Polymorphonuclear Neutrophils Cell Research Articles

Androgen receptor inhibition suppresses anti-tumor neutrophil response against bone metastatic prostate cancer via regulation of TβRI expression

Bone metastatic disease of prostate cancer (PCa) is incurable and progression in bone is largely dictated by tumor-stromal interactions in the bone microenvironment. We showed previously that bone neutrophils initially inhibit bone metastatic PCa growth yet metastatic PCa becomes resistant to neutrophil response. Further, neutrophils isolated from tumor-bone lost their ability to suppress tumor growth through unknown mechanisms. With this study, our goal was to define the impact of metastatic PCa on neutrophil function throughout tumor progression and to determine the potential of neutrophils as predictive biomarkers of metastatic disease. Using patient peripheral blood polymorphonuclear neutrophils (PMNs), we identified that PCa progression dictates PMN cell surface markers and gene expression, but not cytotoxicity against PCa. Importantly, we also identified a novel phenomenon in which second generation androgen deprivation therapy (ADT) suppresses PMN cytotoxicity via increased transforming growth factor beta receptor I (TβRI). High dose testosterone and genetic or pharmacologic TβRI inhibition rescued androgen receptor-mediated neutrophil suppression and restored neutrophil anti-tumor immune response. These studies highlight the ability to leverage standard-care ADT to generate neutrophil anti-tumor responses against bone metastatic PCa.

Methanol Extract of Katuk (Sauropus androgynus) Leaves as an Anti-inflammatory Agent: Animal Study in Carrageenan-induced Rat Models of Inflammation

Background: Inflammation is a response in the human body to survive during infection, injury and tissue damage. Acute inflammation causes edema and polymorphonuclear neutrophils (PMNs) release. Sauropus androgynus leaves which contain flavonoids, tannins, saponins, steroids and triterpenoids may have anti-inflammatory properties. These compounds can be extracted with methanol. This research aimed to determine the anti-inflammatory effect of S. androgynus leaves methanol extract (SALME) on carrageenan induced-rats. Materials and methods: True experimental study was conducted using 12 Wistar rats. Rats were induced with carrageenan subcutaneously on the plantar pedis. Carrageenan-induced rats were treated with/without various doses of SALME. Edema volume was measured with a plethysmometer. The plantar pedis tissues were collected and stained with haematoxylin- eosin (HE) staining, then PMNs were observed and counted under a light microscope. All data were analyzed by one-way ANOVA, Bonferroni post hoc test, Pearson correlation and linear regression. Results: SALME had significant effects on the volume of edema (p=0.000) and the number of PMNs (p=0.000). The most effective dose to reduce the edema volume and decrease the PMNs cell number was 37.80 mg/200 g body weight (BW). SALME doses may affect 91.0% of edema volume and 89.2% of PMNs cell number. Edema volume had a significant, robust correlation (92.3%) with PMNs cell number. Conclusion: SALME is confirmed to have an anti-inflammatory activity by reducing the edema volume and decreasing the PMNs cell number. Keywords: Sauropus androgynus, methanol extract, anti-inflamamatory, carrageenan, plantar pedis, rats

Myeloperoxidase Deficiency Alters the Process of the Regulated Cell Death of Polymorphonuclear Neutrophils.

Polymorphonuclear neutrophils (PMNs) play a key role in host defense. However, their massive accumulation at the site of inflammation can delay regenerative healing processes and can initiate pathological inflammatory processes. Thus, the efficient clearance of PMNs mediated by the induction of regulated cell death is a key process preventing the development of these pathological conditions. Myeloperoxidase (MPO), a highly abundant enzyme in PMN granules, primarily connected with PMN defense machinery, is suggested to play a role in PMN-regulated cell death. However, the contribution of MPO to the mechanisms of PMN cell death remains incompletely characterized. Herein, the process of the cell death of mouse PMNs induced by three different stimuli – phorbol 12-myristate 13-acetate (PMA), opsonized streptococcus (OST), and N-formyl-met-leu-phe (fMLP) – was investigated. MPO-deficient PMNs revealed a significantly decreased rate of cell death characterized by phosphatidylserine surface exposure and cell membrane permeabilization. An inhibitor of MPO activity, 4-aminobenzoic acid hydrazide, did not exhibit a significant effect on PMA-induced cell death compared to MPO deficiency. Interestingly, only the limited activation of markers related to apoptotic cell death was observed (e.g. caspase 8 activation, Bax expression) and they mostly did not correspond to phosphatidylserine surface exposure. Furthermore, a marker characterizing autophagy, cleavage of LC3 protein, as well as histone H3 citrullination and its surface expression was observed. Collectively, the data show the ability of MPO to modulate the life span of PMNs primarily through the potentiation of cell membrane permeabilization and phosphatidylserine surface exposure.

