F-met-leu-phe Research Articles

Novel method for assessing basophil activation by measuring altered expression of membrane-bound and intracellular basogranulin stores

Background Basophil activation tests can provide valuable information on allergic sensitivity to a range of different allergens. The most widely employed methods involve flow cytometric detection of increased expression of membrane-bound CD63 or CD203c following addition of allergen to basophils in vitro. The identification of basogranulin, a unique basic protein stored in basophil granules, has opened the way for new basophil activation methods to be explored. Methods Blood was collected from healthy subjects and patients with a history of allergy to food, drugs, house dust and grass pollen. Basophils were stimulated with specific allergen, anti-IgE antibody, or the peptide f-met-leu-phe (FMLP). Flow cytometry was performed with non-permeabilised and permeabilised cells with antibody specific for basogranulin (BB1) or CD63, and data analysed with CellQuest software. Results Flow cytometry with permeablised cells indicated depletion of intracellular stores of basogranulin following basophil activation. Associated with basogranulin release was the presence of increased quantities of this marker on the basophil membrane following cellular activation. Increased membrane expression of basogranulin mirrored that for CD63; and with allergens and other stimuli tested the measurement of cell surface basogranulin represented a more sensitive means for assessing basophil activation in vitro. The flow cytometric assays for basogranulin were optimised for use with samples of whole blood so as to avoid the need for basophil purification. Conclusions The rapidity, simplicity and sensitivity of basogranulin-based methods for measuring basophil activation will facilitate their application to clinical samples and allow better assessment for allergic sensitivity.

ARACHIDONIC ACID CORRELATION WITH SUSPENDED AND ATTACHED NEUTROPHILS

Background: Neutrophils are the blood cells for which adhesion is one of the main functions. Neutrophils adhered to biological surfaces show different characteristics of activation compared with neutrophils in suspension. The respiratory burst in suspended neutrophils in response to a chemotactic factor is lower when compared with the cells attached to the surface. Objectives: This study aimed to study the role of arachidonic acid release in suspended and attached neutrophils following stimulation of human neutrophils with LPS and PAF. Materials and methods: The suspended and attached neutrophils were primed by LPS and PAF, and inhibited by Anti-CD 14 (MY4). Results: The arachidonic acid release in attached cells is more than in suspended cells. This release is rapid with the increase of incubation time and dose-depended. The Fmet-Leu-Phe (FMLP) potentiates arachidonic acid release in attached cells pretreated with LPS and serum. Arachidonic acid release was less in suspended cells in comparison to attached cells where the addition of low concentration of LPS in presence of serum or platelet activating factor (PAF) to suspended cells produce increase in arachidonic acid release. On the other hand LPS in combination with PAF produce a large and significant increase in arachidonic acid release. The potentiative effect of LPS is mediated via CD14 receptors. The monoclonal antibodies against CD14 had no effect on arachidonic acid release in PAF treated cells where it inhibited greatly the potentiation by LPS or LPS-serum complex with PAF. Conclusion: LPS or PAF alone produced small increase in arachidonic acid release in both suspended and attached human neutrophils, where LPS in combination with PAF induced significant arachidonic acid release in suspended and attached human neutrophils.

Inhibitory effects of N-acetylcysteine on superoxide anion generation in human polymorphonuclear leukocytes.

