Effect of phorbol myristate acetate on processing of formyl peptide receptors by human neutrophils.

We examined the effect of phorbol myristate acetate on the ability of human neutrophils to process formyl peptide receptors. The receptor was affinity-labeled and its extracellular localization assessed over time, by cleavage of extracellular labeled receptor with papain. Neutrophils were capable of internalizing (and/or recycling) affinity labeled formyl peptide receptor in the absence of extracellular calcium. This phenomenon was dependent upon stimulation with phorbol myristate acetate, suggesting a role for protein kinase C in this process.

Investigations on the role of Golgi-mediated, ligand-receptor processing in the activation of granulocytes by chemoattractants: differential effects of monensin.

Human granulocytes were exposed to different concentrations of the ionophore monensin for 20 min at 37 degrees C. Subsequent exposure to 50 nM of the chemoattractant fMet-Leu-[3H]Phe for up to 30 min at 37 degrees C resulted in a receptor-mediated uptake that was inhibited 80% at a monensin concentration of 30 microM. 50% inhibition was observed at 1-10 microM monensin with no significant change in fMet-Leu-Phe dose dependency. Subcellular fractionation of cells treated with monensin, indicated that the low density UDP-galactosyltransferase activity associated with internalized receptor-fMet-Leu-Phe complexes in untreated cells was absent. The high density galactosyltransferase activity cosedimenting with specific granule markers, however, was unaffected. Monensin also inhibited chemotaxis toward fMet-Leu-Phe as measured by migration of granulocytes through millipore filters and fMet-Leu-Phe induction of polarized morphology. Incubation of cell suspensions with up to 30 microM monensin, both before and during measurement of fMet-Leu-Phe stimulated superoxide production, did not affect the magnitude, kinetics, or transiency of the radical generation. Monensin did, however, shift the dose dependency of superoxide production of fMet-Leu-Phe to higher concentrations. These differential effects of monensin suggest that endocytosis of complexes of the chemoattractant and receptor is not involved in the activation or termination of the fMet-Leu-Phe stimulated superoxide production. They also are consistent with a role for receptor modulation and processing in the chemotactic response.

A high-yield, high-purity elutriation method for preparing human granulocytes demonstrating enhanced experimental lifetimes.

A counterflow centrifugal elutriation procedure for preparing human granulocytes is described. This method permits isolation of 1-2 X 10(9) granulocytes with a yield of 70% +/- 7% from a unit of whole blood in 3 h. These cells are 99% +/- 1% pure and 95-99% viable. When compared to cells prepared by a conventional procedure employing hypotonic lysis to remove red cells, the elutriated cells show enhanced oxidative responses resulting from enhanced longevity. Other responses including receptor-mediated uptake of chemoattractant were not significantly different from those of conventionally prepared cells after incubation at 37 degrees C prior to stimulation. The population of elutriated granulocytes, however, was significantly more homogeneous and pure than cells prepared conventionally.

Priming induces functional coupling of N-formyl-methionyl-leucyl-phenylalanine receptors in equine neutrophils.

The synthetic formylpeptide fMLP is widely used as a model chemoattractant and secretagogue for mammalian neutrophils. Despite possessing fMLP receptors, equine neutrophils do not produce superoxide anions in response to fMLP and there is no inflammatory reaction in the horse when fMLP is injected intradermally. The functional capability of these receptors was investigated after pretreatment with recognized priming agents. Purified neutrophils were pretreated with lipopolysaccharide (LPS), platelet-activating factor (PAF), or tumor necrosis factor alpha (TNF-alpha) and superoxide anion generation and shape change quantified by lucigenin-dependent chemiluminescence (LDCL) and flow cytometry, respectively. LPS, TNF-alpha, and PAF pretreatment induced significant LDCL in response to fMLP; similarly LPS pretreatment was a prerequisite for fMLP-stimulated neutrophil polarization in response to fMLP. However, LPS failed to induce fMLP-mediated chemotaxis of equine neutrophils. These data indicate that equine neutrophil fMLP receptors are not vestigial as previously thought but can trigger both respiratory burst activity and cell polarization responses after priming.

Modulation of human neutrophil adherence by beta-endorphin and met-enkephalin.