Differential response of human blood leukocytes to brushite, monetite, and calcium polyphosphate biomaterials.

Calcium phosphate-based biomaterials are extensively used for bone replacement and regeneration in orthopedic, dental, and maxillofacial surgical applications. The injury induced by surgical implantation of bone replacement graft materials initiates a cascade of host responses, starting with blood-biomaterial contact, protein adsorption on the material surface, blood coagulation, and leukocyte responses. During the initial acute inflammatory response, polymorphonuclear neutrophils (PMNs) and monocytes, abundant circulating leukocytes of the myeloid lineage, are recruited to the site of inflammation. In addition to responding to pathogenic challenges, these cells respond to particulate substances within the body including crystals of monosodium urate (MSU). Host responses toward grafts impact short- and long-term success in tissue engineering and regenerative applications. Although multinucleated osteoclasts, formed by monocyte/macrophage fusion, are generally thought to be responsible for resorption of implant biomaterials, the ability of different biomaterials to trigger PMNs, which are invariably present at the early stages after implant surgery, and are abundant in the oral cavity, has never been tested. In this article, we present analysis of the response of human blood-derived PMNs and monocytes toward brushite, monetite, and calcium polyphosphate (CPP) biomaterial substrates and compare this to the response to MSU crystals, the latter serving as a positive control. Employing multicolor flow cytometry to look at PMN and monocyte cell surface markers of activation to gauge the response to different biomaterials, we found that both types of myeloid cells are highly activated after exposure to brushite, monetite, and MSU but not CPP. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:253-262, 2020.

Autophagy in pulmonary macrophages mediates lung inflammatory injury via c-Src tyrosine kinase pathway activation during mechanical ventilation.

It has been clearly demonstrated that autophagy plays a critical role in mechanical ventilation-Induced lung injury (VILI). Herein, we first evaluated the mutual effects of autophagy and c-Src signaling on the lung inflammatory response to mechanical ventilation. Mice were respectively subjected to a lower or higher lung stretch induced by mechanical ventilation with low (7 mL/kg) or high (28 mL/kg) tidal volume, before measuring the activation of autophagy and c-Src signaling through LC3 lipidation and c-Src phosphorylation, respectively. Bone marrow-derived macrophages (BMDMs) were transfected with Atg5 siRNA and administered to AM-depleted mice to generate an autophagy-deficient phenotype, and c-Src signaling was evaluated by Western blot assay to determine the impact of autophagy on c-Src activation during VILI. Afterwards, the c-Src pathway was then blocked using PP2, prior to the evaluation of polymorphonuclear neutrophils (PMN), total cell counts in BAL fluid, and lung injury scores, in order to elucidate the role of the c-Src pathway in autophagy-mediated VILI. Both LC3-II and p-c-Src were remarkably increased after mechanical ventilation, in a time-dependent and tidal volume-dependent manner. Moreover, c-Src phosphorylation induced by ventilation was significantly compromised in autophagy-deficient mice. On the other hand, LC3-II expression did not change due to c-Src signaling abolishment. But the inflammatory response induced by injurious ventilation was markedly attenuated by PP2 or AM-abolishment, shown by PMN and total cell counts in BAL fluid, as well as lung injury scores. Our results suggested that autophagy caused VILI via regulating c-Src activation, which implies that c-Src may serve as a promising therapeutic target in VILI.

Localized Modeling of Biochemical and Flow Interactions during Cancer Cell Adhesion.

