Original Paper Int Arch Allergy Immunol 1992;99:98-106
Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Universität Bochum, FRG; Department Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
Alginate - Its Role in Neutrophil Responses and Signal Transduction towards Mucoid Pseudomonas aeruginosa Bacteria
Key Words Alginate Cystic fibrosis Proteinkinase C G proteins P aeruginosa
Abstract Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mu coid and nonmucoid (NM) P aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aerugi nosa (MEP). In this regard the MEP (alginate) significantly reduced the zymo san-induced leukotriene B4 (LTB4) formation (from 40±7 to 2± 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were prccoatcd with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aerugi nosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate bind ing [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our re sults indicate that the MEP represents an important component which mod ulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacte ria, e.g. the chemiluminescence response, LTB4gencration, and the interaction with components (G proteins. PKC) of the signal transduction pathway.
Introduction
severe burns, cancer, or receiving immunosuppressive therapy [1, 2], Basically, the occurrence of such diverse clinical diseases may cither derive from an altered host defense in these patients and/or a different virulence of taxonomically identical P. aeruginosa strains [2-4].
Pseudomonas aeruginosa is an opportunistic pathogen which causes either localized infections such as pneumo nia in patients with cystic fibrosis (CF) or generalized sep ticemia in immunocompromised hosts suffering from
Correspondence to: Dr. W. König I.ehrstuhl für Med. Mikrobiologie und Immunologie AG Infcktabwehrmechanismcn Ruhr-Universität Bochum. Univcrsitätsstt. 150 D-W-4630 Bochum (FRG)
© 1992 S. Karger AG. Basel 1018-2438/92/0991-0098 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
B. Königa P. FriedP S.S. Pedersenb W. Königa
as leukotrienes from human neutrophils [20, 21]. In addi tion to PKC the G proteins are involved in the complex events of cellular responses [21, 22], Guaninc-nucleotidebinding proteins (G proteins) appear to represent a family of regulatory proteins, all of which serve to transfer in formation from receptor to effector systems of the cell [22, 23]. This paper deals with the question of the suppressive effects of mucoid strains of P aeruginosa and in particular with the question of soluble, purified alginate, the MEP, on neutrophil functions (chemiluminescence, leuko trienes) and on the mechanism of the alginate-induced ef fects on the cellular signalling pathway, e.g. the involve ment of G proteins, PKC. Here, we report that the MEP of mucoid P. aeruginosa is responsible for the inhibitory ef fects on leukotriene release and chemiluminescence re sponse from human neutrophils by mucoid P. aeruginosa strains. We also show that the MEP interacts with compo nents of the signal transduction pathway in a distinct way and that the MEP represents the component which is re sponsible for the altered signal transduction pathway seen in human neutrophils after stimulation with mucoid P ae ruginosa strains as compared to nonmucoid P. aeruginosa strains.
Materials and Methods Materials [gamma-32 P]ATP (5 Ci/mmol) was purchased from New England Nuclear, DE-52 cellulose was from Whatman. Synthetic leukotrienes were a generous gift from Mcrck-Frosst, Pointe Claire, Canada. The solvents used were of high pressure liquid chromatography grade ob tained from local suppliers. The MEP from P. aeruginosa was pre pared by Pedersen as described previously [161. BacteriaI Strains P. aeruginosa from CF patients were isolated from sputa of pa tients suffering from acute exacerbations of pulmonary infections. The strains were identified by their ability to synthesize fluorescent pigments on Pseudomonas agar P and F (Merck, Darmstadt, FRG) and by using the api 20 NE identification system (BIOMerieux, Niirtingen. FRG). Strains CF4 and CF14 showed a nonmucoid growth pattern: strains CF9 and CF7 expressed a mucoid phenotype. Buffer The buffer used for washing the neutrophils and for mediator re lease consisted of 137 mM NaCI. 8 mM Na:H P 0 4. 3 mM KC1. and 3 mM KCI, and 3 mM KH,PQ4, pH 7.4 (modified Dulbecco’s phos phate-buffered saline, PBS). Buffer A consisted of 20 mM Tris-HCI (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA. I mM dithiothreitol, and I mM PMSF.