It has been suggested that reactive oxygen species released by activated polymorphonuclear leukocytes (PMN) in man is one mechanism of tissue injury. Therapeutic action aimed at increasing antioxidant defence mechanisms is still a clinical challenge. This study examines the activity of N-acetylcysteine, a known antioxidant, in the protection of PMN exposed in-vitro to the chemoattractant peptide fMet-Leu-Phe (FMLP), the protein kinase C activator phorbol myristate acetate or the lipid peroxidation promoter t-butyl hydroperoxide. FMLP (3-300 nM) and phorbol myristate acetate (160 pm-160 nM) induced concentration-related superoxide anion generation. Pre-treatment with N-acetylcysteine (33-333 microM) resulted in concentration-related inhibition of superoxide production induced by FMLP (30 nM) or phorbol myristate acetate (16 nM);-log IC50 values were 3.97 +/- 0.07 and 3.91 +/- 0.10, respectively. Changes in intracellular calcium ion concentration ([Ca2+]i) induced by FMLP (30 nM) were studied in fura-2-loaded human PMN. FMLP produced a transient calcium response, i.e. a peak followed by decay to a residual value above baseline. N-Acetylcysteine (333 microM) did not affect either basal [Ca2+]i values or changes in [Ca2+]i values after treatment with FMLP. Activation by phorbol myristate acetate caused a reduction in glutathione levels from 5.94 +/- 0.86 (control) to 1.84 +/- 0.51 nmol/3 x 10(6) cells (P < 0.05 compared with control). Pre-treatment with N-acetylcysteine (333 microM) fully reversed the reduction in glutathione levels induced by phorbol myristate acetate (4.83 +/- 0.68 nmol/3 x 10(6) cells; P > 0.05 compared with control). Exposure to t-butyl hydroperoxide (0.5 mM, 30 min) markedly increased malondialdehyde levels (from 0.03 +/- 0.02 to 0.73 +/- 0.07 nmol/10(6) cells), and index of lipid peroxidation. Malondialdehyde levels were significantly reduced in PMN treated with N-acetylcysteine (333 microM; 0.55 +/- 0.04 nmol/10(6) cells; P < 0.05 compared with untreated cells exposed to t-butyl hydroperoxide). In conclusion, N-acetylcysteine reduces superoxide generation in response to FMLP and phorbol myristate acetate and partially protects against lipid peroxidation in PMN from man. The protection afforded by N-acetylcysteine is not related to alteration of the intracellular calcium signal but might be effected by replenishment of the intracellular glutathione levels.

A Potential Shift From Adaptive Immune Activity to Nonspecific Inflammatory Activation Associated With Higher Depression Symptoms in Chronic Heart Failure Patients

Background Chronic heart failure (CHF) patients with elevated depression symptoms are at greater risk of morbidity and mortality. The mechanisms linking symptoms of depression with disease progression in CHF are unclear. However, research studies have found evidence of alterations in immune activity associated with depression symptoms that may influence heart function. The present study sought to determine the relationship between depression symptoms and chemotaxis of peripheral blood mononuclear cells (PBMCs) in CHF patients, both at rest and in response to moderate exercise. Methods and Results Sixty-five patients diagnosed with CHF (mean age, 59.8 ± 14.5 years) and 45 non-CHF control subjects (mean age, 52.1 ± 11.6) completed the Beck Depression Inventory (BDI) before undergoing a moderate 20-minute bicycle exercise task. Chemotaxis of PBMCs was examined in vitro to a bacterial peptide f-met leu phe (fMLP) and a physiologic chemokine, stromal cell derived factor-1 (SDF-1) immediately before and after exercise. CHF patients had reduced chemotaxis to SDF-1 ( P = .025) compared with non-CHF subjects. Higher BDI scores were associated with reduced baseline chemotaxis to SDF-1 in both CHF and non-CHF subjects ( P = .027). In contrast, higher BDI scores were associated with increased chemotaxis to fMLP ( P = .049) and SDF-1 ( P = .018) in response to exercise in the CHF patients. Conclusion The present study suggests a shift in immune cell mobility in CHF patients with greater depression symptom severity, with reduced chemotaxis to a physiologically specific chemokine at rest but increased chemotaxis to both nonspecific and specific chemical attractants in response to physical activity. This could have implications for cardiac repair and remodeling in CHF patients and therefore may affect disease progression.

Involvement of protein kinase Cδ in the activation of NADPH oxidase and the phagocytosis of neutrophils

This experiment was performed to clarify the role of protein kinase C (PKC) δ in NADPH oxidase-dependent production and actin polymerization followed by phagocytosis in neutrophils. Bovine neutrophils and human neutrophil-like differentiated HL-60 (dHL-60) cells were stimulated with serum-opsonized zymosan (OZ) and fMet–Leu–Phe (fMLP), respectively. Rottlerin, a specific inhibitor of PKCδ, attenuated the production of from NADPH oxidase in both neutrophils and dHL-60 cells. However, it did not inhibit the translocation of p47phox from the cytosol to the membrane in either type of cell or the phosphorylation of p47phox in dHL-60 cells. GF109203X (GFX), an inhibitor of cPKC, attenuated not only the production of but also the translocation of p47phox in both cells. Furthermore, rottlerin significantly attenuated the ingestion of opsonized particles and the formation of F-actin in OZ-stimulated neutrophils, whereas, GFX did not affect those phagocytic processes. These results suggest that both PKCδ and cPKC regulate NADPH oxidase through different pathways, but only PKCδ regulates the phagocytic function in neutrophils.