Early events in an acute inflammatory response include the adherence of neutrophils (PMN) to capillary endothelial cells and the migration of these cells into tissues. This study was designed to determine if the opioid peptides beta-endorphin or met-enkephalin would induce a concentration-dependent increase in PMN adherence to serum-coated glass. Results show that PMN adherence is increased with both beta-endorphin and met-enkephalin and this increase may be partially blocked by the opiate antagonist naloxone. Binding of opioid peptides to the formyl peptide receptor was ruled out as a mechanism of increased adherence by showing that the opioids failed to block the binding of f-met-leu-phe-lys to PMN. These studies suggest that alterations in circulating opioid concentrations may modulate the adherence of PMN and thereby influence acute inflammatory reactions.

Diclofenac binding to human polymorphonuclear neutrophils: effect on respiratory burst and N-formylated peptide binding.

The respiratory burst of human polymorphonuclear neutrophils (PMN) induced by particle or soluble stimuli was measured in the presence of the nonsteroidal anti-inflammatory drug, diclofenac sodium (Voltaren). Diclofenac (25-100 micrograms/ml) inhibited the oxygen consumption of PMN stimulated by 5 X 10(-7) M of N-formyl-methionyl-leucyl-phenylalanine (FMLP). The inhibition was linearly correlated to diclofenac concentration. By contrast, diclofenac did not affect the rate of heat-killed Klebsiella pneumoniae ingestion of PMN, or the PMN O2-uptake induced by (0.67 microgram/ml) serum-opsonized zymosan or (1 microgram/ml) phorbol myristate acetate (PMA). The PMN production of superoxide anion induced by various FMLP concentrations (10(-7), 10(-6) and 10(-5) M) was also decreased by diclofenac. However, this inhibition declined when the formylated peptide concentration was raised suggesting that diclofenac could alter FMLP binding to the PMN membrane. Binding experiments of tritiated FMLP to intact PMN performed at 22 degrees and 4 degrees showed high- and low-affinity FMLP sites with dissociation constant (Kd) values of approximately 2 X 10(-8) M and 10(-5) M respectively. Diclofenac did not significantly alter the low-affinity component but induced modifications of the high-affinity component which were different at 22 degrees and 4 degrees. At 22 degrees only the dissociation constant value was enhanced by diclofenac (competitive inhibition) whereas at 4 degrees both binding parameters (i.e. dissociation constant and number of available binding sites) were modified (mixed inhibition). Diclofenac was also shown to bind to PMN with a low affinity. This binding was not diminished at 4 degrees by various concentrations of FMLP which even increased the number of diclofenac binding sites on PMN at 22 degrees. These data suggest that diclofenac binding to PMN may decrease FMLP-induced PMN respiratory burst by interfering with the peptide recognition by specific FMLP receptors.

Endotoxemia and enhanced generation of oxygen radicals by neutrophils from patients undergoing cardiopulmonary bypass.

Plasma endotoxin concentrations and oxidative burst response of peripheral blood polymorphonuclear leukocytes were examined in 12 patients undergoing coronary artery bypass. The measurements were made just before the operation, 5 minutes after removal of the aortic crossclamp, and 24 hours after the operation. Endotoxin was quantitated by a combination of a sensitive Limulus amebocyte lysate assay and rocket immunoelectrophoresis measuring picogram amounts of endotoxin. Peripheral blood neutrophils were purified by a two-step dextran sedimentation and metrizoate sodium Ficoll (Lymphoprep., Nyegaard, Oslo, Norway) centrifugation. The oxidative burst response of these cells was measured for their ability to generate superoxide anion and was determined by a cytochrome c reduction assay. Preoperatively, all the plasma samples except one were free of endotoxin. The endotoxin levels reached 100 pg/ml 5 minutes after removal of the aortic crossclamp, and except in one sample they had decreased 24 hours after the operation. Studies on the generation of superoxide by neutrophils showed a decline in the response 5 minutes after removal of the aortic crossclamp and an enhancement of the response to f-Met-Leu-Phe by cells obtained from 11 of 12 patients 24 hours postoperatively. In vitro addition of bacterial lipopolysaccharide to blood from healthy individuals also enhanced the superoxide response of the neutrophils. We conclude that during cardiopulmonary bypass the circulating blood is contaminated by endotoxin and the neutrophils are primed for enhanced generation of oxygen radicals. The released oxygen radicals may be involved in the tissue damage observed in these patients.

Altered chemotactic peptide binding in tobacco smokers' neutrophils.

