Decreased affinity for a chemotactic factor in stored granulocyte concentrates

Previous studies in this laboratory demonstrated decreased migration of neutrophils after storage for 24 hours at room temperature. The current work was undertaken to identify a possible mechanism for decreased migration after storage. Initial studies ruled out the possibility that chemotaxis was decreased due to impaired ability to sense a chemotactic factor gradient since chemokinesis was decreased in addition to chemotaxis. Dose-response curves to the synthetic chemotactic agent Formyl-Methionyl-Leucyl-Phenylalanine (FMLP) showed decreased response to submaximal chemokinetic stimuli in stored neutrophils. This suggested the possibility of altered FMLP receptor binding in stored neutrophils. Neutrophil FMLP receptors were measured in 11 fresh and stored granulocyte concentrates. Although there was a small increase in total FMLP receptors per neutrophil after storage, the affinity of FMLP receptors in fresh neutrophils was significantly greater than that in neutrophils stored 24 hours at room temperature. Thus, decreased migration toward submaximal chemotactic stimuli in stored neutrophils may be due to altered membrane FMLP binding. However, decreased migration of stored neutrophils to maximal stimuli cannot be explained by altered FMLP binding affinity.     

Generation of superoxide radicals by human peripheral neutrophils activated by chemotactic factor. Evidence for the role of calcium.

In response to activation by the synthetic chemotactic factor FMLP, human peripheral neutrophils generated superoxide radicals as assessed by ferricytochrome C reduction. A dose-dependent increase in the amount of superoxide induced by FMLP over the concentration range of 1 X 10(-8) M to 1.6 X 10(-7) M was observed. Examination of the kinetics of the response revealed large amounts of superoxide generated by 1 min of incubation at 37 degrees C at an optimal dose of FMLP and a plateau effect after 5 min of incubation. Divalent cations did not influence the binding of 3H-FMLP to the cell, but superoxide generation by FMLP-activated neutrophils was observed to be dependent on the presence of divalent cations in the medium. In the absence of Mg2+, increasing Ca2+ ion concentration in the medium led to progressive increases in superoxide generation up to 4 mM, after which the response declined slightly. Mg2+, 0.25 to 4 mM, increased FMLP-induced superoxide generation to a much lower extent than did Ca2+. Lanthanum ion, 0.1 to 1 mM, in the presence of 1 mM Ca2+ inhibited the production of superoxide by FMLP 4 X 10(-8 ) M. Over the concentration range 3.3 X 10(-5 M to 3 X 10(-4 M, verapamil, a drug which selectively blocks the calcium channel, caused a dose-dependent inhibition of superoxide production and calcium-45 uptake in response to FMLP. This effect of verapamil could be overcome by increasing the concentration of Ca2+ in the medium. These observations suggest that a calcium influx plays an important role in the superoxide-generating capacity of the neutrophil.     

Failure of neutrophil chemotactic function in septic patients

OBJECTIVE: To investigate the in vitro chemotactic function of neutrophils obtained from patients with sepsis. DESIGN: Prospective study in which purified neutrophils obtained from septic patients and nonseptic control volunteers were assayed for chemotactic function induced by N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP) and leukotriene B4. The sera nitrate concentrations also were quantified. SETTING: University hospital. PATIENTS: Twenty patients with sepsis caused by different infectious foci. INTERVENTIONS: Routine blood tests, blood or other site cultures, blood collection for neutrophil purification sera collection for nitrate assay. MEASUREMENTS AND MAIN RESULTS: Neutrophils from septic patients exhibited significantly less chemotactic activity than neutrophils obtained from healthy volunteers, in response to FMLP (93.4 +/- 6.6 vs. 51 +/- 8.3 migrated neutrophils) and leukotriene B4 (90.2 +/- 10 vs. 42.4 +/- 11.6 migrated neutrophils) stimuli, in a microchemotaxis chamber assay. The impaired chemotaxis occurred mainly in neutrophils from nonsurvivor patients. The extent of neutrophil chemotaxis inhibition (survivor/nonsurvivor) was 33.43%/61.67% and 43.4%/86.98%, in response to FMLP and leukotriene B4, respectively. Increased serum nitrate (micromoles of NO2 + NO3) concentrations were detected in septic patients, compared with controls, but no differences were found between survivor (91.84 +/- 14.12) and nonsurvivor (102.6 +/- 17.36) groups. CONCLUSIONS: Septic patients present suppressed neutrophil chemotactic responses to FMLP and leukotriene B4 stimuli compared with healthy controls. This is accompanied by increased serum concentrations of nitrate. The impairment of neutrophil chemotaxis was observed mainly in the cells obtained from nonsurvivor patients and may thus be an additional factor contributing to disease outcome.     