99
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
We have recently shown that clinical isolates from burn patients induce inflammatory mediator release from mast cells and neutrophil granulocytes [5]; the release of media tor substances correlated with the expression of the en zymatic (phospholipase C) or the nonenzymatic (glycolipids) hemolysins from P. aeruginosa. In addition. Meyers and Berk [6] also showed that soluble phospholipase C in duced I.TB4 formation from human granulocytes. In con trast P. aeruginosa strains isolated from CF patients in duced only insignificant levels of inflammatory mediators from human neutrophils [unpubi. data]. In the past we demonstrated that mucoid unlike nonmucoid bacteria led to a defined modulation of the neutrophil responses, e.g. a decrease in the chemiluminescence response, a reduced leukotriene B4 (LTB4) formation on subsequent cellular stimulation as well as changes on the C3bi receptor ex pression [7]. However, these studies did not prove the role of alginate. A predominant feature of P. aeruginosa in CF is the production of alginate, the mucoid exopolysaccharide (MEP) [8-11]. Alginate has been described to exhibit sev eral features such as an increased binding of P. aeruginosa to epithelial cells, inhibition of phagocytosis of mucoid Pseudomonas bacteria by human granulocytes as well as by macrophages, inhibition of the chemotaxis of granulocytes to the inflammatory area, and inhibition of opsonization of bacteria [12-15]. Nonetheless, up to now there has been no explanation for the fatal outcome of a chronic P. aerugi nosa infection in CF as well as the local destruction of the lung tissue [16]. Neutrophils play a crucial role in host de fense mechanisms [17]. Upon stimulation with microbial pathogenicity factors neutrophils are triggered for the production of O5 metabolites, the release of enzymes and of arachidonic acid metabolites such as LTB4 [18]. LTB4 is strongly chemotactic for neutrophil granulocytes. The generation and the appropriate control of inflammatory mediator release, e.g. the generation of arachidonic acid metabolites and oxygen radicals is a prerequisite for a nor mal host defense. Alginate has been made responsible for a variety of pathological cellular immune functions. In this regard, it has been reported by Mai ct al. [19] that the MEP interferes with a broad range of neutrophil functions, in cluding adherence, locomotion, oxygen metabolism, de granulation, and bactericidal activity. Cellular responses are mediated by a complex signal transduction cascade which is dependent on the stimulus and the cell type. Substantial evidence was obtained for the fact that protein kinase C (PKC) played an essential role in the cellular activation process leading to reactive oxygen metabolites and mediators of inflammation such
Preparation o f Cells Human neutrophils were prepared from heparinized blood (15 U/ ml) of healthy donors and separated on a Ficoll metrizoate gradient followed by dextran sedimentation [24], The cells were washed at 300 # to remove the platelets. The erythrocytes were lysed by exposing the cells to hypotonic conditions. This method results in more than 97% pure neutrophils. Cell Viability’ Cell viability was tested by trypan blue exclusion as well as by the analysis of lactate dehydrogenase (LDH) release from stimulated and nonstimulated cells. Analysis of LDH (EC 1.1.1.27) was carried out as described previously [21). Chemiluminescence Oxygen-dependent respiratory activity was measured as de scribed earlier [21]. In brief: neutrophils (107150 pi) in Ca/Mg (1/0.5 mM) were incubated with bacteria (50 pi) in the presence of 40 pi of 0.25 mM luminol. In several experiments the neutrophils were preincubated with bacteria for 20 min at 37°C and subsequently stimu lated with PMA ( 1 0 M) for a further 20 min. Leukotriene Release from Human Neutrophils The assay was performed as described previously [21]. In prcincubation experiments neutrophils were treated