This work focuses on one component of a larger research effort to develop a simulation tool to model populations of flowing cells. Specifically, in this study a local model of the biochemical interactions between circulating melanoma tumor cells (TC) and substrate adherent polymorphonuclear neutrophils (PMN) is developed. This model provides realistic three-dimensional distributions of bond formation and attendant attraction and repulsion forces that are consistent with the time dependent Computational Fluid Dynamics (CFD) framework of the full system model which accounts local pressure, shear and repulsion forces. The resulting full dynamics model enables exploration of TC adhesion to adherent PMNs, which is a known participating mechanism in melanoma cell metastasis. The model defines the adhesion molecules present on the TC and PMN cell surfaces, and calculates their interactions as the melanoma cell flows past the PMN. Biochemical rates of reactions between individual molecules are determined based on their local properties. The melanoma cell in the model expresses ICAM-1 molecules on its surface, and the PMN expresses the β-2 integrins LFA-1 and Mac-1. In this work the PMN is fixed to the substrate and is assumed fully rigid and of a prescribed shear-rate dependent shape obtained from micro-PIV experiments. The melanoma cell is transported with full six-degrees-of-freedom dynamics. Adhesion models, which represent the ability of molecules to bond and adhere the cells to each other, and repulsion models, which represent the various physical mechanisms of cellular repulsion, are incorporated with the CFD solver. All models are general enough to allow for future extensions, including arbitrary adhesion molecule types, and the ability to redefine the values of parameters to represent various cell types. The model presented in this study will be part of a clinical tool for development of personalized medical treatment programs.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide.

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN.

Glycoengineered CD20 Antibody Obinutuzumab Activates Neutrophils and Mediates Phagocytosis Through CD16B More Efficiently Than Rituximab

Background Obinutuzumab (GA101) is a third generation, glycoengineered defucosylated anti-CD20 antibody which shows higher binding affinity for CD16A than fully glycosylated rituximab (RTX). This higher binding leads to stronger NK mediated antibody dependent cellular cytotoxicity activity (ADCC) by obinutuzumab compared to that induced by RTX. The GPI-anchored CD16B molecule is highly homologous to CD16A and is the major FcγR on polymorphonuclear neutrophils (PMN). We have therefore investigated the binding of obinutuzumab to CD16B and its functional activity on human PMN compared to parent rituximab (RTX) or to defucosylated rituximab (G2 antibody). Methods Binding to CD16B of glycoengineered or fully glycosylated anti-CD20 antibodies was measured by surface plasmon resonance (Biacore). For PMN activation and phagocytosis, we have used either purified PMN or analyzed PMN function in unmanipulated whole blood assays from normal donors or CLL patients. PMN activation was measured as CD11b upregulation and CD62L downmodulation by flow cytometry. Phagocytosis by PMN of chronic lymphocytic leukemia (CLL) cells was measured by triple fluorescence (PKH26, CD15-FITC and CD19-APC) and flow cytometry. Results Obinutuzumab or glycoengineered defucosylated rituximab (called G2) bound CD16B with about 7 fold higher affinity, compared to non-glycoengineered wild type parental antibodies. This was true either using surface plasmon resonance or measuring antibody binding to live PMN. Furthermore obinutuzumab activated PMN, either purified or in whole blood, more efficiently than RTX. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on PMN and in release of TNFα, IL-6 and IL-8. Activation was not accompanied by generation of reactive oxygen species or ADCC, but led to phagocytosis of anti-CD20 antibody opsonized CLL targets by purified PMN. Indeed up to 50% phagocytic PMN could be observed in presence of obinutuzumab or G2 antibodies after 6-24 hours incubation of purified PMN with CLL targets. Significant phagocytosis (15%) was also observed in whole blood, but only in presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using blocking F(ab) and F(ab’)2 fragments specific for CD16B and CD32A, that both these receptors are involved in PMN activation, phagocytosis and cell death induced by glycoengineered anti-CD20 antibodies. The possible effect of NA1 and NA2 polymorphisms of CD16B on obinutuzumab binding and phagocytosis is under further investigation. Conclusions We conclude that phagocytosis by PMN is an additional mechanism of action of obinutuzumab, mediated through its higher binding affinity for CD16B compared to RTX. Phagocytosis takes place in whole blood and is followed by PMN death. This effect may in part explain the neutropenia observed after treatment of B-CLL patients with GA101. Disclosures: Golay: Roche Glycart AG: Research Funding. Ferrara Koller:Roche Glycart AG: Employment. Rambaldi:Roche Italia: Consultancy, Honoraria. Klein:Roche Glycart AG: Employment. Introna:Roche Glycart AG: Research Funding.