Promotion of neutrophil adherence to human osteoblasts by microcrystals and f-Met–Leu–Phe

Human osteoblast-like cells (hOB) stimulated by monosodium urate monohydrate (MSUM) or calcium pyrophosphate dihydrate (CPPD) microcrystals produce the neutrophil chemoattractant IL-8. We investigated whether human neutrophils can adhere to hOB and respond to hOB preactivated by MSUM, CPPD, or by f-Met–Leu–Phe (fMLP). Confluent hOB were coincubated with human blood neutrophils in the presence of MSUM, CPPD or fMLP. MSUM, CPPD, and fMLP stimulated a significant adherence of neutrophils to hOB after a 1 h incubation. This effect was not abrogated by pretreating the cells with an anti-CD18 mAb. MSUM stimulated more efficiently the adherence of neutrophils to non-preactivated hOB while CPPD were more efficient when hOB were preactivated. Crystal-free conditioned media from MSUM- or CPPD-stimulated hOB mobilized intracellular free calcium in human neutrophils. Thus, microcrystals were powerful promoters of neutrophil adherence to hOB via a CD18-independent mechanism. The bacterial peptide fMLP also stimulated the adherence of neutrophils to hOB. Functional neutrophil–hOB interactions could be important in bone pathophysiology of crystal- or infection-associated arthritis.

The N-formylpeptide receptor (FPR) and a second G i-coupled receptor mediate fMet–Leu–Phe-stimulated activation of NADPH oxidase in murine neutrophils

N-Formylypeptides such as fMet–Leu–Phe (fMLF) potently induce superoxide production through NADPH oxidase activation. The receptors that mediate this response have not been defined. Here, we provide definitive proof using a mouse model that formyl peptide receptor (FPR) is a receptor, but not the only receptor, that mediates fMLF-induced oxidase activation. In wild-type (FPR +/+) mouse neutrophils, superoxide production is dependent on the concentration of fMLF with an EC 50 of ∼5 μM and a peak at ∼50 μM. In contrast, FPR-deficient (FPR −/−) mouse neutrophils produced markedly less superoxide with an EC 50 of ∼50 μM and a peak at ∼200 μM. Yet, FPR +/+ and FPR −/− neutrophils showed similar oxidase activation kinetics and G i protein-dependent pharmacological sensitivities. These results suggested that a second receptor, likely FPR2, mediates superoxide production at high concentrations of fMLF. This less sensitive second pathway may permit continued oxidant generation in response to formyl peptides when FPR is desensitized in high concentrations of the chemotactic gradient.

Lipopolysaccharide-induced priming of the human neutrophil is not associated with a change in phosphotyrosine phosphatase activity

The activation of the neutrophil respiratory burst is a two-step process involving an initial `priming' phase followed by a `triggering' event. The biochemical mechanisms which underlie these events are yet to be fully elucidated, but the evidence suggests a crucial role for stimulus-induced tyrosine phosphorylation. The enhanced tyrosine phosphorylation observed upon triggering primed cells may reflect an increase in tyrosine kinase activity or a reduction in the levels of the opposing phosphotyrosine phosphatases (PTPases). We have investigated the latter by examining the possibility that lipopolysaccharide (LPS)-induced priming of the neutrophil respiratory burst involves the suppression of cellular PTPase activity. Purified human neutrophils were incubated for 60 min with and without LPS. Priming of the respiratory burst was confirmed by fMet–Leu–Phe-induced cytochrome c reduction. The level of PTPase activity was assessed by dephosphorylation of [ 32P]RR- src peptide as substrate. Pretreatment of human neutrophils with 200 ng/ml LPS induced a 2.9±0.3 (mean±SEM, n=3, P=0.022) fold increase in the fMet–Leu–Phe-triggered respiratory burst. In the same cells, LPS did not induce a significant change in the total cellular PTPase activity (1.02±0.02-fold, mean±SEM, n=3, P=0.63). Similarly, stimulation of neutrophils with fMet–Leu–Phe or phorbol myristate acetate did not significantly affect the cellular PTPase activity ( P=0.94 and 0.68, respectively). Our results suggest that suppression of PTPase activity is not the mechanism underlying the priming and/or triggering of the neutrophil respiratory burst.