with alginate for the in dicated time intervals and then stimulated with opsonized zymosan. Analysis o f Leukotriene Release Cell supernatants were analyzed for leukotrienes as described [21], 2 ml of methanol-acetonitrile (50:50, vol/vol) were added to the culture supernatants. After centrifugation at 1,900# for 15 min (Cryofuge 6-4 Heraeus Christ) the supernatants were evaporated to dry ness by lyophilization (Modulyo, Edwards-Kniesc, Marburg. FRG). The residues were dissolved in 600 pi of methanol/water (30:70) and 200 pi were analyzed by reversed-phase high pressure liquid chroma tography. The column was packed (4.6x200 mm) was packed with Nueleosil Cls (particle 5 pm: Macherey-Nagcl. Diiren, FRG). HPLC equipment consisted of a CM4000 pump, a SM4000 detector (both Laboratory Data Control/Milton Roy, Hasselroth. FRG), and an au tomatic sample injector (WISP 7I0B, Waters, Eschborn. FRG). Leukotriencs were analyzed using a mobile phase consisting of methanol/ water/acctonitrile/phosphoric acid (48:24:28:0.03, v/v) including 0.04% EDTA and 0.15% K ,H P 04. pH 5.0. The flow rate was main tained at 0.9 ml/min. The A ^ of the column effluent was determined. Quantification and identification of leukotrienes were performed with synthetic standard solutions. LTB., generation was calculated as the combined amounts of LTB4 and the LTB4 omega-oxidation prod ucts (20-hydroxy-LTB4 and 20-carboxy-LTB4). PKC Assay Human neutrophils (4 xl()7) were prcincubated for 10 min at 37 °C in PBS with Ca/Mg. The cells were subsequently incubated with the indicated stimuli. Activation of the cells was stopped by centrifu gation at 1,000g for 10 min: the cells were then resuspended in I ml
mo
buffer A, and sonicated (amplitude 50 pm, sonificr 250, Branson Power Company, USA) for 2 x 10 s. After centrifugation of the crude homogenate at 9,700# for 10 min the cytosolic fractions were chroma tographed on DEAE-cellulose columns (0.5x3 cm), equilibrated and washed with buffer A (3 ml). The protein kinase C was eluted with 1.5 ml of buffer A containing 100 mM NaCI. The reaction mixture (190 pi) contained 20 mM Tris-HCI (pH 7.5). 10 mM magnesium ace tate, 5(H) pg/ml histone type 11IS. 10 pA/ [gamma- >:P]ATP (10' cpm/ nmol), 2.5 pg diolein, 25 pg phosphatidylserine, 1 mM CaCL, and the enzyme preparation under study. All reagents were dissolved in dis tilled water. The quantitation of background phosphotransferase ac tivity was performed by analyzing histone phosphorylation in the ab sence of calcium and phospholipid. After 10 min at 30 °C. the reaction was stopped by the addition of 0.75 ml of trichloroacetic acid (25%). Acid-prccipitable materials were collected on Millipore membrane filters (0.45-pm pore size, Milliliter HA filtration plates. Millipore, Eschborn. FRG). The filters were washed with 2x1 ml of trichloro acetic acid (10%). The dried filters were transferred into scintillation vials, distilled water was added and the radioactivity was measured by Ccrcncov radiation (Rack beta 1209, LKB, Turku, Finnland). Protein kinase C activity was calculated by substracting background activities, values are reported as percent of cytosolic protein kinase C activity from untreated cells. Binding o f Guanylylimidodiphosphate and GTPase Activity Human neutrophils were incubated in the presence of alginate, NaF, bacterial suspensions, or in the absence of any stimulus for 10 min at 37 °C. In preincubation experiments, human neutrophils were first treated with buffer or alginate: then NaF was added and the stimulation of the cells proceeded for a further 10 min. The assay was performed as described previously 121]. Binding of guanylylimido diphosphate [Gpp(NH)p] was expressed in cpm bound to the membranes. GTPase activity was reported as [,:P] released from [gamma-':P]GTP. Statistics Data show mean values ± standard deviation (SD) of at least three individual experiments with cells from different donors. Signif icance was examined with the Student t test for independent means.
