the labeling index of proliferating cell nuclear antigen was observed to be increased by immunochemical staining, the mRNA and protein expression levels of p21/WAF1 were strongly upregulated in reverse transcription-polymerase chain reaction and western blot analyses, whereas p53 gene expression was not significantly changed. In PMNs to which naringenin (50~80 μmol/l) was added 1 h subsequent to ADM, the cell damage induced by ADM was significantly reduced, coincident with reductions in the levels of reactive oxygen species (ROS) and malondialdehyde (MDA) and increases in the activity of superoxide dismutase and glutathione peroxidase. However, the cytotoxic effect of ADM in K562 cells was not significantly altered by naringenin, and the oxidative stress indices in K562 cells remained stable. In conclusion, the present study revealed the promising value of naringenin in leukemia treatment. Naringenin demonstrated a significant inhibitory effect on the growth of K562 cells but not on normal PMNs. Furthermore, naringenin protected PMNs from ADM-induced oxidative damage at low concentrations. Cell cycle arrest and apoptosis-inducing effects, achieved through p53-independent p21/WAF1 upregulation, are likely to be the mechanism of the antileukemic effects of naringenin, and the protective effect against ADM chemotherapy-induced damage in PMNs may be due to the antioxidant capability of this agent at low concentrations.

Antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis

Human peripheral blood polymorphonuclear leucocytes (PMN) were examined in vitro for antifungal activity against yeast cells of Paracoccidioides brasiliensis. The yeast cell of this fungus was resistant to killing by PMN. However, PMN exhibited a fungistatic effect on the fungal isolates employed in a long-term (∼72 h) assay. Lysates of PMN did not show a fungistatic or fungicidal effect, indicating that live PMN are necessary for the antifungal effect. Interferon-Γ (IFN-Γ) enhanced the antifungal activity of PMN. IFN-Γ-treated PMN killed isolate Bt-4 within 2 h of coculture, and after 24 h a still greater killing effect was observed. By contrast, IFN-Γ-treated PMN did not show a significant killing effect on isolate Tatu, but did exhibit an enhanced fungistatic effect on this isolate. In contrast, tumor necrosis factor-α and interleukin 8 had no effect on the antifungal activity of PMN. Scanning electron microscopy revealed that the surface structure of the fungal cell was apparently damaged by PMN within 24 h of cocultures. Based on these novel findings, we speculate that human PMN might play a role in host resistance in early infection with this fungus due to their antifungal activity.

Immunomodulatory properties of Achyrocline satureioides (Lam.) D.C. infusion: A study on human leukocytes

Aim of the study Achyrocline satureioides (Lam.) D.C. is a South American native medicinal herb known by the popular name of “Marcela”. Its infusion is widely utilized for the treatment of several digestive ailments, as an anti-inflammatory preparation, as a sedative and anti-atherosclerotic. Circumstantial evidence suggests that extracts of Achyrocline satureioides may have immunomodulatory properties. The present study was therefore devised to investigate the in vitro effects Achyrocline satureioides infusion on human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs). Materials and methods Experiments were performed on cells isolated from venous blood obtained from healthy donors. PBMC proliferation and cytokine production were assessed by standard ELISA methods. Reactive oxygen species (ROS) production by PMNs was evaluated by spectrofluorimetry. Results In PBMCs, Achyrocline satureioides infusion in the 0.06–0.24 μg/ml quercetin equivalent (QE) concentration range concentration-dependently reduced PHA-induced proliferation and production of interferon (IFN)-γ and interleukin (IL)-4. Lower concentrations of the infusion (0.006–0.03 μg/ml QE), which were ineffective on cell proliferation, significantly increased the production of both IFN-γ and IL-4 and decreased the ratio IFN-γ/IL-4. In PMNs, Achyrocline satureioides infusion slightly increased the spontaneous generation of ROS only at concentrations ≥0.06 μg/ml QE. On the contrary, in the 0.0012–0.03 μg/ml QE concentration range the infusion profoundly inhibited fMLP-induced ROS generation as well as spontaneous and fMLP-induced IL-8 production. Conclusions The present results provide evidence that Achyrocline satureioides infusion may exert several immunomodulatory effects, in line with its traditional use as an anti-inflammatory agent in many disease conditions. Further studies are warranted to better characterize such effects and to assess their therapeutic relevance.