Glycoengineered CD20 antibody obinutuzumab activates neutrophils and mediates phagocytosis through CD16B more efficiently than rituximab

AbstractObinutuzumab (GA101) is a glycoengineered type 2 CD20 antibody with enhanced CD16A-binding and natural killer–mediated cytotoxicity. CD16B is highly homologous to CD16A and a major FcγR on human polymorphonuclear neutrophils (PMNs). We show here that glycoengineered obinutuzumab or rituximab bound CD16B with approximately sevenfold higher affinity, compared with nonglycoengineered wild-type parental antibodies. Furthermore, glycoengineered obinutuzumab activated PMNs, either purified or in chronic lymphoblastic leukemia whole blood, more efficiently than wild-type rituximab. Activation resulted in a 50% increase in CD11b expression and 70% down-modulation of CD62L on neutrophils and in release of tumor necrosis factor alpha, IL-6, and IL-8. Activation was not accompanied by generation of reactive oxygen species or antibody-dependent cellular cytotoxicity activity, but led to up to 47% phagocytosis of glycoengineered anti-CD20 opsonized chronic lymphoblastic leukemia targets by purified PMNs. Significant phagocytosis was observed in whole blood, but only in the presence of glycoengineered antibodies, and was followed by up to 50% PMN death. Finally we show, using anti-CD16B and anti-CD32A Fab and F(ab')2 fragments, that both of these receptors are involved in PMN activation, phagocytosis, and cell death induced by glycoengineered antibodies. We conclude that phagocytosis by PMNs is an additional mechanism of action of obinutuzumab mediated through its higher binding affinity for CD16B.

Detection of anti‐CD32 alloantibody in donor plasma implicated in development of transfusion‐related acute lung injury

Transfusion-related acute lung injury (TRALI) occasionally causes serious symptoms that may be fatal to recipients. Polymorphonuclear neutrophils (PMNs) and alloantibodies specific to PMN cell surface antigens are suspected to cause TRALI. The aim of this study is to establish a sensitive and stable procedure of detecting alloantibodies not only in donor blood, but also in recipient's plasma. We have introduced a new method of detecting alloantibodies based on double-determinant enzyme-linked immunosorbent assay (DD-ELISA) and a monoclonal antibody-immobilized granulocyte antigen (MAIGA) test (arbitrarily designated as modified DD-ELISA). We verified the specificity of alloantibodies against PMN cell surface antigens in plasma samples from three normal healthy donors of blood that induced respiratory distress in recipients after a blood transfusion. Anti-CD32 (Fc gamma RIII) alloantibodies were detected in all the plasma samples using two different clones of the monoclonal anti-CD32 antibody. The specificities of these plasma samples could not be identified by the granulocyte immunofluorescence test (GIFT) using typed test cells. Except for the anti-CD32 alloantibodies, one plasma sample was proved to have the anti-HNA-1a alloantibodies. In another plasma sample, the anti-HNA-2a alloantibodies were detected. By modified DD-ELISA, we could clearly specify the presence of alloantibodies in the three plasma samples. Our results also suggest that the anti-CD32 alloantibodies can be generated in vivo and may play some roles in the development of TRALI.

Dose-Related Inhibition of Proinflammatory Cytokine Release from Neutrophils of the Newborn by Dexamethasone, Betamethasone, and Hydrocortisone