Role of Ca 2+-ATPase inhibitors in activation of cytosolic phospholipase A 2 in human polymorphonuclear neutrophils

In the present study, we investigated the involvement of Ca 2+-signaling and protein kinases in the effect of Ca 2+-ATPase inhibitors on the activation of cytosolic phospholipase A 2 (cPLA 2) in human polymorphonuclear neutrophils. We found that activity and mobility on electrophoresis gels of the cPLA 2 protein were significantly increased by f-Met–Leu–Phe (fMLP), 12-myristate 13-acetate (PMA) and the Ca 2+-ATPase inhibitors, thapsigargin and cyclopiazonic acid. This effect was completely suppressed by staurosporine. Calphostin C partially inhibited the fMLP- and PMA-induced cPLA 2 activation, but had no influence on thapsigargin- and cyclopiazonic acid-treated cells. Thapsigargin and cyclopiazonic acid also showed no effect on protein kinase C activity. However, the thapsigargin- and cyclopiazonic acid-induced cPLA 2 activation was completely inhibited by the tyrosine kinase inhibitor, erbstatin, and Ca 2+ chelator, EGTA. In addition, the cPLA 2 activity was reduced after pretreatment with the mitogen-activated protein kinase kinase inhibitor PD98059. The arachidonic acid release was significantly reduced in cells pretreated with the cPLA 2 inhibitor, AACOCF 3. Furthermore, we found that the human neutrophil cPLA 2 cDNA contain a Ca 2+-dependent-lipid binding domain which shares homology to several other enzymes such as protein kinase C and phospholipase C. Our results suggest that tyrosine kinases and the MAP kinase cascade are involved in Ca 2+-ATPase inhibitor-induced activation and phosphorylation of cPLA 2. Protein kinase C is not required in this event.

Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

The expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (&gt; 98% purity), PKCβΙ, βΙΙ, δ, and  were expressed, PKC was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and , respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKC was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.© 1998 by The American Society of Hematology.

Expression of Protein Kinase C Isozymes in Human Basophils: Regulation by Physiological and Nonphysiological Stimuli

AbstractThe expression of protein kinase C (PKC) isozymes in human basophils and the regulation of PKC isozymes during basophil activation by phorbol 12-myristate 13-acetate (PMA) ± ionomycin, f-met-leu-phe (FMLP), and anti-IgE antibody were examined. In human basophils (> 98% purity), PKCβI, βII, δ, and ε were expressed, PKCα was difficult to detect, and PKCγ and η were undetectable. In unstimulated basophils, PKCβI and βII were found primarily in the cytosol fraction (95% ± 3% of total and 98% ± 1%, respectively). Within 5 minutes of stimulation with PMA (100 ng/mL), both PKCβI and βII were translocated to the membrane fraction (85% ± 4% and 83% ± 6%, respectively). In resting cells, 48% ± 3% and 61% ± 10% of PKCδ and ε, respectively, existed in the membrane fraction. Within 1 minute of stimulation with PMA, 90% ± 6% of PKCα was found in the membrane fraction, however, no translocation of PKCδ was apparent. Stimulation with FMLP caused modest translocation (≈20%) of all PKC isozymes by 1 minute, whereas stimulation with anti-IgE antibody led to no detectable changes in PKC location throughout a 15-minute period of measurement. However, concentrations of PMA and ionomycin that alone caused no PKC translocation and little histamine release, together caused significant histamine release but no apparent PKC translocation. Studies with bis-indolylmaleimide analogs showed inhibition of PMA-induced, but not anti–IgE-induced, histamine release. These pharmacological studies suggest that PKC does not play a prodegranulatory role in human basophil IgE-mediated secretion.

The effect of interleukin-8 and granulocyte macrophage colony stimulating factor on the response of neutrophils to formyl methionyl leucyl phenylalanine

Neutrophils isolated from patients with chronic bronchitis and emphysema have been shown to have enhanced responses to formyl peptides when assessed in vitro compared to age, sex matched controls. It is currently unclear whether the observed differences are due to a ‘priming’ effect by a second agent in vivo, or whether this is a primary difference in the neutrophils. We have studied the effects of interleukin-8, which is thought to be one of the major pro-inflammatory cytokines in chronic lung disease and granulocyte macrophage colony stimulating factor (GMCSF), in order to assess their effects on neutrophil chemotaxis and connective tissue degradation. In addition, we have assessed the effect of preincubation of these agents with neutrophils for 30 min followed by stimulation with F-Met-Leu-Phe (FMLP) to investigate any possible ‘priming’ effect that may be relevant to our clinical data. We report suppression of neutrophil chemotaxis to FMLP following incubation of the neutrophils with both IL-8 and GMCSF. However, we have observed an additive effect of IL-8 and FMLP for neutrophil degranulation leading to fibronectin degradation. The results suggest that IL-8 does not ‘prime’ neutrophils for subsequent FMLP stimulation as observed in vivo. Although the results for GMCSF were similar for the chemotactic response, the agent also had a synergistic effect on connective tissue degradation. However, it is concluded that neither agent could explain the enhanced neutrophil responses seen in our patients.

