Absence Of fMLP Research Articles

Leukadherin-1 ameliorates endothelial barrier damage mediated by neutrophils from critically ill patients

BackgroundMulti-organ failure occurs during critical illness and is mediated in part by destructive neutrophil-to-endothelial interactions. The β2 integrin receptor, CR3 (complement receptor 3; Mac-1; CD11b/CD18), which binds endothelial intercellular adhesion molecule-1 (ICAM-1), plays a key role in promoting the adhesion of activated neutrophils to inflamed endothelia which, when prolonged and excessive, can cause vascular damage. Leukadherin-1 (LA-1) is a small molecule allosteric activator of CR3 and has been shown to promote adhesion of blood neutrophils to inflamed endothelium and restrict tissue infiltration. Therefore, LA-1 offers a novel mechanism of anti-inflammatory action by activation, rather than inhibition, of the neutrophil CR3 integrin. However, whether promotion of neutrophil-to-endothelial interaction by this novel therapeutic is of benefit or detriment to endothelial barrier function is not known.MethodsCritically ill septic and trauma patients were prospectively enrolled from the surgical and the trauma ICU. Blood was collected from these patients and healthy volunteers. Neutrophils were isolated by dextran sedimentation and adhered to TNF-α (tumor necrosis factor-α)-activated human umbilical vein endothelial (HUVEC) monolayers in the presence or absence of fMLP (formylmethionine-leucine-phenylalanine) and/or LA-1. Electric cell-substrate impedance sensing (ECIS) and exposure of underlying collagen were used to quantify endothelial barrier function and permeability.ResultsNeutrophils from critically ill trauma and septic patients caused similar degrees of endothelial barrier disruption which exceeded that caused by cells obtained from healthy controls both kinetically and quantitatively. LA-1 protected barrier function in the absence and presence of fMLP which served as a secondary stimulant to cause maximal loss of barrier function. LA-1 protection was also observed by quantifying collagen exposure underlying endothelial cells challenged with fMLP-stimulated neutrophils. LA-1 treatment resulted in decreased migration dynamics of neutrophils crawling on an endothelial monolayer with reduced speed (μm/s = 0.25 ± 0.01 vs. 0.06 ± 0.01, p < 0.05), path length (μm = 199.5 ± 14.3 vs. 42.1 ± 13.0, p < 0.05), and displacement (μm = 65.2 ± 4.7 vs. 10.4 ± 1.3; p < 0.05).ConclusionNeutrophils from patients with trauma or sepsis cause endothelial barrier disruption to a similar extent relative to each other. The CR3 agonist LA-1 protects endothelial barrier function from damage caused by neutrophils obtained from both populations of critically ill patients even when exposed to secondary stimulation.

Inhibition of Glycan Shedding from the Endothelial Glycocalyx and Adhesion of Leukocytes by Doxycycline in Post‐capillary Venules

Previous studies have demonstrated that shedding of glycans from the endothelial cell (EC) glycocalyx and leukocyte (WBC)‐EC adhesion in response to the chemoattractant fMLP could be attenuated by doxycycline (Doxy) through the inhibition of matrixmetalloproteases (MMPs). Given that Doxy is a strong calcium chelator, the present studies were undertaken to determine if the calcium chelator EDTA could produce a similar effect. To this end, WBC‐EC adhesion and adhesion of lectin (BS‐1) coated microspheres (0.1 um diameter) in post‐capillary venules of mesentery (rat) were studied during superfusion of the tissue with Ringers solution plus EDTA in the presence or absence of fMLP. In the absence of fMLP, with 1 mM EDTA sequestration of microspheres steadily increased with time, and with 3 mM sequestration reached levels 50–100% greater than without EDTA. In contrast, with 0.5 uM Doxy alone, microsphere adhesion remained constant under control conditions. Calcium chelation at levels (1 mM EDTA) that inhibited fMLP induced WBC adhesion to the same extent as Doxy (0.5 uM) caused a disruption of the venular permeability barrier that resulted in increased microsphere sequestration in the venular wall. Thus, the inhibition of microsphere shedding and WBC adhesion in response to fMLP by doxycycline appear to be caused by inhibition of MMPs in contrast to calcium chelation. Supported by R01 HL39286.