The objective of this study was to determine the doses of dexamethasone (DEX), betamethasone (BET), and hydrocortisone (HC) that effectively inhibit the release of two potent proinflammatory chemokines, interleukin 8 (IL-8) and macrophage inflammatory protein alpha (MIP), from polymorphonuclear neutrophils (PMNs) of the newborn. Human PMNs were isolated from cord blood (n = 18). Chemokines were measured from PMN cell culture supernatants after 18 h of stimulation using tumor necrosis factor (1 ng/ml), with and without pretreatment by DEX (10^–10 to 10^–6 M) versus HC or BET (10^–10 to 10^–5 M). Maximal inhibitions of IL-8 release by BET, DEX, and HC were 97, 91, and 91%, respectively. For MIP, the maximal inhibitions by BET, DEX, and HC were 88, 69, and 70%, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for IL-8 release were 3.4 ± (SE) 1.6 × 10^–9, 1.8 ± 7.4 × 10^–8, and 1.8 ± 0.5 × 10^–7 M, respectively. The 50% inhibitory concentrations by DEX, BET, and HC for MIP release were 1.0 ± (SE) 0.5 × 10^–8, 3.8 ± 3.1 × 10^–8, and 4.8 ± 1.6 × 10^–7 M, respectively. In vitro, these corticosteroids effectively inhibited the release of two structurally different chemokines that are found in the airway lavage fluids of infants developing bronchopulmonary dysplasia. When compared to plasma DEX levels previously reported during the treatment of bronchopulmonary dysplasia, our results suggest that the doses of DEX, and potentially BET, needed to treat chronic lung disease may be more than five to ten times lower than those of current DEX regimens.

Differences in neutrophil death among beta-lactam antibiotics after in vitro killing of bacteria.

Antibiotic therapy is an essential treatment for gram-negative bacterial infections. Antibiotic-induced endotoxin release and subsequent production of inflammatory cytokines reportedly depend on the type of antibiotic action. This study examined the effects of various beta-lactam antibiotics on cell death of human polymorphonuclear neutrophils (PMNs) cocultured with Escherichia coli (E. coli) in vitro. E. coli morphology after antibiotic treatment was determined. PMNs and E. coli were cocultured with antibiotics for 0, 4, or 12 h. Levels of endotoxin and cytokines (TNF-alpha, IL-1beta, and IL-6) in the supernatants were measured. The filtrates of antibiotic-treated E. coli supernatants were cocultured with PMNs for 0, 4, or 12 h. In all experiments, ampicillin (ABPC), cefazolin sodium (CEZ), cefoperazone sodium (CPZ), latamoxef sodium (LMOX), imipenem (IPM), and polymyxin B sulfate (PLB) were used at 30 microg/mL. PMNs were isolated from healthy volunteers. PMN cell death was assessed by flow cytometry and light microscopy. ABPC, CEZ, CPZ, and LMOX, which induce bacterial filament formation with lysis, caused PMN necrosis when cocultured with E. coli. In contrast, IPM, which induces bacterial spheroplast formation with lysis, caused PMN apoptosis. Levels of endotoxin, TNF-alpha and IL-6 in the supernatants with IPM and PLB were significantly lower than in those with other beta-lactam antibiotics. The filtrates of IPM- and PLB-treated E. coli supernatants induced PMN apoptosis, whereas those treated with other beta-lactam antibiotics increased PMN necrosis. Beta-lactam antibiotics have different impacts on the types of PMN cell death after E. coli killing. Underlying mechanisms and the clinical relevance of IPM-induced PMN apoptosis in severe gram-negative infection warrant further investigation.

Monoclonal anti-double stranded DNA antibody is a leucocyte-binding protein to up-regulate interleukin-8 gene expression and elicit apoptosis of normal human polymorphonuclear neutrophils.

To determine whether anti-double stranded DNA (anti-dsDNA) autoantibody could bind and affect the functions of normal human polymorphonuclear neutrophils (PMN). Normal human PMN were incubated with different concentrations of a monoclonal mouse anti-dsDNA antibody (12B3) or mouse isotype-matched IgG2a. The binding of anti-dsDNA and PMN was measured by flow cytometry and interleukin-8 (IL-8) gene expression in PMN was detected by enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR). PMN apoptosis was justified by morphological changes. The cognate antigen(s) of anti-dsDNA on the PMN surface was identified by membrane biotinylation, immunoprecipitation and Western blot. The binding of PMN with anti-dsDNA was much higher than with non-specific mouse IgG2a (70.8 vs 2.0%). Anti-dsDNA at concentrations higher than 12.5 ng/ml significantly enhanced the production and mRNA expression of IL-8 by PMN. However, anti-dsDNA facilitated PMN apoptosis after 3 h incubation. Western blot analysis of biotinylated PMN cell lysates demonstrated that a 50-52 kDa membrane molecule is the cognate antigen of anti-dsDNA. Anti-dsDNA autoantibody up-regulates IL-8 gene expression and elicits activation-induced cell death (AICD) of human PMN via binding to a 50-52 kDa membrane-expressed molecule.