RANTES is a pro-inflammatory chemokine and chemoattracts basophil cells to extravascular sites.

RANTES (regulated upon activation normal T expressed and secreted) is another member of the intercrine beta subfamily which acts as a selective chemoattractant for human monocytes and CD4-positive lymphocytes and increases the adherence of monocytes to endothelial cells. In this work, the effect of RANTES was studied on rat skin injection sites. Rats were intradermally injected with 50 microliters of RANTES, at different concentrations, fMet-Leu-Phe (FMLP), or LPS (positive controls) or PBS vehicle (negative control). The animals were then injected with 0.6 ml of Evans' blue in the tail vein in order to obtain a blue colour in the areas where the compounds were injected. After 4 h the rats were killed and the maximum diameter of the blue extravasation area was measured. The coloured areas were then excised and optical and electron microscopic studies were performed. In addition, in some of the excised tissue, a Northern blot analysis for histidine decarboxylase (HDC) mRNA was performed along with an estimation of the amount of histamine generated in the tissue injection sites. In these studies it was found that intradermal injections of 5, 2.5, and 1.25 x 10(-5) M RANTES produced a strong inflammatory response with the accumulation of a great number of basophil cells compared with the PBS (50 microliters) negative control, or FMLP (10(-6) M/50 microliters) or LPS (10 ng/50 microliters) positive control, after 4 h. Moreover, 5, 2.5, 1.25 x 10(-5) M RANTES produced a dose-response stimulation of HDC mRNA in the tissues of skin injection sites. The increasing number of basophils in the RANTES inflamed tissues led to augmentation of histamine content, compared with the PBS control. In conclusion, the pro-inflammatory chemokine RANTES stimulates the generation of HDC mRNA in skin injection sites.

Inhibition by pulmonary surfactant Curosurf of secretory phospholipase A2 expression in guinea-pig alveolar macrophages

Replacement therapy with exogenous surfactant has been proven successful in animal models of acute respiratory distress syndrome (ARDS). Here, we investigated the effect of seminatural surfactant Curosurf ® on the expression of secretory phospholipase A2 (sPLA2) in guinea-pig alveolar macrophages (AM). The latter produced an sPLA2 activity whose level was markedly reduced when culture medium was supplemented with Curosurf ®. This effect was concentration-dependent and was accompanied by a decrease in sPLA2 mRNA levels. By contrast, when AM were first cultured for 20 hr and then incubated with Curosurf ®, no significant change was observed in their sPLA2 activity. Finally, f-Met-Leu-Phe (FMLP)-induced thromboxane B 2 release from AM was not altered by Curosurf ®, indicating that the inhibition of sPLA2 expression cannot be attributed to a nonspecific membraneous effect of Curosurf ®. These findings show that pulmonary surfactant modulates the expression of sPLA2 in AM and suggest that this effect may account for the clinical efficacy of surfactant replacement therapy in ARDS.

Chemotactic Peptide-induced Activation of Ras in Human Neutrophils Is Associated with Inhibition of p120-GAP Activity

The monomeric G-protein Ras is now considered to function as an initial regulator of multiple signaling pathways in both normal and transformed cell types. Adhesion and chemoattractant receptors are known to trigger activation of Ras in human neutrophils, but the signaling mechanism that activates Ras has only been partially elucidated. The present results show that in neutrophils, a time- and dose-dependent f-Met-Leu-Phe (FMLP)-induced activation of Ras is mediated by Gi2-proteins, because such activation is inhibited by pertussis toxin and because direct stimulation of heterotrimeric G-proteins with AlF4- is sufficient to activate Ras. Pretreatment of neutrophils with tyrosine kinase inhibitors, i.e. genistein or erbstatin that completely block FMLP-stimulated protein tyrosine phosphorylations, did not affect the FMLP-induced activation of Ras. Moreover, FMLP did not induce any detectable translocation of Grb2 and Sos to the plasma membrane of neutrophils. Other signaling molecules, such as protein kinase C, phosphatidylinositol 3-kinase and Ca2+, do not appear to be involved in the FMLP-induced Ras activation. Instead, stimulation of neutrophils with FMLP or C5a, the latter of which also activates Gi2-proteins, resulted in transient inhibition of the activity of Ras GTPase-activating proteins (GAP) with kinetics that correlated well with the kinetics of Ras activation. Moreover, decreased Ras-GAP activity was found in p120-GAP but not in neurofibromin immunoprecipitates of FMLP-stimulated cells. These results suggest that tyrosine kinase-dependent Ras exchange factors do not contribute to the FMLP-induced activation of Ras but that such activation is mediated via inhibition of p120-GAP in neutrophils.