Modification of membrane markers on THP-1 cells after ozone exposure in the presence or absence of fMLP

The impacts of ozone and N-formyl-methionyl-leucyl-phenylalanine (fMLP) on the detection of membrane markers on non-differentiated THP-1 cells were evaluated for in vitro exposures. Several markers, specific for monocytes and macrophages, were identified on the THP-1 cells, allowing their use as a model for alveolar macrophages. Ozone exposure modified not only the detection of membrane markers, especially CD13 and CD14, monocyte and macrophage markers, but also the detection of the specific receptor for fMLP, formyl peptide receptor (FPR). Activation by fMLP also reduced the detection of the CD antigens, and a combined exposure to ozone and fMLP amplified this decrease, probably due to an additive effect of these chemicals. Overall, these results suggest important membrane rearrangements for short-term treatments to ozone and/or fMLP.

Leukocyte spreading behavior on vascular biomaterial surfaces: consequences of chemoattractant stimulation

Chemoattractant-induced phenomena of polarity and migration of polymorphonuclear leukocytes (PMN) are believed to play a key physiological role in controlling bacterial infections on implantable vascular biomaterials. Our study targeted the spreading behavior of human PMN adherent to expanded polytetrafluoroethylene (ePTFE), pretreated with various plasma proteins, in response to the chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMLP). To this end, a novel imaging configuration was developed to allow in situ reconstructive analysis of PMN 3-D morphology on opaque ePTFE surfaces, using optical sectioning confocal microscopy. Following fMLP stimulation, PMN morphological polarity was enhanced on all substrates studied except fibrinogen treated ePTFE. 3-D PMN morphometry revealed that in the absence of fMLP, overall cell spreading was minimized on albumin-treated ePTFE and maximized on fibrinogen and immunoglobulin-G-treated ePTFE. Following fMLP stimulation, overall PMN spreading increased markedly on untreated and albumin-coated ePTFE, while it stayed invariant on IgG and plasma treated ePTFE, and decreased on fibrinogen-treated ePTFE. Spatial analysis of PMN spreading following fMLP stimulation revealed enhanced PMN attachment on untreated and albumin treated ePTFE and diminished attachment on fibrinogen and plasma treated ePTFE. Thus, chemoattractant stimulation altered a wide range of PMN spreading attributes on ePTFE, including morphological polarity, substrate attachment, and 3-D membrane spreading, in a substrate dependent manner. These chemoattractant-induced spreading responses may also have important consequences for PMN phagocytosis. We report that fMLP stimulation led to enhanced unopsonized particulate phagocytosis on untreated and albumin treated ePTFE, but caused no discernible change in phagocytosis on other protein substrates. Thus, chemoattractant modulation of PMN spreading on ePTFE is highly substrate-regulated, and manifests in concerted effects on PMN phagocytosis.

Effect of three polychlorinated biphenyls on f-met-leu-phe-induced degranulation in rat neutrophils

It has been known that polychlorinated biphenyl (PCB) mixtures and individual congeners produce degranulation of rat neutrophils. Structure–activity relationships for congeners PCB 8 (2,4′-dichlorobiphenyl), PCB 126 (3,3′,4,4′,5-pentachlorobiphenyl) and PCB 128 (2,2′,3,3′,4,4′-hexachlorobiphenyl) were examined by correlating the extent of degranulation and cytotoxicity with molecular and physico-chemical parameters. Neutrophils were exposed to PCB congeners and then to the neutrophil activator f-met-leu-phe (fmlp). Degranulation and cytotoxicity were quantified by measuring released myeloperoxidase and lactate dehydrogenase activities, respectively. Degranulation in the absence of fmlp, that is in quiescent neutrophils, was detected only for PCB 8. Inhibition of fmlp-induced degranulation was observed for both PCB 8 (50 μM) and PCB 128 (10 and 50 μM). PCB 126 did not affect degranulation of quiescent or fmlp-stimulated neutrophils. Thus, effects on degranulation were observed only for ortho-substituted congeners. Cytotoxicity was observed under all conditions with PCB 8, in quiescent neutrophils with PCB 128, and in activated neutrophils with PCB 126. Structure–activity relationships revealed that effects of PCBs on neutrophil degranulation correlate with the energy of the lowest unoccupied molecular orbital but not with torsional angle 2,1,1′,2′. This study demonstrates the importance of molecular, electronic parameters in PCB-induced effects on neutrophil degranulation.