The functions of polymorphonuclear leukocytes from tobacco smokers are altered compared to those from nonsmokers. Since neutrophil chemotaxis and oxidative metabolism are mediated by surface receptors, we studied the association of the chemotactic peptide formyl Met-Leu-Phe with neutrophils from smokers and non-smokers. An apparently single class of binding sites was observed in neutrophils from all the non-smokers, whereas upwardly curving Scatchard plots were obtained for binding to smokers' cells. Thus changes at the receptor level may be responsible for the previously observed alterations in smoker neutrophil function.

Study of human serum albumin structure by dynamic light scattering: two types of reactions under different pH and interaction with physiologically active compounds.

The effect of pH and binding of ten physiologically active compounds (isoproterenol, yohimbine, propranolol, clonidine, phenylephrine, carbachol, tripeptide fMLP, diphenhydramine, chlorpromazine and atropine) on the molecular structure of human serum albumin (HSA) has been studied using the dynamic light scattering. It was found that albumin globule has the most compact configuration (Stokes diameter 59-62 A) at physiological pH 7.4. The changes in pH, both increase to 8.0 and decrease to 5.4, result in the growth of globule size to 72-81 A. At acidic shift of pH an additional peak arises in the correlation spectra caused by the light scattering on the structures with the Stokes diameters of 29-37 A. Those conform to the sizes of the albumin subdomains. The indicated peak is not displayed at basic shift of pH. The interaction with propranolol, clonidine, phenylephrine, carbachol and tripeptide fMLP which hinder adenylate cyclase (AdC) and activate Ca-polyphosphoinositide (Ca-PPI) signaling system of a cell initiates structural rearrangements similar to acidic transitions. Isoproterenol, yohimbine diphenhydramine, chlorpromazine and atropine, which activate AdC and hinder Ca-PPI, cause conformational changes of HSA similar to basic transitions.

Effects of tenoxicam on superoxide anion formation, beta-glucuronidase release and fMLP binding in human neutrophils: comparison with other NSAIDs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are considered to exert their activity by interfering with the generation of arachidonate metabolites in various cells, mainly in neutrophils and monocytes. The inhibition of cellular cyclooxygenase enzyme, however, does not always correlate with the in vivo activity of these drugs. Recent evidence indicates that several NSAIDs may interfere with the stimulus-response coupling of inflammatory cells. In this study, the effects of tenoxicam, an oxicam derivative with a thienothiazine structure, on neutrophil activation were evaluated by the assessment of the following parameters: (1) superoxide anion generation by neutrophils and whole blood stimulated with N-formyl-methionyl-leucyl-phenylalanine (fMLP), the calcium ionophore A23187 and serum treated zymosan (STZ); (2) beta-glucuronidase release from neutrophils stimulated with fMLP, A23187 and STZ; (3) binding of [3H]fMLP to intact neutrophils. The results were compared to those obtained using piroxicam and diclofenac. Tenoxicam, added in vitro to whole blood, at concentrations ranging between 10(-5) and 3 x 10(-4) M, significantly inhibited the generation of superoxide anion induced by fMLP, A23187 and STZ. The activity of tenoxicam on whole blood was similar to that of piroxicam, whereas diclofenac had only minimal effects on this experimental system. In isolated cells tenoxicam inhibited the generation of superoxide anion induced by A23187 and STZ. In addition, at the 3 x 10(-4) M concentration, tenoxicam and diclofenac similarly inhibited O2- generation by neutrophils stimulated with fMLP, whereas piroxicam only minimally affected this parameter. Tenoxicam also slightly, but not significantly, inhibited beta-glucuronidase release by isolated neutrophils induced by all the agonists used. Specific binding of [3H]fMLP to neutrophils was inhibited by the three NSAIDs tested in a dose-dependent fashion and tenoxicam was the most potent. The affinities (Kd) of tenoxicam, piroxicam and diclofenac were 1.11, 1.80 and 2.70 x 10(-5) M, respectively. The mechanism of inhibition of [3H]fMLP binding by tenoxicam was non-competitive. It is concluded that tenoxicam, at concentrations achievable in plasma at steady state, effectively inhibits some of the processes involved in neutrophil activation, which bear some relevance in the inflammatory disease.

Inhibition of chemotactic factor-activated Na+/H+ exchange in human neutrophils by analogues of amiloride: structure-activity relationships in the amiloride series.