Effect of cyclosporin A on human neutrophil and monocyte function.

The effect of various concentrations of cyclosporin A (CyA), ranging from below peak blood levels to 20 times higher than blood levels of human peripheral blood polymorphonuclear and mononuclear leukocytes, was examined. CyA was found to bind to neutrophils with Kd values in the range of 20-50 nM. CyA at clinically obtainable blood level concentrations had no effect on neutrophil and monocyte chemotaxis, neutrophil oxidative burst, monocyte phagocytosis, or neutrophil bactericidal activity. The data on the release of lactoferrin, a secondary granule substance, from activated neutrophils showed that the calcium ionophore A 23187-induced lactoferrin release was inhibited by treatment of cells with 4 microM CyA, whereas release of lactoferrin from zymosan- or phorbol myristate acetate-activated neutrophils was not affected by the same concentration of CyA. This effect could either be due to differences in the degree of cell membrane perturbation by the various activators or to calcium dependence of neutrophil activation. A third possibility may be that CyA acts at some subsequent steps in the release process of neutrophils. It is concluded that CyA does not interfere with important functions of human phagocytes, the cells that play a major role in the defence against invading microorganisms.

Effects of resveratrol, piceatannol, tri-acetoxystilbene, and genistein on the inflammatory response of human peripheral blood leukocytes

Inflammatory processes are involved in the etiology of diseases. We analyzed the effect of resveratrol, piceatannol, synthetic tri-acetoxystilbene (TAS), and genistein (Bonistein(TM)) on the production of inflammatory mediators including prostaglandin E2 (PGE2), tumor necrosis factor-alpha (TNF-alpha), interleukins, and chemokines, which participate in the progression of inflammation. In order to induce inflammatory responses, human peripheral blood mononuclear and/or polymorphonuclear leukocytes were stimulated with lipopolysaccharide (LPS) and interferon-gamma (IFN(gamma)) and the production of PGE2, interleukin-8 (IL-8), and TNF-alpha was determined. In response to the stimuli, genes were substantially activated within < 2 h (e. g., TNF-alpha, IL-1alpha), or at a later stage, (e. g., COX-2, IL-6, IL-8). Unlike genistein, resveratrol and related compounds dose-dependently reduced PGE2 production. Genistein, piceatannol, and TAS diminished secretion of TNF-alpha, and IL-8. TAS reduced mRNA levels of COX-2, TNF-alpha, IL-8, IL-6, and IL-1alpha, while resveratrol impaired early expression of IL-8 and TNF-alpha. Piceatannol out-performed resveratrol, yet without matching TAS. Genistein downregulated TNF-alpha and IL-8 expression. These substances altered the LPS/IFNgamma-induced gene expression in mononuclear cells rather than in polymorphonuclear leukocytes. Immunoblot analyses corroborated the distinct activity pattern of resveratrol and genistein. In conclusion, resveratrol and their derivatives attenuated the inflammatory response of PBLs at several levels, whereas genistein acts on cytokines and pro-inflammatory interleukins.

Inhibitory effect of furosemide on activation of human peripheral blood polymorphonuclear leukocytes stimulated with n-formyl-methionyl-leucyl-phenylalanine

The clinical efficacy of inhalatory furosemide (Fu) has been extensively studied in bronchial asthma patients but there are only a few studies addressing its action on cells participating in the underlying inflammatory process. Therefore, we investigated the effect of Fu on human peripheral blood polymorphonuclear leukocytes (PMNL) at concentrations that can be achieved in the bronchial lining fluid by inhalation, i.e. 10 −5, 10 −4 and 10 −3 M. The influence of Fu on the following PMNL parameters was investigated: intracellular calcium changes ([Ca 2+] i) as a part of signal transduction and luminol dependent chemiluminescence (LCL) as an indirect measure of NADPH-oxidase activation upon n-formyl-methionyl-leucyl-phenylalanine (fMLP) stimulation; chemotaxis to fMLP, phagocytosis and intracellular killing of Staphylococcus aureus. Incubation with Fu resulted in a concentration dependent reduction of Ca 2+ influx and Fu (10 −3 M) decreased the main Ca 2+ parameters to one half of the control values and to the level obtained in calcium-free buffer. In contrast, Fu had no effect if preincubated with the cells and then removed by washing. The LCL signal was reduced by Fu (10 −3 M) from 2000±870 to 550±440 arbitrary units [aU] ( p<0.05). In contrast to the [Ca 2+] i measurements, a slightly diminished LCL was also observed following preincubation with Fu and washing. No effect of Fu was found on phagocytosis and intracellular killing of St. aureus. Fu diminished chemotaxis to fMLP but at 10 −3 M it also displayed weak chemoattractant properties. The differential action of Fu on human PMNL may add to the understanding of its topical and restricted efficacy in bronchial asthma.