Endotoxin-Induced, Neutrophil-Mediated Endothelial Cytotoxicity Is Enhanced by T-Lymphocytes

The role of T-lymphocytes (T cells) and interferon-γ (IFN-γ) in the pathogenesis of sepsis-induced microvascular endothelial injury remains unclear. We sought to determine whether the syngeneic coculture of human T cells in the presence of LPS promoted subsequent neutrophil (PMN)-mediated endothelial cytotoxicity. Syngeneic T cells were cocultured with51Cr-loaded human adipose microvascular endothelial cell (HAMVEC) monolayers in the absence and presence of LPS. Subsequent PMN-mediated HAMVEC cytotoxicity (measured as percent specific51Cr release) was absent in cultures that contained T cells but no LPS and was significantly increased when T cells were cocultured in the presence of LPS. This was true both following addition of unstimulated PMNs (−0.8 ± 3.0% vs 4.9 ± 4.7% for T cells alone vs T cells plus LPS, respectively) and PMNs stimulated with f-Met–Leu–Phe (−0.4 ± 3.1% vs 10.7 ± 3.0% for T cells alone vs T cells plus LPS, respectively). Increased cytotoxicity was associated with increased expression of the endothelial adhesion molecules ICAM-1 and VCAM-1. Control experiments failed to demonstrate cytotoxicity when HAMVEC were cultured in the presence of IFN-γ alone, LPS alone, or T cells without LPS. It appears that there is a necessary requirement of both LPS and (presumably activated) T cells or their products (other than IFN-γ) for enhanced PMN-mediated endothelial cytotoxicity. This phenomenon may also be mediated by increased expression of endothelial adhesion molecules that promote subsequent PMN adhesion.

FMLP actions and its binding sites in isolated human coronary arteries.

The chemoattractant f-Met-Leu-Phe (FMLP) can modulate human coronary arterial tone without the involvement of peripheral leukocytes. We investigated the actions of FMLP and its cellular mechanism in human coronary arteries isolated 2-3 h after death. A single dose of FMLP (0.01-10 microM) produced transient contraction (or, followed by relaxation) responses in most human coronary rings examined. These responses to FMLP were in large part mediated by the generation of cyclooxygenase products, mainly thromboxane A2 (TXA2) and prostaglandin I2 (PGI2). Radiolabeled N-formyl hexapeptide. 125I-f-Nle-Leu-Phe-Nle-Tyr-Lys bound densely to intimal and adventitial sites that accumulated macrophages (CD68-positive) with a Kd of 14-29 nM and, further, weakly to the media with a Kd of 2.4-3.6 microM. Several cell types including macrophages, endothelial cells and smooth muscle cells were positively immunostained for both TXA2 synthase and PGI2 synthase. However, there was no significant relation between the magnitude of the responses to FMLP and dense macrophage accumulation in the intimal plaques or the adventitia. A reverse transcription-polymerase chain reaction showed predominant expression of FMLP receptor homologues, FPRH1 and FPRH2 mRNA, in human coronary medial tissues relative to that in leukocytes. In conclusion. FMLP produced transient tension changes in human coronary arteries, mainly via the generation of TXA2 and PGI2. This effect of FMLP did not appear to be mediated by the activation of densely accumulated intimal and/or adventitial macrophages, but by the activation of unidentified medial tissue cells which might have functional FMLP receptor homologues.