The ability of a number of analogues of the diuretic, amiloride, to inhibit chemotactic factor-stimulated Na+/H+ exchange in human neutrophils was investigated. Intracellular pH (pHi) changes were measured from the equilibrium distribution of 14C-labeled 5,5-dimethyloxazolidine-2,4-dione (DMO). Exposure of cells to 10 nm N-formyl-methionyl-leucyl-phenylalanine (FMLP) caused activation of Na+/H+ exchange: in 140 mM Na+ medium (extracellular pH 7.40), the pHi rose from a resting value of approximately 7.25 to reach a new steady state of approximately 7.75 by 10-15 min. This intracellular alkalinization was sensitive to amiloride (apparent Ki approximately 75 microM), a known inhibitor of Na+/H+ countertransport. The structure-activity relationships in the amiloride series were characterized by testing the effect of these compounds on the DMO-derived pHi changes and on the FMLP-stimulated rate of 22Na+ efflux from the cells. Substitutions of the guanidino group of amiloride resulted in relatively inactive products (Ki greater than or equal to 1 mM). Replacement of the 6-Cl group of amiloride by other halogen atoms had only modest effects on drug efficacy. However, replacement of one or both H atoms of the 5-amino group by short alkyl groups led to a 10-500-fold increase in potency for inhibition of Na+/H+ exchange. Amiloride and three of its more potent derivatives (compounds I, O, and MM, the 5-N,N-dimethyl, 5-N,N-diethyl, and 5-N,N-hexamethylene analogues, respectively) caused parallel inhibition of FMLP-activated 22Na+ efflux and the rate of intracellular alkalinization, with apparent Ki values of approximately 75, 8, 1, and 0.2 microM, respectively. In each instance, the inhibitory effects of the drugs were readily reversible on washing the cells. None of the compounds altered the binding of 3H-labeled FMLP to its cell surface receptors. The development of potent derivatives of amiloride should provide powerful tools for assessing the role of FMLP-activated Na+/H+ exchange and the resultant pHi transients on stimulated neutrophil functions.

The role of 5-hydroperoxyeicosatetraenoic acid in neutrophil activation.

5-Hydroperoxyeicosatetraenoic acid (5HPETE) has been recently reported to play an important role in regulating and modulating neutrophil function. In order to clarify the mechanism of neutrophil activation by 5HPETE, we have measured the cytosolic free calcium, which is thought to be necessary for neutrophil activation using fura-2-loaded human neutrophils. Low concentration of 5HPETE, which is thought to be produced during cell activation, had minimal effect on cytosolic free calcium by itself but dose-dependently augmented FMLP-stimulated increase in cytosolic free calcium in the presence or absence of extracellular calcium without converting to LTB4. 5HPETE had no effect on 3H-FMLP binding to human neutrophils. The present data suggested that 5HPETE would augment FMLP-stimulated increase in cytosolic free calcium by enhancing the influx of extracellular calcium and/or the release of calcium from intracellular pool, which resulted in augmentation of neutrophil activation by primary agonist such as FMLP.

Extensive hydrolysis of N-formyl-L-methionyl-L-leucyl-L-[3H] phenylalanine by human polymorphonuclear leukocytes. A potential mechanism for modulation of the chemoattractant signal.

Chemoattractant receptors on human polymorphonuclear leukocytes (PMNs) stimulate a series of important biological responses. In an attempt to better understand the mechanism of stimulus response coupling of chemoattractant receptors, the kinetics of N-formyl-L-methionyl-L-leucyl-L-[3H]phenylalanine (fMet-Leu-[3H]Phe) binding to PMNs was evaluated. Unexpectedly, extensive degradation of the ligand was found to occur rapidly at 37 degrees C. Exposure of 10(7) cells/ml to 10 nM fMet-Leu-[3H]Phe led to the specific uptake of approximately 1% of the ligand within 10 min, while approximately 50% of the extracellular chemoattractant was hydrolyzed to free amino acids. Under the same conditions, isolated plasma membranes equivalent to 2.5 X 10(7) PMNs/ml bound specifically approximately 1% of the ligand and degraded about one-half of it primarily to Leu-[3H]Phe. The fMet-Leu-[3H]Phe hydrolysis commenced with no apparent latency, yet disobeyed first order kinetics as a 100-fold excess unlabeled ligand enhanced the initial consumption rate of 10 nM fMet-Leu-[3H]Phe by 500-fold, yielding an enhancement of the relative hydrolysis to approximately 70%. 1-butanol at 0.25%, which accelerates chemotaxis but inhibits superoxide anion and lysosomal enzyme secretion, reduced the hydrolysis to about 15% independent of the fMet-Leu-[3H]Phe specific activity. Lysosomal secretion could not mediate the hydrolysis process, since the supernatants of PMNs exposed either to 10 nM of 1 microM fMet-Leu-Phe reveal no degradation capacity toward fMet-Leu-[3H]Phe. These data indicate that the hydrolysis of the chemoattractant occurs at the cell surface and is dependent on the plasma membrane physical state. This phenomenon may well modulate the chemotactic signal due to its ability to profoundly alter the level of the chemoattractant proximate to the cell surface.