Effects of remifentanil on neutrophil adhesion, transmigration, and intercellular adhesion molecule expression.

Anaesthetic drugs are used for pain therapy and anaesthesia. Neutrophils play a significant role during the process of inflammation. The aim of the current study was to investigate the effects of remifentanil and fentanyl on neutrophil migration through endothelial cell monolayers, and on adhesion molecule expression. After isolation of polymorphonuclear neutrophils (PMNL) we used a currently described migration assay. PMNL and/or endothelial cell monolayers (ECM) were pre-treated with remifentanil using clinically relevant, as well as higher and lower concentrations or relevant concentrations of fentanyl. Concentrations of remifentanil (50 ng/mL) similar to the relevant plasma concentration were able to inhibit PMNL migration through ECM significantly (migration compared to the control 82+/-7% SD; P<0.05), when both cell types were treated with the synthetic narcotic remifentanil. Fentanyl (30 ng/mL) showed a stronger inhibitory effect (migration compared to the control 67+/-9.2%; P<0.05). Endothelial cell adhesion molecule expression was reduced after either remifentanil or fentanyl. The results of the present investigation indicate that remifentanil influences interaction of ECM against human neutrophils. Compared to fentanyl, remifentanil seems to exhibit minor inhibitory effects on neutrophil migration.

Decreased Leukotriene C4 Synthesis Accompanies Adherence-Dependent Nuclear Import of 5-Lipoxygenase in Human Blood Eosinophils

The enzyme 5-lipoxygenase (5-LO) catalyzes the synthesis of leukotrienes (LTs) from arachidonic acid (AA). Adherence or recruitment of polymorphonuclear neutrophils (PMN) induces nuclear import of 5-LO from the cytosol, which is associated with enhanced LTB4 synthesis upon subsequent cell stimulation. In this study, we asked whether adherence of human eosinophils (EOS) causes a similar redistribution of 5-LO and an increase in LTC4 synthesis. Purified blood EOS examined either in suspension or after adherence to fibronectin for 5 min contained only cytosolic 5-LO. Cell stimulation resulted in activation of 5-LO, as evidenced by its translocation to membranes and LTC4 synthesis. As with PMN, adherence of EOS to fibronectin for 120 min caused nuclear import of 5-LO. Unexpectedly, however, adherence also caused a time-dependent decrease in LTC4 synthesis: EOS adhered for 120 min produced 90% less LTC4 than did cells adhered for 5 min. Adherence did not diminish the release of [3H]AA from prelabeled EOS or reduce the synthesis of the prostanoids thromboxane and PGE2. Also, inhibition of LTC4 production caused by adherence could not be overcome by the addition of exogenous AA. Adherence increased, rather than decreased, LTC4 synthase activity. However, the stimulation of adherent EOS failed to induce translocation of 5-LO from the nucleoplasm to the nuclear envelope. This resistance to activation of the nuclear pool of 5-LO with diminished LT production represents a novel mode of regulation of the enzyme, distinct from the paradigm of up-regulated LT synthesis associated with intranuclear localization of 5-LO observed in PMN and other cell types.