Human peripheral blood polymorphonuclear leukocytes at the ovulatory period are in an activated state

The ovulatory process has been compared with inflammation. We investigated the state of female peripheral polymorphonuclear leukocytes (PMN) during menstrual cycles, and found that PMN contained high levels of superoxide, hydrogen peroxide and nitric oxide (NO) during at the peri-ovulatory period. Assuming the cause of this elevation to be a luteinizing hormone (LH), the surge of which preceded the ovulation, we examined the responsiveness of PMN to pituitary LH. The results revealed that this hormone elevated dose-dependently the production of reactive oxygen intermediates (ROI). Furthermore, we demonstrated the mRNA expression of LH receptors and their presence on PMN. The data indicated that the LH surge before on the ovulatory day resulted in general activation of PMN, suggesting that this state of PMN may be a necessary step for initiation of ovulation, rather than a defensive role against infection.

Lack of transmission to polymorphonuclear leukocytes and human umbilical vein endothelial cells as a marker of attenuation of human cytomegalovirus.

The development of a human cytomegalovirus (HCMV) vaccine for the prevention of perinatal disease is urgently needed. However, markers of HCMV attenuation are not defined at present. An in vitro model for the study of interactions of HCMV-infected human fibroblasts and peripheral blood polymorphonuclear leukocytes showed that (1) known laboratory-adapted HCMV strains as well as cell culture-adapted HCMV isolates were not transmitted to polymorphonuclear leukocytes; (2) each of the 80 HCMV recent isolates was consistently transmitted to polymorphonuclear leukocytes as both infectious virus and viral components (nucleic acid and proteins); and (3) all 15 polymorphonuclear leukocyte-tropic strains tested thus far were also endothelial cell-tropic. The in vitro transmissibility to polymorphonuclear leukocytes (and endothelial cells) is proposed as a surrogate marker of pathogenicity of HCMV strains. It seems reasonable to assume that a HCMV strain candidate for a vaccine be verified as deprived of the transfer property to polymorphonuclear leukocytes (and endothelial cells) before involvement in clinical trials assessing safety and immunogenicity.

Isolation of human polymorphonuclear leukocytes (granulocytes) from a leukocyte-rich fraction.

Human peripheral blood polymorphonuclear leukocytes (PMNs) or granulocytes from a leukocyte-rich plasma (LRP) are banded at an interface between two layers of iodixanol. If the denser layer of iodixanol is omitted the PMNs may alternatively be pelleted. The procedure can be adapted to blood from other species by small changes to the density of the two iodixanol layers. The method works optimally with EDTA- or citrate-anticoagulated blood.

Synergistic action of beta-glucan and platelets on interleukin-8 production by human peripheral blood leukocytes.

The effects of human platelets on interleukin (IL)-8 production from human peripheral blood mononuclear cells (PBMCs) and polymorphonuclear leukocytes (PMNs) stimulated with the fungal (1-->3)-beta-D-glucan schizophyllan (SPG) were examined using ELISA. PBMCs/PMNs in the presence of platelets and SPG enhanced IL-8 production in comparison with those in the presence of either platelets or SPG. IL-8 production was dependent on the concentration of platelets and incubation time, and the activity reached the maximal level at 18 h of incubation. These activities were also observed with the addition of platelets prestimulated with SPG to PBMCs. Addition of SPG directly enhanced expression of P-selectin on platelet membrane surfaces. These results suggest that platelets play a key role in the cytokine production of leukocytes induced by fungal (1-->3)-beta-D-glucans and might be mediated, at least in part, by P-selectin.