Anti-inflammatory lipocortin-derived peptides.

Peptide Ac2-26, drawn from the sequence of human lipocortin 1, inhibited the release of elastase activity from cytoplasmic granules of human neutrophils, and neutrophil adhesion to monolayers of endothelial cells, in a concentration-dependent manner (approximate IC50 of 100 micrograms/ml, 33 microM). The effect of peptide Ac2-26 was not restricted to a specific neutrophil activator, being effective against formyl-Met-Leu-Phe (FMLP), leukotriene B4 (LTB4) and platelet-activating factor (PAF). Peptide Ac2-26 did not alter FMLP binding to its receptor. These in vitro observations complement in vivo data obtained with this peptide and may enable a better understanding of its pharmacology and, perhaps, that of of lipocortin 1 too.

The interactive effects of fluoride and N-formyl-L-methionyl-L-leucyl-L-phenylalanine on superoxide production and cAMP levels in human neutrophils.

The kinetics of superoxide (O2-) production and intracellular cAMP levels were monitored in human neutrophils incubated in vitro with sodium fluoride and the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP). F- activation of both the O2- -generating system, NAD(P)H oxidase, and adenylate cyclase was characterized by a prolonged lag period of 8 to 10 min at 37 degrees C. Adenylate cyclase agonists or cAMP analogues which inhibited FMLP-induced O2- bursts did not affect O2- production of F- -activated cells. Prior treatment of cells with F- suppressed the short rapid burst elicited by FMLP but not the binding of the tripeptide. FMLP reciprocally decreased the lag period of the F- -induced burst by 40 to 50% and, in the case of cells incubated at temperatures below 37 degrees C, increased the rate of O2- production. A similar potentiating effect of FMLP on F- -induced elevation of intracellular cAMP levels was observed.

Neutrophil dysfunction in sepsis. III. Degranulation as a mechanism for nonspecific deactivation.

Prominent and global abnormalities in chemotactic, oxidative, and microbicidal activity have been identified in neutrophils from patients with severe sepsis. To evaluate the possible contribution of degranulation as the basis for the observed abnormalities, 12 patients with intrabdominal infection were serially studied and neutrophil chemotaxis, enzyme content, and receptors for FMLP were evaluated. There was a significant correlation between chemotactic response to both activated serum and FMLP with the granular enzymes beta-glucuronidase and lysozyme. For FMLP-directed migration, r = 0.73, P less than 0.001 for lysozyme, and r = 0.59, P less than 0.001 for beta-glucuronidase. There was a similarly significant correlation between loss of lysozyme and an increase in FMLP receptors, previously shown to be a marker for degranulation. These data support the concept that in vivo degranulation, possibly due to effects of circulating chemoattractants on adherent neutrophils, is responsible for the enzymatic and chemotactic loss seen in cells from septic patients. This hypothesis also provides a mechanism to explain the respiratory distress syndrome if degranulation were to occur in the pulmonary capillary bed.

Abnormal membrane fluidity as a cause of impaired functional dynamics of chemoattractant receptors on neonatal polymorphonuclear leukocytes: lack of modulation of the receptors by a membrane fluidizer.