The role of receptors for tumour necrosis factor-α in the induction of human polymorphonuclear neutrophil chemiluminescence

Tumour necrosis factor-α (TNF-α) is a potent mediator of inflammation, which exerts profound effects on polymorphonuclear neutrophils (PMN). TNF-α binds to distinct cell surface receptors termed p55 and p75, expressed in approximately equal amounts on the PMN surface. We have studied the effects of TNF-α on the priming of F-Met-Leu-Phe (FMLP)-stimulated oxidative metabolism of PMN, using a luminol-enhanced chemiluminescence assay, and have examined the relative roles of PMN receptors for TNF-α in priming this oxidative metabolism, using antibodies with p55 and p75 receptor-specific agonistic and antagonistic activities. We have obtained the following results: (1) Antibody Htr-9 with agonistic activity at the p55 receptor mimicked the effect of TNF-α; however, a combination of Htr-9 and TNF-α did not result in any further increase in chemiluminescence relative to the response observed with TNF-α alone. The p75 agonistic antibody MR2-1 actually decreased basal and FMLP-enhanced chemiluminescence. Additionally, MR2-1 substantially inhibited the effects of both TNF-α itself and of the p55 agonist Htr-9. (2) Addition of antibodies with antagonistic activities at the p55 (antibody TBP-2) and p75 (antibody Utr-1) receptors resulted in a marked inhibition of the PMN response to TNF-α. A combination of both Utr-1 and TBP-2 was most effective at inhibiting the action of TNF. We have confirmed previously published observations that TNF-α alone effectively stimulates the oxidative metabolism of PMN in vitro, and that pre-incubation of PMN with TNF-α enhances subsequent generation of oxidative metabolites in response to FMLP. We conclude that both p55 and p75 receptors play a critical role in mediating the activation of PMN by TNF-α.

Phosphatidylinositol-3-kinase pathway is stimulated by fungal lipid containing cocoa butter equivalents (CBE) in FMet-Leu-Phe (FMLP)- and phorbolester (PMA)-challenged human neutrophils.

It is well established that fungi produce fats and oils, although a commercial utilization of these processes with rare exception could not yet be achieved. The reason hereto lied primarily on the high costs of production. Recently, a novel biotechnological process for the production of fungal lipids from Mucor circinelloides by using acetic acid (a cheap carbon source) at sub-toxical levels as the starting substrate has been described (1). The products obtained were oil- and butter-like compounds depending upon the dissolved oxygen tension maintained (Figure 1). The analyses of the lipid demonstrated a γ-linolenic acid (GLA)-rich lipid (so-called fungal oil) and a stearic acid-rich lipid (so-called fungal butter) (2). This first report of low cost production of high value lipids thus raised a caveat for more consideration.KeywordsCocoa ButterHuman NeutrophilDissolve Oxygen TensionCocoa Butter EquivalentMucor CircinelloidesThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Heat-Aggregated IgA Prepared from Patients with IgA Nephropathy Increases Priming of Human Neutrophils to Produce Inositol Triphosphate following FMet-Leu-Phe Stimulation in vitro

It is envisaged that circulating IgA complexes play a primary role in the glomerular injury of IgA nephropathy, the most common glomerulonephritis worldwide. In this study, we examined the pathophysiological effects of IgA and IgG isolated from IgA-nephritic patients on the signal transduction of human neutrophils. Heat-aggregated forms and monomers of IgA and IgG were prepared from sera of 11 IgA-nephritic patients and 11 healthy controls. Signal transduction was studied by measuring the inositol triphosphate (IP3) production in neutrophils incubated with the immunoglobulin preparations. Different forms of IgA or IgG from IgA-nephritic patients failed to induce a significant increase in IP3 production directly as compared with control IgA or IgG. However, neutrophils preincubated with heat-aggregated IgA (HAA) from IgA-nephritic patients demonstrated a significant rise in IP3 production upon subsequent stimulation by a chemotactic peptide, FMet-Leu-Phe (FMLP); a similar finding was not observed with heat-aggregated IgG. HAA pretreatment of neutrophils increased FMLP-induced IP3 production in a dose-dependent manner. The raised IP3 production was not due to increased FMLP receptors, as HAA preincubation of neutrophils did not increase the binding of tritiated FMLP. The increased IP3 production upon FMLP stimulation in HAA-primed neutrophils was completely abolished by pertussis toxin in a dose-dependent manner. These findings tend to refute a direct stimulatory effect of HAA on phospholipase C, but, instead, may suggest that HAA prepared from IgA-nephritic patients upregulates the activation of G proteins in the plasma membrane.(ABSTRACT TRUNCATED AT 250 WORDS)