Induction of beta2 integrin-dependent neutrophil adhesion to human alveolar epithelial cells by type 1 Streptococcus pneumoniae and derived soluble factors.

Pneumonia caused by Streptococcus pneumoniae is characterized by neutrophil infiltration and variable epithelial injury. Neutrophil adhesion to alveolar epithelial pneumocytes (A549) was measured and demonstrated to be dose-dependent following preincubation of these (A549) pneumocytes with type 1 S. pneumoniae. Adhesion peaked at a bacteria-to-epithelial cell ratio of 5:1 after a 4-h incubation but was absent after 2 h and without FMLP. Filtered conditioned media (CM) from pneumococci cultured with (CM+) or without (CM-) epithelial cells were tested. CM+ induced significant adhesion in the absence of FMLP (P < .001); CM- had no effect. In the presence of FMLP, adhesion induced by both media was significantly greater than by FMLP alone (P < .001) and was significantly blocked (P < .01) by antibodies to CD11b and CD18. CM+ upregulated epithelial intercellular adhesion molecule 1 but CM- did not. These data provide new information concerning the interactions of S. pneumoniae, alveolar epithelial cells, and neutrophils.

Chemoattractant-induced respiratory burst: increases in cytosolic Ca2+ concentrations are essential and synergize with a kinetically distinct second signal.

The role of the cytosolic free Ca2+ concentration ([Ca2+]c) and its relationship to other second messengers in the signalling between chemoattractant [e.g. N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP)] receptors and the NADPH oxidase is still poorly understood. In this study, we have used thapsigargin, an inhibitor of the Ca2+-ATPase of intracellular stores, as a tool to selectively manipulate Ca2+ release from intracellular stores and Ca2+ influx across the plasma membrane. We thereby temporarily separated the Ca2+ signal from other signals generated by fMLP and analysed the consequences on the respiratory burst. Under all conditions investigated, the extent of fMLP-induced respiratory burst activation was critically determined by [Ca2+]c elevation. fMLP was unable to activate the respiratory burst without [Ca2+]c elevation. Thapsigargin-induced Ca2+ influx activated the respiratory burst in the absence of fMLP, but only to approx. 20% of the values observed in the presence of fMLP. The second signal generated by fMLP did not activate the respiratory burst by itself, but acted in synergy with [Ca2+]c elevation. The second signal was long lasting (>15 min) provided that there was no rise in [Ca2+]c and that the receptor was continuously occupied. The second signal was inactivated by high [Ca2+]c elevation. Our results demonstrate that [Ca2+]c elevations are an essential step in the signalling between the fMLP receptor and NADPH oxidase. They also provide novel information about the properties of the second Ca2+-independent signal that activates the respiratory burst in synergy with [Ca2+]c.

Farnesyl-L-cysteine analogs can inhibit or initiate superoxide release by human neutrophils

A series of farnesylcysteine analogs was studied with respect to their abilities to interfere with fMet-Leu-Phe (fMLP)-stimulated superoxide (O2-.) release by human neutrophils. Simple acyl derivatives of farnesyl-L-cysteine, such as the N-acetyl (L-AFC) and N-isobutyryl derivatives (L-iBFC), which are substrates for the isoprenylated protein methyltransferase, can block O2-. release. The N-butyryl analog (L-BFC), which is an isomer of L-iBFC and also a substrate for the methyltransferase, does not inhibit O2-. release but actually stimulates it in the absence of fMLP. Other analogs, including the N-pivaloyl derivative, which has been found to be neither a substrate nor an inhibitor of methyltransferase, also stimulate very large quantities of O2-. production. The stimulatory effects of these derivatives are saturable and exquisitively sensitive to small structural changes in the analogs. The signal transduction pathway(s) utilized by pivaloyl derivatives for triggering O2-. generation is very similar to that employed by fMLP. These data make it clear that farnesyl-L-cysteine analogs do not produce their pharmacological effects in neutrophils via methyltransferase blockade. This could be further demonstrated by showing that sinefungin and S-adenosylhomocysteine, both powerful and general methyltransferase inhibitors which bind at the S-adenosylmethionine site, had no effect in preventing the increased oxygen consumption associated with O2-. production in permeabilized neutrophils. These studies reveal that farnesyl-L-cysteine analogs interact with a hitherto undefined target in neutrophils that may be exploited for inhibiting or stimulating the inflammatory or antimicrobial responses of these cells.