Results Inflammatory’ Mediator Release Effect o f Alginate on the Chemiluminescence Response. In a first set of experiments we analyzed the role of the MEP on the bacteria-induced chemiluminescence re sponse from human neutrophils. For this purpose nonmu coid R aeruginosa bacteria (CF14; CF4) were cultured in the absence or presence of alginate (MEP) (1 mg/ml) for the last hour of growth at 37 °C and were then used as stimulus. As is apparent from figure 1 for strain CF4, algi nate-coated bacteria of both P. aeruginosa strains induced a chemiluminescence response from human neutrophils to a lesser degree as compared to the nonmucoid bacteria. In subsequent experiments neutrophils were preincubated
König/Fricdl/Pedersen/König
Pseudomonas aeruginosa Alginate and Neutrophil Function
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
Bacterial Growth Bacterial growth was carried out in peptone medium (1% NaCI, 1% peptone, 1% glycerol). The broth (10 ml) was inoculated with 100 ul of an overnight culture. Bacterial growth proceeded on a shaker (180 rpm) at 37 °C for the indicated times.
Table 1. Effect of alginate on the zymosan-induced LTB, generation
Neutrophils preincubated with
LTB,
Buffer (control) Alginate (1(X) pg/nil)
40 ±7 2±5*
LTB., values are given in mean values ±SD (ng/107 neutrophils) for three inde pendent experiments. * p
Medizinische Mikrobiologie und Immunologie, AG Infektabwehrmechanismen, Ruhr-Universität Bochum, FRG; Department Clinical Microbiology, Rigshospitalet, Copenhagen, Denmark
Alginate - Its Role in Neutrophil Responses and Signal Transduction towards Mucoid Pseudomonas aeruginosa Bacteria
Key Words Alginate Cystic fibrosis Proteinkinase C G proteins P aeruginosa
Abstract Mucoid Pseudomonas aeruginosa bacteria impaired neutrophil functions, e.g. chemiluminescence response, and leukotriene formation to a significantly higher degree as compared to nonmucoid P. aeruginosa bacteria. To study the cell biological requirements for the different cellular response pattern by mu coid and nonmucoid (NM) P aeruginosa bacteria, further experiments were performed with purified alginate, the mucoid exopolysaccharide of P. aerugi nosa (MEP). In this regard the MEP (alginate) significantly reduced the zymo san-induced leukotriene B4 (LTB4) formation (from 40±7 to 2± 4 ng). The chemiluminescence response induced by NM bacteria was abolished when the bacteria were prccoatcd with the MEP. Mucoid and NM P. aeruginosa bacteria interacted with components of the cellular signal transduction pathway to a different degree. Mucoid bacteria induced a 2-fold enhanced GTPase activity but activated the protein kinase C (PKC) to a lesser degree than NM P. aerugi nosa bacteria. Prior exposure of neutrophils to the MEP increased the sodium fluoride (NaF)-induced GTPase activity and guanylylimidodiphosphate bind ing [Gpp(NH)p] by approximately 60 and 30%, respectively. The phorbol myristic acid-induced PKC activation was inhibited by 30-40% in the presence of the MEP. However, the MEP by itself was inactive in all assay systems. Our re sults indicate that the MEP represents an important component which mod ulates neutrophil responses of mucoid as compared to NM P. aeruginosa bacte ria, e.g. the chemiluminescence response, LTB4gencration, and the interaction with components (G proteins. PKC) of the signal transduction pathway.
Introduction
severe burns, cancer, or receiving immunosuppressive therapy [1, 2], Basically, the occurrence of such diverse clinical diseases may cither derive from an altered host defense in these patients and/or a different virulence of taxonomically identical P. aeruginosa strains [2-4].