Suppressive Effects of Cyclosporin A and FK-506 on Superoxide Generation in Human Polymorphonuclear Leukocytes Primed by Tumor Necrosis Factor α

Most previous studies have found no effects of cyclosporin A and FK-506 on active oxygen generation in human polymorphonuclear leukocytes. Recently various differences in biologic properties have been reported between unprimed peripheral blood human polymorphonuclear leukocytes and tissue or primed human polymorphonuclear leukocytes. In this study, we investigated the effects of cyclosporin A and FK-506 on superoxide (O(2)(-)) generation induced by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine in human peripheral blood polymorphonuclear leukocytes primed or unprimed with tumor necrosis factor alpha. Neither cyclosporin A nor FK-506 suppressed N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced O(2)(-) generation in unprimed human polymorphonuclear leukocytes at concentrations between 0.1 nM and 10 microM, as in previous studies. Only at 1 microM of cyclosporin A and 100 nM of FK-506 were marginal suppressive effects observed. On the other hand, cyclosporin A and FK-506 both suppressed N-formyl-L-methionyl-L-leucyl-L-phenylalanine-induced O(2)(-) generation in tumor-necrosis-factor-alpha-primed human polymorphonuclear leukocytes, strongly and dose dependently, at concentrations between 1 nM and 1 microM. Neither cyclosporin A nor FK-506 influenced tyrosyl phosphorylation of 115 kDa protein, which is inducible during the priming process, suggesting that neither cyclosporin A nor FK-506 influenced the tumor-necrosis-factor-alpha-induced priming process itself, and instead modified the biologic response of primed human polymorphonuclear leukocytes.

Effect of cytokines on antifungal activity of human polymorphonuclear leucocytes against yeast cells of Paracoccidioides brasiliensis.

In our previous study, it was observed that human peripheral blood polymorphonuclear leucocytes (PMNs) exhibited a fungistatic effect on yeast cells of Paracoccidioides brasiliensis, and that interferon-gamma (IFN-gamma), but not tumor necrosis factor-alpha or interleukin-8 (IL-8), enhanced the antifungal activity of PMNs. In the present study, granulocyte-macrophage colony-stimulating factor (GM-CSF) also enhanced the PMN activity. GM-CSF-activated PMNs exhibited a killing effect on P. brasiliensis isolate Bt-4 and an enhanced fungistatic effect on isolate Aoki. IL-1beta activated PMNs to kill isolate Bt-4. Granulocyte colony-stimulating factor had no effect. Combinations of IFN-gamma with GM-CSF or IL-1beta, but not a combination of GM-CSF and IL-1beta, exhibited a synergistic effect in enhancing the antifungal activity of PMNs. These results strongly suggest that PMNs activated with IFN-gamma, GM-CSF and/or IL-1beta might play an important role in host defense in early infection with P. brasiliensis due to their enhanced antifungal activity.

Effect of water extracts of Rhizopus delemar on the stimulus coupled responses of neutrophils and their modulation by various protein kinase inhibitors

Human peripheral blood polymorphonuclear leukocytes were preincubated with water extracts from Rhizopus delemar. The water extracts significantly inhibited arachidonic acid induced superoxide generation, whereas enhanced superoxide generation induced by phorbol 12-myristate 13-acetate but not those induced by N-formyl-methionyl-leucyl-phenylalanine. Superoxide generation induced by water extracts was inhibited by staurosporine, an inhibitor of protein kinase C, and was enhanced by genistein, an inhibitor of tyrosine kinase. The water extracts incubated with phorbol 12-myristate 13-acetate markedly increased phosphorylation of serine residue of 28.5 kDa protein with time and the phosphorylation depended on the concentration of the water extracts, whereas the water extracts incubated with arachidonic acid decreased the phosphorylation of serine residue of 38 and 42 kDa proteins. The phosphorylation of 28.5 kDa protein induced by the water extracts was inhibited by staurosporine, an inhibitor of protein kinase C, but was not inhibited by genistein, an inhibitor of tyrosine kinase.

Superoxide anion scavenging properties of fluvastatin and its metabolites.