Phosphorylation down-regulates the store-operated Ca2+ entry pathway of human neutrophils.

We have reported previously that the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) inhibits transiently Ca2+ entry through the plasma membrane Ca2+ pathway activated by emptying the intracellular Ca2+ stores (Montero, M., García-Sancho, J., and Alvarez, J. (1993) J. Biol. Chem. 268, 13055-13061). We show here that calyculin A and okadaic acid, inhibitors of protein phosphatases 1 and 2A, prevent the spontaneous reversion of the fMLP-induced inhibition of the entry of Ca2+ and Mn2+ (used as a Ca2+ surrogate), leading to a permanently inhibited Ca2+ entry pathway. At high concentrations or long incubation times the phosphatase inhibitors were even able to inhibit the store-operated Ca2+ entry pathway (SOCP) in the absence of fMLP. Inhibition of SOCP by phorbol dibutyrate, which is not reversible, was not modified by phosphatase inhibitors. These results provide additional support for the view that fMLP inhibits SOCP through phosphorylation of either the SOCP protein or a regulatory protein and indicate that dephosphorylation mediated by protein phosphatases 1 and/or 2A restores the activity of SOCP after inhibition by fMLP. The time course of the inhibition of SOCP by fMLP was similar to the one reported previously for the transient fMLP-induced phosphorylation of a 47-kDa protein involved in the generation of respiratory burst, which was similarly affected by the phosphatase inhibitors.

Agonist-dependent phosphorylation of N-formylpeptide and activation peptide from the fifth component of C (C5a) chemoattractant receptors in differentiated HL60 cells.

Attenuation of signaling is a key step in controlling the cytotoxic potential of leukocyte responses to chemotactic factors. Antipeptide antibodies, directed against the N-formyl chemotactic peptide receptor (FPR) and the activation peptide from the fifth component of C (C5a) anaphylatoxin receptor (C5aR) of human neutrophils, were used to analyze the ability of these receptors to be phosphorylated. Our data show that, in granulocyte-like differentiated HL-60 cells, both FPR and C5aR undergo an agonist dose-dependent phosphorylation that reaches completion in less than 2 to 3 min, consistent with the rate and the dose-dependent attenuation of signaling in phagocytes. Therefore, phosphorylation might be one of the possible mechanisms involved in the desensitization process of FPR and C5aR. Addition of either C5a or the protein kinase C activator (PMA) did not appear to induce the phosphorylation of FPR in the absence of FMLP or to modulate the phosphorylation of the latter at low concentrations of agonist. In contrast, although FMLP at a saturating concentration barely stimulated the phosphorylation of unoccupied C5aR, it markedly potentiated C5aR phosphorylation in cells exposed to low concentrations of C5a. Moreover, PMA was able to induce C5aR phosphorylation in the absence of agonist, indicating that protein kinase C or protein kinase C-activated kinase(s) could be involved in the phosphorylation of C5aR. Pretreatment of cells with staurosporine, a potent but nonspecific inhibitor of protein kinase C, resulted in the partial inhibition of both FPR and C5aR phosphorylation induced by saturating concentrations of agonist, suggesting that a kinase different from protein kinase C might be mainly responsible for the phosphorylation of these chemotactic receptors.

Functional behaviour of mononuclear blood cells from patients with hypercholesterolemia

Mononuclear cells were prepared from venous blood obtained from 20 patients with a newly diagnosed hypercholesterolemia and without clinical signs of vascular disease, and from 19 age and sex matched controls. Adhesiveness to plastic surface, phagocytic activity measured as ingestion of zymosan particles, and spontaneous motility of mononuclear cells from patients were significantly higher by 57 %, 19 % and 50 %, respectively, when compared to controls. In controls chemotaxis induced by the chemotactic peptide FMLP was slightly higher than spontaneous motility measured in absence of FMLP, whereas in patients FMLP significantly inhibited cell motility by about 47 %. With the exception of FMLP-induced chemotaxis the results indicate that mononuclear cells are hyperreactive in hypercholesterolemia.