Pseudomonas aeruginosa is an opportunistic pathogen which causes either localized infections such as pneumo nia in patients with cystic fibrosis (CF) or generalized sep ticemia in immunocompromised hosts suffering from
Correspondence to: Dr. W. König I.ehrstuhl für Med. Mikrobiologie und Immunologie AG Infcktabwehrmechanismcn Ruhr-Universität Bochum. Univcrsitätsstt. 150 D-W-4630 Bochum (FRG)
© 1992 S. Karger AG. Basel 1018-2438/92/0991-0098 $ 2.75/0
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
B. Königa P. FriedP S.S. Pedersenb W. Königa
as leukotrienes from human neutrophils [20, 21]. In addi tion to PKC the G proteins are involved in the complex events of cellular responses [21, 22], Guaninc-nucleotidebinding proteins (G proteins) appear to represent a family of regulatory proteins, all of which serve to transfer in formation from receptor to effector systems of the cell [22, 23]. This paper deals with the question of the suppressive effects of mucoid strains of P aeruginosa and in particular with the question of soluble, purified alginate, the MEP, on neutrophil functions (chemiluminescence, leuko trienes) and on the mechanism of the alginate-induced ef fects on the cellular signalling pathway, e.g. the involve ment of G proteins, PKC. Here, we report that the MEP of mucoid P. aeruginosa is responsible for the inhibitory ef fects on leukotriene release and chemiluminescence re sponse from human neutrophils by mucoid P. aeruginosa strains. We also show that the MEP interacts with compo nents of the signal transduction pathway in a distinct way and that the MEP represents the component which is re sponsible for the altered signal transduction pathway seen in human neutrophils after stimulation with mucoid P ae ruginosa strains as compared to nonmucoid P. aeruginosa strains.
Materials and Methods Materials [gamma-32 P]ATP (5 Ci/mmol) was purchased from New England Nuclear, DE-52 cellulose was from Whatman. Synthetic leukotrienes were a generous gift from Mcrck-Frosst, Pointe Claire, Canada. The solvents used were of high pressure liquid chromatography grade ob tained from local suppliers. The MEP from P. aeruginosa was pre pared by Pedersen as described previously [161. BacteriaI Strains P. aeruginosa from CF patients were isolated from sputa of pa tients suffering from acute exacerbations of pulmonary infections. The strains were identified by their ability to synthesize fluorescent pigments on Pseudomonas agar P and F (Merck, Darmstadt, FRG) and by using the api 20 NE identification system (BIOMerieux, Niirtingen. FRG). Strains CF4 and CF14 showed a nonmucoid growth pattern: strains CF9 and CF7 expressed a mucoid phenotype. Buffer The buffer used for washing the neutrophils and for mediator re lease consisted of 137 mM NaCI. 8 mM Na:H P 0 4. 3 mM KC1. and 3 mM KCI, and 3 mM KH,PQ4, pH 7.4 (modified Dulbecco’s phos phate-buffered saline, PBS). Buffer A consisted of 20 mM Tris-HCI (pH 7.5), 0.5 mM EGTA, 0.5 mM EDTA. I mM dithiothreitol, and I mM PMSF.
99
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
We have recently shown that clinical isolates from burn patients induce inflammatory mediator release from mast cells and neutrophil granulocytes [5]; the release of media tor substances correlated with the expression of the en zymatic (phospholipase C) or the nonenzymatic (glycolipids) hemolysins from P. aeruginosa. In addition. Meyers and Berk [6] also showed that soluble phospholipase C in duced I.TB4 formation from human granulocytes. In con trast P. aeruginosa strains isolated from CF patients in duced only insignificant levels of inflammatory mediators from human neutrophils [unpubi. data]. In the past we demonstrated that mucoid unlike nonmucoid bacteria led to a defined modulation of the neutrophil responses, e.g. a decrease in the chemiluminescence response, a reduced leukotriene B4 (LTB4) formation on subsequent cellular stimulation as well as changes on the C3bi receptor ex pression [7]. However, these studies did not prove the role of alginate. A predominant feature of P. aeruginosa in CF is the production of alginate, the mucoid