Leukocyte Rolling In Vivo Is Mediated by P-Selectin Glycoprotein Ligand-1

Leukocyte rolling, an early and important step in the inflammatory response, is mediated by the selectin family of adhesion molecules. The selectins bind with low affinity to sialylated and fucosylated glycans such as sialyl Lewisx (sLex), but bind with high affinity to only a few specific glycoproteins on cell surfaces. One such glycoprotein is P-selectin glycoprotein ligand-1 (PSGL-1). The relative contributions of low- and high-affinity ligands to leukocyte rolling in vivo are unknown. We show here that a monoclonal antibody to PSGL-1 (PL1) dramatically reduces rolling of human polymorphonuclear neutrophils (PMN) and promyelocytic HL-60 cells in venules of acutely exteriorized rat mesentery. Control PMN and HL-60 cell rolling flux fractions were 39% +/- 3% and 33% +/- 5%, which were reduced by PL1 to 7% +/- 2% and 6% +/- 2%, respectively. Similar reductions were seen with F(ab) fragments of PL1. PL1-treated PMN rolled at significantly higher mean velocities than untreated PMN owing to intermittent rather that continuous interactions. These findings show that interaction of P-selectin with PSGL-1 is required for rolling of myeloid cells in mesenteric venules at physiologic shear stress in vivo.

Hydrophobic characterisation of Helicobacter (Campylobacter) pylori

Cell-surface hydrophobicity of Helicobacter (formerly Campylobacter) pylori was tested by aqueous two-phase partitioning and hydrophobic interaction chromatography. The hydrophobicity of H. pylori greatly exceeded that of Campylobacter fetus subsp. fetus, C. jejuni and Bacillus subtilis. A partition coefficient (PC) of hydrophobicity in the two-phase system was determined for H. pylori. PC was dependent on pH and the PC value was increased by greater than 20-fold at pH 2.5. Lithium cations increased PC, indicating a net negative surface charge. The presence of urea prevented the relative loss of hydrophobicity at raised pH. Exposure of H. pylori to proteolytic enzymes reduced the ability of the bacteria to adhere to human polymorphonuclear neutrophils (PMN). These findings suggest that H. pylori possesses protein-associated hydrophobic factors that are responsible for the non-opsonic adherence to PMN cell membranes.

Density changes in leukocytes following hemodialysis or exposure to chemotactic factors.

Analysis of standard Ficoll-Hypaque separation profiles of peripheral WBC from patients undergoing hemodialysis (HD) demonstrated that dialysis caused a marked alteration in the number of cells found at both the interface between the Ficoll-Hypaque and plasma which normally contains mononuclear cells and the cell pellet which normally contains granulocytes. By 30 min into dialysis, there was a 175% increase in white blood cells in the mononuclear band with a corresponding decrease in the number of cells obtained from the cell pellet. When peripheral blood samples from normal donors were pumped through various types of hemodialyzers, a shift in the cell separation profiles similar to that of patients undergoing HD was observed. Differential analysis of the cells obtained from both the interface between the Ficoll-Hypaque and plasma and the cell pellet showed that by 30 min into dialysis, the 'mononuclear' band contained 40-50% polymorphonuclear neutrophils (PMN). To ascertain whether the cell separation changes were possibly due to C5a generation resulting from complement activation by the HD membrane, whole blood was incubated with the purified chemotactic factors C5a, C5ades arg, and formyl-methionyl-leucyl-phenylalanine. This resulted in similar alterations in PMN densities. This study demonstrates that both in vivo and in vitro exposure of human peripheral blood to HD membranes as well as the chemotactic factors C5a, C5ades arg, and formyl-methionyl-leucyl-phenylalanine results in density changes in PMN.(ABSTRACT TRUNCATED AT 250 WORDS)

Polymorphonuclear neutrophil-mediated antibody-dependent cell cytotoxicity of herpesvirus-infected cells: ultrastructural studies.

Bovine polymorphonuclear neutrophils (PMN) can mediate antibody-dependent cell cytotoxicity (ADCC) of herpesvirus-infected cells. Since cytotoxicity occurs only in the presence of PMN and specific antiviral antibody, but not until viral membrane antigens are expressed on the target cell, it is concluded that antibody must recognize viral membrane antigens before cytotoxicity can occur. Cytotoxicity also requires very close contact between the target cell and the PMN cell. These interactions occur as early as 1 h after incubating antibody, infected cells, and PMN, but the actual lysis and release of intracellular components occur over an extended period. It was assumed that degranulation was not involved in the initiation of cytotoxicity, but was involved in the final stage of destruction. The mechanism of lysis is proposed to involve the interaction of PMN membranes with target cell membranes with subsequent reorganization and activation of the PMN plasma membrane at points of contact with the target cell. This results in possible production of transmembrane channels which allows for the release of target cell contents.