We investigated the in vitro superoxide anion scavenging activities of fluvastatin and its metabolites. Fluvastatin showed dose-dependent superoxide anion scavenging activity in the NADH/phenazine methosulphate (PMS)/nitroblue tetrazolium (NBT) system, and the effect was as potent as the reference antioxidant, trolox, which is a water-soluble alpha-tocopherol derivative. The superoxide anion scavenging activities of the major metabolites of fluvastatin (M2, M3, M4, M7) were also determined in this system. All of these metabolites showed the activity. In particular, M2 and M3, which possess a phenolic hydroxyl group at the 5 or 6-position of the indole moiety, respectively, showed 3 times stronger activities than that of fluvastatin. Further, we also determined the effects of fluvastatin, M2 and M3 on phorbol myristate acetate (PMA)-induced superoxide anion generation in human peripheral blood polymorphonuclear leukocytes (PMN). The compounds tested also showed a depressing effect on the amount of superoxide anion in this system. We suggest that fluvastatin and its metabolites have the potential to protect cells or lipids from oxidative modification mediated by superoxide anion.

Antifungal activity of human polymorphonuclear leucocytes against yeast cells ofParacoccidioides brasiliensis

Human peripheral blood polymorphonuclear leucocytes (PMN) were examined in vitro for antifungal activity against yeast cells of Paracoccidioides brasiliensis. The yeast cell of this fungus was resistant to killing by PMN. However, PMN exhibited a fungistatic effect on the fungal isolates employed in a long-term ( approximately 72 h) assay. Lysates of PMN did not show a fungistatic or fungicidal effect, indicating that live PMN are necessary for the antifungal effect. Interferon-Gamma (IFN-Gamma) enhanced the antifungal activity of PMN. IFN-Gamma-treated PMN killed isolate Bt-4 within 2 h of coculture, and after 24 h a still greater killing effect was observed. By contrast, IFN-Gamma-treated PMN did not show a significant killing effect on isolate Tatu, but did exhibit an enhanced fungistatic effect on this isolate. In contrast, tumor necrosis factor-alpha and interleukin 8 had no effect on the antifungal activity of PMN. Scanning electron microscopy revealed that the surface structure of the fungal cell was apparently damaged by PMN within 24 h of cocultures. Based on these novel findings, we speculate that human PMN might play a role in host resistance in early infection with this fungus due to their antifungal activity.

Inhibition of leukotriene B4-INDUCED increase in intracellular calcium ion level of human peripheral blood polymorphonuclear leukocytes by Y-24180, an antagonist of platelet-activating factor receptor

Y - 24180 ( (±) - 4 - (2-chlorophenyl ) - 2 - [2 - (4-isobutylphenyl ) ethyl] - 6,9 - dimethyl - 6 H - thieno [3,2 - f ] [1,2,4] triazolo [4,3- a] [1,4] diazepine) , an antagonist of platelet-activating factor (PAF) receptor, has already been reported to inhibit leukotriene B 4 (LTB 4) -induced activation of polymorphonuclear leukocytes. In this article, to clarify the mechanism of inhibition of LTB 4-induced activation by Y-24180, we examined the effect of Y-24180 on LTB 4-induced increase in intracellular calcium ion ( [Ca 2] i) level of human polymorphonuclear leukocytes at a single cell level using a laser scanning confocal microscope. Preincubation with Y-24180 significantly inhibited the increase in [Ca 2] i level at 0.3–3 μM, while WEB 2086 (4- [3- [4- (2-chlorophenyl ) -9-methyl-6 H-thieno [3,2- f ] [1,2,4] triazolo [4,3- a] [1,4] diazepin-2-yl] propionyl] morpholine) , another PAF receptor antagonist, did not show the inhibitory effect at 3–30 μM, indicating that the difference in potency between these compounds was more than 100-fold. The LTB 4-induced increase in [Ca 2] i level was suppressed by microinjection of anti-PAF antibody, but not control antibody, into leukocytes. Microinjection of Y-24180 at 0.003–0.03 μM or WEB 2086 at 0.03–0.3 μM into leukocytes also inhibited the LTB 4-induced increase. The microinjection of WEB 2086 at the 10-fold greater concentrations than those of Y-24180 inhibited to the almost equal level to that obtained by Y-24180. In addition, microinjection of PAF into leukocytes induced the increase in [Ca 2] i level. Preincubation with Y-24180 at 1 or 3 μM significantly attenuated the PAF microinjection-induced increase, but WEB 2086 showed little effect at 3–30 μM. These results indicate that Y-24180 inhibits LTB 4-induced activation of leukocytes by suppressing the increase in [Ca 2] i level and the inhibitory effect is mediated by antagonistic action against intracellular PAF-induced up-regulation of [Ca 2] i level. The difference in inhibitory activity for the increase in [Ca 2] i level between Y-24180 and WEB 2086 when these are added in the culture medium may depend upon their aptitude for the transmembrane influx.