exopolysaccharide (MEP) [8-11]. Alginate has been described to exhibit sev eral features such as an increased binding of P. aeruginosa to epithelial cells, inhibition of phagocytosis of mucoid Pseudomonas bacteria by human granulocytes as well as by macrophages, inhibition of the chemotaxis of granulocytes to the inflammatory area, and inhibition of opsonization of bacteria [12-15]. Nonetheless, up to now there has been no explanation for the fatal outcome of a chronic P. aerugi nosa infection in CF as well as the local destruction of the lung tissue [16]. Neutrophils play a crucial role in host de fense mechanisms [17]. Upon stimulation with microbial pathogenicity factors neutrophils are triggered for the production of O5 metabolites, the release of enzymes and of arachidonic acid metabolites such as LTB4 [18]. LTB4 is strongly chemotactic for neutrophil granulocytes. The generation and the appropriate control of inflammatory mediator release, e.g. the generation of arachidonic acid metabolites and oxygen radicals is a prerequisite for a nor mal host defense. Alginate has been made responsible for a variety of pathological cellular immune functions. In this regard, it has been reported by Mai ct al. [19] that the MEP interferes with a broad range of neutrophil functions, in cluding adherence, locomotion, oxygen metabolism, de granulation, and bactericidal activity. Cellular responses are mediated by a complex signal transduction cascade which is dependent on the stimulus and the cell type. Substantial evidence was obtained for the fact that protein kinase C (PKC) played an essential role in the cellular activation process leading to reactive oxygen metabolites and mediators of inflammation such
Preparation o f Cells Human neutrophils were prepared from heparinized blood (15 U/ ml) of healthy donors and separated on a Ficoll metrizoate gradient followed by dextran sedimentation [24], The cells were washed at 300 # to remove the platelets. The erythrocytes were lysed by exposing the cells to hypotonic conditions. This method results in more than 97% pure neutrophils. Cell Viability’ Cell viability was tested by trypan blue exclusion as well as by the analysis of lactate dehydrogenase (LDH) release from stimulated and nonstimulated cells. Analysis of LDH (EC 1.1.1.27) was carried out as described previously [21). Chemiluminescence Oxygen-dependent respiratory activity was measured as de scribed earlier [21]. In brief: neutrophils (107150 pi) in Ca/Mg (1/0.5 mM) were incubated with bacteria (50 pi) in the presence of 40 pi of 0.25 mM luminol. In several experiments the neutrophils were preincubated with bacteria for 20 min at 37°C and subsequently stimu lated with PMA ( 1 0 M) for a further 20 min. Leukotriene Release from Human Neutrophils The assay was performed as described previously [21]. In prcincubation experiments neutrophils were treated with alginate for the in dicated time intervals and then stimulated with opsonized zymosan. Analysis o f Leukotriene Release Cell supernatants were analyzed for leukotrienes as described [21], 2 ml of methanol-acetonitrile (50:50, vol/vol) were added to the culture supernatants. After centrifugation at 1,900# for 15 min (Cryofuge 6-4 Heraeus Christ) the supernatants were evaporated to dry ness by lyophilization (Modulyo, Edwards-Kniesc, Marburg. FRG). The residues were dissolved in 600 pi of methanol/water (30:70) and 200 pi were analyzed by reversed-phase high pressure liquid chroma tography. The column was packed (4.6x200 mm) was packed with Nueleosil Cls (particle 5 pm: Macherey-Nagcl. Diiren, FRG). HPLC equipment consisted of a CM4000 pump, a SM4000 detector (both Laboratory Data Control/Milton Roy, Hasselroth. FRG), and an au tomatic sample injector (WISP 7I0B, Waters, Eschborn. FRG). Leukotriencs were analyzed using a mobile phase consisting of methanol/ water/acctonitrile/phosphoric acid (48:24:28:0.03, v/v) including 0.04% EDTA and 0.15% K ,H P 04. pH 5.0. The flow rate was main tained at 0.9 ml/min. The A ^ of the column effluent was determined. Quantification and identification of leukotrienes were performed with synthetic standard solutions. LTB., generation was calculated as the combined amounts of LTB4 and the LTB4 omega-oxidation prod ucts (20-hydroxy-LTB4 and 20-carboxy-LTB4). PKC Assay Human neutrophils (4 xl()7) were prcincubated for 10 min at 37 °C in PBS with Ca/Mg. The cells were subsequently incubated with the indicated stimuli. Activation of the cells was stopped by centrifu gation at 1,000g for 10 min: the cells were then resuspended in I ml