Membrane properties associated with chemoattractant-mediated cellular responsiveness of neonatal polymorphonuclear leukocytes (PMN) were analyzed using n-formylmethionyl-leucyl-phenylalanine. Inasmuch as aliphatic alcohols as a membrane fluidizer can enhance the chemoattractant binding and affect subsequent cellular responsiveness in adult PMN, neonatal PMN were studied for such properties by their treatment with iso-propyl alcohol, an aliphatic alcohol. The alcohol (less than 2.5%) treatment enhanced the N-formylmethionyl-leucyl-phenylalanine binding to adult PMN, but there were no changes in the N-formylmethionyl-leucyl-phenylalanine binding to neonatal PMN. Although the N-formylmethionyl-leucyl-phenylalanine-induced subsequent responsiveness including migration, lysosomal enzyme release and superoxide anion production were modulated by the alcohol treatment in adult PMN, there was no such modulation in neonatal PMN. Because membrane fluidity is largely involved in the regulation of the receptor functions, the membrane fluidity of neonatal PMN was next measured by an excimer-forming lipid technique in flow cytometry. The membrane fluidity value (0.45 +/- 0.037) of neonatal PMN was lower than that (0.74 +/- 0.072) of adult PMN (p less than 0.01). Although the aliphatic alcohol enhanced the membrane fluidity of adult PMN, it did not affect the membrane fluidity of neonatal PMN. We conclude that there is abnormal membrane fluidity as a cause of impaired functional dynamics of the chemoattractant receptors, which appears to underlie the defective modulation of cell functions by the membrane fluidizer in neonatal PMN.

The effect of enkephalins and prostaglandins on O-2 release by neutrophils.

Derivatives of superoxide (O-2), produced by phagocytic cells, are thought to play a role in the adult respiratory distress syndrome (ARDS) and other disease states. Control of the release of O-2 may prove beneficial. Using human neutrophils as a source of O-2, and an assay for O-2 based upon the reduction of cytochrome C, we found that prostaglandin D2 (PGD2), leucine enkephalin (LE), and methionine enkephalin (ME) inhibited O-2 release. The Escherichia coli product, N-formyl methionyl leucyl phenylalanine (FMLP), was employed to stimulate O-2 release. PGD2 was most potent while there was no significant difference between LE and ME. Another peptide, thyrotropin releasing hormone (TRH), had no effect on O-2 release. There was no correlation between the potency of the inhibitory effect on O-2 release and the effect of these agents on the binding of [3H] FMLP to human neutrophils. Comparison of different but structurally related prostaglandins (PGD2, PGE2, and PGF2 alpha) revealed that PGD2 was more potent than PGE2 in inhibiting O-2 and that PGF2 alpha had no effect. This result suggested that the presence and position of the carbonyl group was an important determinant of the magnitude of inhibition.

Increased plasma chemoattractant in Sweet's syndrome.

The neutrophil function and plasma leukotactic activity of a patient with Sweet's syndrome and cystonodular acne were evaluated during a 2 1/2-year period. These studies demonstrated that chemotaxis was frequently slightly increased, especially during an exacerbation of Sweet's syndrome, but showed some decrease during isotretinoin therapy. Other functions, such as phagocytosis, metabolic activation, and bacterial killing, also were slightly increased. In addition, the patient's serum contained a heat-stable, nonlipid chemoattractant that was present at all times except during a course of isotretinoin. Although his symptoms responded to aspirin, the plasma continued to show this chemoattractant. These findings are consistent with the hypothesis that excess chemoattractant in Sweet's syndrome attracts neutrophils, which then mediate an inflammatory response. In addition, aspirin may be used to control Sweet's syndrome symptoms, although it does not suppress the plasma chemoattractant.

Directed neutrophil migration to IL-8 is increased in cystic fibrosis: a study of the effect of erythromycin.

BACKGROUND: The aim of this study was to compare neutrophil migration in cystic fibrosis (CF) and non-CF populations and to investigate the effect of erythromycin on directed migration of neutrophils (PMNs) in CF. METHODS: PMNs were isolated and their migratory capacity in response to interleukin-8 (IL-8) or f-Met-Leu-Phe (fMLP) in the presence or absence of erythromycin (1-100 microg/ml) was assessed. RESULTS: CF derived PMNs showed significantly increased migration to IL-8 but not to fMLP compared with non-CF PMNs. Erythromycin had no significant effect on migration responses to IL-8 and in vitro exposure of PMNs to erythromycin had no effect. CONCLUSIONS: CF derived PMNs show higher migratory responsiveness to IL-8 but not to fMLP, suggesting that CF PMNs may be "primed" to IL-8 which is significantly increased in CF serum compared with non-CF serum. Treatment with erythromycin had no significant effect on PMN migration in vitro.