Changes in neutrophil aggregation and chemotaxis response during 18- hour cold storage

Providing neutrophil transfusions to septic neonatal patients with depleted neutrophil reserves can be troublesome and require unscheduled blood donations. Buffy coats from stored whole blood are a potential source of neutrophils provided they remain viable during the interval between the whole blood collection and the buffy coat production. This study determined neutrophil function during storage of whole blood at 4 degrees C for 18 hours. Whole blood pH, hematocrit, platelet, and white cell counts remained unchanged. Chemotactic response to formyl methionyl leucyl penylalanine (FMLP) declined from 172.5 +/- 29 units (mean +/- SD) to 125 +/- 48 at 9 hours and 63.6 +/- 48 (p less than 0.05) at 18 hours. Aggregation response to FMLP remained normal for 9 hours but dropped to 15.5 percent of normal after 18 hours. Neutrophil response to cytaxins was maintained for at least 9 hours during storage of whole blood at 4 degrees C but seriously declined within 18 hours. Limiting 4 degrees C storage of whole blood to 9 hours prior to preparing buffy coats may provide flexibility needed for urgent provision of neutrophil transfusions.     

Adenosine: a physiological modulator of superoxide anion generation by human neutrophils

The effects of adenosine were studied on human neutrophils with respect to their generation of superoxide anion, degranulation, and aggregation in response to soluble stimuli. Adenosine markedly inhibited superoxide anion generation by neutrophils stimulated with N-formyl methionyl leucyl phenylalanine (FMLP), concanavalin A (Con A), calcium ionophore A23187, and zymosan-treated serum; it inhibited this response to PMA to a far lesser extent. The effects of adenosine were evident at concentrations ranging from 1 to 1,000 microM with maximal inhibition at 100 microM. Cellular uptake of adenosine was not required for adenosine-induced inhibition since inhibition was maintained despite the addition of dipyridamole, which blocks nucleoside uptake. Nor was metabolism of adenosine required, since both deoxycoformycin (DCF) and erythro-9-(2-hydroxy-3-nonyl) adenine did not interfere with adenosine inhibition of superoxide anion generation. The finding that 2-chloroadenosine, which is not metabolized, resembled adenosine in its ability to inhibit superoxide anion generation added further evidence that adenosine metabolism was not required for inhibition of superoxide anion generation by neutrophils. Unexpectedly, endogenously generated adenosine was present in supernatants of neutrophil suspensions at 0.14-0.28 microM. Removal of endogenous adenosine by incubation of neutrophils with exogenous adenosine deaminase (ADA) led to marked enhancement of superoxide anion generation in response to FMLP. Inactivation of ADA with DCF abrogated the enhancement of superoxide anion generation. Thus, the enhancement was not due to a nonspecific effect of added protein. Nor was the enhancement due to the generation of hypoxanthine or inosine by deamination of adenosine, since addition of these compounds did not affect neutrophil function. Adenosine did not significantly affect either aggregation or lysozyme release and only modestly affected beta-glucuronidase release by neutrophils stimulated with FMLP. These data indicate that adenosine (at concentrations that are present in plasma) acting via cell surface receptors is a specific modulator of superoxide anion generation by neutrophils.     

Adenosine promotes neutrophil chemotaxis

We have previously (1-4) demonstrated that adenosine, by engaging specific receptors on the surface of neutrophils, inhibits generation of toxic oxygen metabolites by activated neutrophils and prevents these activated neutrophils from injuring endothelial cells. We now report the surprising observation that engagement of these same neutrophil adenosine receptors promotes chemotaxis to C5 fragments (as zymosan- activated plasma [ZAP]) or to the bacterial chemoattractant FMLP. When chemotaxis was studied in a modified Boyden chamber, physiologic concentrations of adenosine promoted chemotaxis by as much as 60%. Adenosine receptor analogues, 5'N-ethylcarboxamidoadenosine (NECA) and N6-phenylisopropyladenosine (PIA), also promoted chemotaxis; the order of agonist potency was consistent with that of an A2 adenosine receptor (NECA greater than PIA greater than or equal to adenosine). A potent antagonist at adenosine receptors, 8-p-sulfophenyltheophylline (10 microM), completely reversed NECA enhancement of chemotaxis but did not affect chemotaxis by itself. Neither NECA nor 2-chloroadenosine, a nonselective adenosine receptor agonist, alone was chemotactic or chemokinetic by checkerboard analysis. NECA also promoted chemotaxis quantitated by a different technique, chemotaxis under agarose, to the surrogate bacterial chemoattractant FMLP. These data suggest that engagement of adenosine A2 receptors uniquely modulates neutrophil function so as to promote migration of neutrophils to sites of tissue damage while preventing the neutrophils from injuring healthy tissues en route.     

The effects of irradiation on blood components

The functional properties of formed elements of whole blood were studied following irradiation doses of 500 to 20,000 rads. Irradiated lymphocytes retained only 1.5 per cent of their 3H thymidine uptake after a 5,000-rad exposure and none after 7,500 rads. Red blood cells stored for 21 days and then irradiated with 5,000 rads had the same survival as nonirradiated controls. In contrast, 5,000 rads reduced platelet yields. However, transfused irradiated platelets produced the expected increases in platelet counts and controlled hemostasis in thrombocytopenic patients. After 5,000 rads, granulocytes had normal bacterial killing capacity, chemotactic mobility, and normal superoxide production after high-dose stimulation. Nitroblue tetrazolium reduction and ingestion stimulated by complement opsonized oil droplets were not diminished by 5,000- and 10,000-rad irradiation. The functional qualities of cellular blood components other than lymphocytes are not compromised by 5,000 rads. This irradiation dose may be an effective means of controlling incidence of graft-vs-host disease in immunosuppressed patients.     