Immunomodulatory activities ofTrichilia glabra leaf aqueous extracts

Human peripheral blood monocytes and polymorphonuclear leukocytes treated with leaf aqueous extracts of the Meliaceae tree Trichilia glabra showed a diminution in both their phagocytic and respiratory burst activities. A decrease in the haemolytic capacity of the human complement was also observed. The significance of the inhibitory effect observed over some components of the human immune system closely related with the inflammatory process is discussed. © 1998 John Wiley & Sons, Ltd.

Priming effect of N-acetyl-S-(3-oxo-3-carboxy- n-propyl)cysteine in human neutrophils and tyrosyl phosphorylation of 45 kDa protein

Human peripheral blood polymorphonuclear leukocytes were preincubated with N-acetylcystathionine and N-acetyl-S-(3-oxo-3-carboxy- n-propyl)cysteine (NAc-OCPC) found in the urine of a patient with cystathioninuria. NAc-OCPC significantly enhanced the N-formyl-methionyl-leucyl-phenylalanine-induced superoxide generation, whereas N-acetylcystathionine did not enhance the superoxide generation. When the cells were incubated with NAc-OCPC, the tyrosyl phosphorylation of 45 kDa protein of the cell was markedly increased with time. The phosphorylation process was dependent on the concentration of NAc-OCPC. Both the superoxide generation and the tyrosyl phosphorylation of 45 kDa protein increased by NAc-OCPC were inhibited by genistein and herbimycin A, the inhibitors of protein tyrosine kinase, but were rather enhanced by staurosporine, an inhibitor of protein kinase C.

Binding of human peripheral blood polymorphonuclear leukocytes to E-selectin (CD62E) does not promote their activation.

E-selectin (CD62E) is a cytokine-inducible endothelial cell adhesion molecule that tethers polymorphonuclear leukocytes (PMNs) and supports PMN rolling under conditions of flow. We examined whether interaction of PMNs with E-selectin also leads to activation of CD11b/CD18 (Mac-1, alphaMbeta2), an event that can promote firm adhesion. PMNs were added to monolayers of IL-1beta-activated HUVECs and Chinese hamster ovary (CHO) cells transfected with E-selectin cDNA. PMN activation was assessed by 1) increased CD11b/CD18 surface expression, 2) appearance of activation epitope on CD11b/CD18 (CD11b*) detected by mAb CBRM1/5, and 3) decreased L-selectin (CD62L) expression, as determined by flow cytometry. Both adherent and nonadherent supernatant PMNs became activated on IL-1beta-pretreated HUVECs. This activation was not affected by CD62E-blocking mAb P6E2. The activation state of PMNs adhered to CHO cells transfected with E-selectin cDNA was not increased over background and was similar to that of PMNs exposed to parent CHO cells. The findings were confirmed using confocal microscopy, which allowed staining of the cells for CD11b* in situ. In concert, the results suggest that PMN binding to E-selectin does not elicit inter-receptor signaling that could result in strengthening of PMN adhesion to endothelium.

Effect of Cystathionine and Cystathionine Metabolites on the Phosphorylation of Tyrosine Residues in Human Neutrophils

The effect of cystathionine and cystathionine metabolites found in the urine of patients with cystathioninuria on the phosphorylation of tyrosine residues was studied with human peripheral blood polymorphonuclear leukocytes. Among the cystathionine metabolites, cystathionine ketimine markedly increased phosphorylation of a 45 kDa protein with time and the phosphorylation depended on the concentration of cystathionine ketimine, while cystathionine and the reduced form of cystathionine ketimine (cyclothionine) did not increase the phosphorylation of the 45 kDa protein. The phosphorylation of the 45 kDa protein induced by cystathionine ketimine was inhibited by genistein and herbimycin A, inhibitors of tyrosine kinase, but was not inhibited by 1-(5-isoquinolinesulfonyl)-2-methylpiperazine and staurosporine, inhibitors of protein kinase C.