6 MERCAPTOPURINE (6MP) REDUCES FORMYL-METHIONYL-LEUCYL-PHENYLALANINE (fMLP) INDUCED MACROMOLECULAR UPTAKE IN RAT ILEUM

Increased ileal permeability may play a role in the pathogenesis of Crohn's disease (CD). fMLP, a common bacterial peptide, initiates inflammation and increased permeability in the rat ileum via neutrophil activation and chemotaxis. We investigated the effect of 6MP often used to treat CD, upon fMLP-induced changes in ileal macromolecular uptake. METHODS: Using in situ ileal perfusion, permeability to 0.5%. horseradish peroxidase (HRP), ± simultaneous luminal perfusion of 10 −5M fMLP, was measured by blood levels over 90 min, in each of 3 groups of male rats: Group I = daily 48 mg 6MP m−2 for 14 days; Group II = vehicle-injected, pair fed controls; Group III = ad libitum controls. RESULTS: In the absence of fMLP, HRP uptake was low in all Groups (90 min blood HRP <37 ± 5 units)(mean ± SE). With fMLP, HRP uptake was markedly elevated over basal in rats not receiving CMP: Group II = 210 ± 12; Group III = 263 ± 24 units, (p<0.01). Conversely, 6MP treatment (Group I) ameliorated the fMLP response (106 ± 8 units, p<0.01). 6MP therapy decreased the circulating neutrophil count, but did not alter mucosal myeloperoxidase, a neutrophil marker. CONCLUSION: 6MP therapy decreases macromolecular uptake induced by fMLP. SPECULATION: In CD, CMP may be therapeutic through diminished neutrophil inflammatory response and reduced antigen uptake.

Activation of electropermeabilized neutrophils by adenosine 5′-[γ-thio]triphosphate (ATP[S]). Role of phosphatases in stimulus-response coupling

Electrically permeabilized human neutrophils were used to study the mechanism of activation of the NADPH oxidase by chemotactic factors. The respiratory burst elicited by formyl-methionyl-leucyl-phenylalanine (fMLP) was strictly dependent on the addition of ATP. The response was also supported by adenosine 5'-[gamma-thio]triphosphate (ATP[S]), but not by the non-hydrolysable analogue (p[NH]ppA). In the presence of ATP, displacement of fMLP from its receptor by antagonist peptides resulted in the abrupt termination of the O2-consumption burst. In contrast, the response persisted after displacement of fMLP when ATP[S] was present. This finding is consistent with the formation of biologically active thiophosphoproteins which are resistant to cleavage by cellular phosphatases. Accordingly, lower concentrations of ATP[S], as compared with ATP, were required to support the fMLP response. The data indicate that protein phosphatases control the extent and duration of the response in cells stimulated with chemoattractants. Unlike ATP, sub-millimolar concentrations of ATP[S] elicited a spontaneous respiratory burst in the absence of fMLP or other stimuli. This effect was inhibited by p[NH]ppA and was not observed in intact (non-permeabilized) cells, indicating interaction of ATP[S] with an intracellular adenine-nucleotide-binding site, possibly a protein kinase. These results suggest that protein kinases are active in neutrophils in the absence of exogenous stimuli, but that accumulation of the essential phosphoprotein(s) is normally prevented by the ongoing vigorous phosphatase activity. It is conceivable that control of the respiratory burst is exerted by inhibition of phosphatase activity, instead of or in addition to the more commonly postulated activation of protein kinases.

Migration of neutrophils across monolayers of cultured microvascular endothelial cells. An in vitro model of leucocyte extravasation.