mo
buffer A, and sonicated (amplitude 50 pm, sonificr 250, Branson Power Company, USA) for 2 x 10 s. After centrifugation of the crude homogenate at 9,700# for 10 min the cytosolic fractions were chroma tographed on DEAE-cellulose columns (0.5x3 cm), equilibrated and washed with buffer A (3 ml). The protein kinase C was eluted with 1.5 ml of buffer A containing 100 mM NaCI. The reaction mixture (190 pi) contained 20 mM Tris-HCI (pH 7.5). 10 mM magnesium ace tate, 5(H) pg/ml histone type 11IS. 10 pA/ [gamma- >:P]ATP (10' cpm/ nmol), 2.5 pg diolein, 25 pg phosphatidylserine, 1 mM CaCL, and the enzyme preparation under study. All reagents were dissolved in dis tilled water. The quantitation of background phosphotransferase ac tivity was performed by analyzing histone phosphorylation in the ab sence of calcium and phospholipid. After 10 min at 30 °C. the reaction was stopped by the addition of 0.75 ml of trichloroacetic acid (25%). Acid-prccipitable materials were collected on Millipore membrane filters (0.45-pm pore size, Milliliter HA filtration plates. Millipore, Eschborn. FRG). The filters were washed with 2x1 ml of trichloro acetic acid (10%). The dried filters were transferred into scintillation vials, distilled water was added and the radioactivity was measured by Ccrcncov radiation (Rack beta 1209, LKB, Turku, Finnland). Protein kinase C activity was calculated by substracting background activities, values are reported as percent of cytosolic protein kinase C activity from untreated cells. Binding o f Guanylylimidodiphosphate and GTPase Activity Human neutrophils were incubated in the presence of alginate, NaF, bacterial suspensions, or in the absence of any stimulus for 10 min at 37 °C. In preincubation experiments, human neutrophils were first treated with buffer or alginate: then NaF was added and the stimulation of the cells proceeded for a further 10 min. The assay was performed as described previously 121]. Binding of guanylylimido diphosphate [Gpp(NH)p] was expressed in cpm bound to the membranes. GTPase activity was reported as [,:P] released from [gamma-':P]GTP. Statistics Data show mean values ± standard deviation (SD) of at least three individual experiments with cells from different donors. Signif icance was examined with the Student t test for independent means.
Results Inflammatory’ Mediator Release Effect o f Alginate on the Chemiluminescence Response. In a first set of experiments we analyzed the role of the MEP on the bacteria-induced chemiluminescence re sponse from human neutrophils. For this purpose nonmu coid R aeruginosa bacteria (CF14; CF4) were cultured in the absence or presence of alginate (MEP) (1 mg/ml) for the last hour of growth at 37 °C and were then used as stimulus. As is apparent from figure 1 for strain CF4, algi nate-coated bacteria of both P. aeruginosa strains induced a chemiluminescence response from human neutrophils to a lesser degree as compared to the nonmucoid bacteria. In subsequent experiments neutrophils were preincubated
König/Fricdl/Pedersen/König
Pseudomonas aeruginosa Alginate and Neutrophil Function
Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/4/2018 10:07:21 PM
Bacterial Growth Bacterial growth was carried out in peptone medium (1% NaCI, 1% peptone, 1% glycerol). The broth (10 ml) was inoculated with 100 ul of an overnight culture. Bacterial growth proceeded on a shaker (180 rpm) at 37 °C for the indicated times.
Table 1. Effect of alginate on the zymosan-induced LTB, generation
Neutrophils preincubated with
LTB,
Buffer (control) Alginate (1(X) pg/nil)
40 ±7 2±5*
LTB., values are given in mean values ±SD (ng/107 neutrophils) for three inde pendent experiments. * p