Monolayers of bovine microvascular endothelial cells (BMECs) grown on connective tissue derived from human amniotic membrane were used to examine the transendothelial migration of human neutrophils in vitro. Neutrophils placed above these cultures migrated in response to a chemotactic gradient generated by placing 10(-7) M-formyl-methionyl-leucyl-phenyl-alanine (fMLP) below the cultures. Under these conditions, an average of 29 +/- 12% of the total population of neutrophils migrated beneath the endothelium after 1 or 2 h of incubation. Neutrophil migration in the absence of fMLP or in the presence of equal concentrations of fMLP above and below the cultures was less than 8% of the response to a 10(-7) M-fMLP gradient. Migration was a rapid event. Neutrophils began adhering to the apical surface of the endothelium within 2 min following exposure to an fMLP gradient; Ca2+ was required for this initial adhesion. Within 10 min, the majority of neutrophils associated with the BMEC-amnion cultures had migrated beneath the endothelial monolayer. Ultrastructural studies revealed that the initial adhesion between migrating neutrophils and endothelium was characterized by close contact between the two types of cell in focal areas. This close association was maintained as the neutrophils traversed the clefts between endothelial cells. Following their migration across the endothelium, neutrophils often were observed lying between the endothelium and its basement membrane. With time, the neutrophils penetrated the basement membrane and moved into the underlying amniotic connective tissue. To test the role of neutrophil proteinases in breaching endothelial and subendothelial barriers, migration was allowed to proceed in the presence of a variety of proteinase inhibitors, including p-nitrophenyl p'-guanidinobenzoate, soybean trypsin inhibitor, 6-aminocaproic acid, alpha 1-proteinase inhibitor, leupeptin, antipain and methoxysuccinyl alanine-alanine-proline-valine chloromethyl ketone. None of these had a significant effect on the number of neutrophils that migrated or the depth to which they penetrated the amniotic tissue as compared with controls. In contrast, pepstatin and chymostatin reduced migration in response to fMLP to 7% and 52% of control values, respectively. However, these two inhibitors did not affect migration in response to another chemoattractant, leukotriene B4. Migration was neither enhanced nor inhibited by the following treatments: (1) removal of plasminogen from the calf serum used in the assay medium and addition of polyclonal antibody to plasminogen; (2) addition of monoclonal or polyclonal antibody to plasminogen activator.(ABSTRACT TRUNCATED AT 400 WORDS)

Intracellular pH modulates the generation of superoxide radicals by human neutrophils.

The relationship of intracellular pH (pHi) to superoxide radical (O2-) generation was investigated in chemotactic factor-stimulated human neutrophils. Exposure of cells to 100 nM N-formylmethionyl-leucyl-phenylalanine (FMLP) caused activation of Na/H exchange which, in 140 mM Na medium (pH0 7.40), led to a rise in pHi from 7.22 to 7.80. This pHi change was sensitive to amiloride (apparent Ki 78 microM), an inhibitor of Na/H countertransport. The time course of the alkalinization was similar to that of FMLP-stimulated O2- production, which was complete by 5 min. In the presence of 1 mM amiloride, which nearly blocked the pHi transient elicited by FMLP, or in the absence of external Na, where intracellular acidification was observed in FMLP-stimulated cells, O2- release was still roughly 25-45% of normal. Thus, an alkalinization cannot be an obligatory requirement for O2- generation. By independently varying either pH0, pHi, or the internal or external concentrations of Na, both the direction and magnitude of the FMLP-induced pHi transients could be altered. In each instance, the amount of O2- release correlated directly with pHi and was enhanced by intracellular alkalinization. In the absence of FMLP, a rise in pHi to 7.7-7.8 by exposure of cells to 30 mM NH4Cl, 10 microM monensin (a Na/H exchanging ionophore), or after a prepulse with 18% CO2 did not result in O2- generation. Thus, these results imply that an alkalinization per se is not a sufficient trigger. Neutrophils exposed to 4 nM FMLP exhibited a threefold slower rate of alkalinization (reaching pHi approximately 7.80 by 20-30 min) as compared to that obtained with 100 nM FMLP and did not release significant amounts of O2- under normal incubation conditions. However, these cells could be induced to generate O2- when the degree of alkalinization was enhanced by internal Na depletion or by pretreatment with 18% CO2. Together, these results indicate a modulating effect of pHi on O2- production and suggest that other functional responses of neutrophils may be regulated by their pHi.