Impaired neutrophil killing in a patient with defective degranulation of myeloperoxidase

A case of recurrent, superficial abscesses in an 18 year old girl, is described. Staphylococcus aureus was the pathogen most often implicated and on several occasions the abscesses required surgical drainage. Defects in humoral immunity, neutrophil chemotaxis or opsonophagocytosis were not observed. However, her neutrophil's ability to kill ingested S. aureus in vitro was impaired. This was associated with impaired luminol-dependent chemiluminescence in response to stimulation by either latex beads, or the chemotactic peptide FMLP plus cytochalasin B. Oxygen uptake and superoxide anion production were normal but release of myeloperoxidase by this patient's neutrophils occurred more slowly and to a lower extent than in control cells. These data suggest that the recurrent infections and diminished in vitro neutrophil bactericidal activity observed in this patient are associated with impaired degranulation of myeloperoxidase.     

Neutrophil-endothelial cell interactions. Modulation of neutrophil adhesiveness induced by complement fragments C5a and C5a des arg and formyl-methionyl-leucyl-phenylalanine in vitro.

Neutrophil adherence to vascular endothelial cells is the initial event in the emigration of neutrophils through blood vessel walls to tissue sites of inflammation; this process is attributed to the generation of extravascular chemotactic factors. To investigate the effect of chemotactic factors on neutrophil adherence to endothelium, we developed a sensitive, reproducible in vitro microtiter adherence assay. Base-line nonstimulated adhesion of human neutrophils to cultured human umbilical vein endothelial cell monolayers was 35.2 +/- 0.9%, which is equivalent to three to four neutrophils per endothelial cell. Addition of either purified complement fragment C5a des arg, or formyl-methionyl-leucyl-phenylalanine (FMLP), in concentrations ranging from 10(-10) to 10(-6) M, increased neutrophil adherence to endothelium in a dose-dependent manner. Purified C5a and C5a des arg were essentially equal in their ability to enhance neutrophil adherence, in contrast to the previously described greater in vitro potency of C5a compared with C5a des arg in stimulating neutrophil chemotaxis and enzyme release. Nonstimulated neutrophils adhered preferentially to human endothelial cells compared with fibroblasts or smooth muscle cells, suggesting that endothelial cells may make a unique contribution to the base-line adhesive interaction. However, chemotactic factors appear to enhance neutrophil adherence to endothelium by exerting an effect primarily on the neutrophil. In the presence of chemotactic factor, neutrophils adhered equally well to different cell types or to protein-coated plastic. Pretreatment of endothelial cells with chemotactic factor for as long as 4 h failed to increase subsequent neutrophil adherence. In contrast, pretreatment of neutrophils with chemotactic factor increased adherence to endothelium. Chemotactic factor-stimulated neutrophil adherence to endothelium occurred rapidly (within 2 min), diminished upon removal of stimulus, but could be rapidly and maximally restimulated upon readdition of the original dose of chemotactic factor. Thus, adherence to endothelium stimulated by chemotactic factor would appear to be a dynamic neutrophil response capable of rapid modulation, possibly important to the ability of neutrophils to adhere to and then migrate through vessel walls to localize at sites of inflammation.     

Defective neutrophil chemotaxis in bone marrow transplant patients.

Infection is a frequent cause of death in patients receiving bone marrow transplants. Although lymphocyte dysfunction has been observed in a few such patients, no systematic study of neutrophil function has yet been reported. Neutrophil chemotaxis was evaluated by a 51Cr-radioassay after bone marrow transplantation in 34 patients with acute leukemia or aplastic anemia. The response to a chemotactic stimulus (C5a) was severely depressed (less than 35% of normal) in 18 patients, moderately depressed (35-65% of normal) in an additional 6, and normal in 10 subjects. The mean response in the absence of graft vs. host disease and antithymocyte globulin administration was 73.3+/-9.2% (SE) in contrast to 29.7+/-9.6% (P is less than 0.01) in patients with graft vs. host disease treated with antithymocyte golbulin. Both graft vs. host disease and antithymocyte globulin were implicated since the presence of either factor alone was associated with depressed chemotaxis (31.1+/-4.9% for graft vs. host disease, P is less than 0.01; 17.0+/-7.8% for antithymocyte globulin, P is less than 0.02). When normal neutrophils were incubated with antithymocyte globulin in vitro, their chemotactic response was markedly suppressed in the absence of a cytotoxic effect. Transplant patients with defective chemotaxis experienced significantly more infections than those with normal chemotaxis, and analysis of specific etiologic agents showed that this was predominantly related to bacterial pathogens. Chemotactic inhibitors were detected in the sera of seven patients and elevated IgE levels were found in nine subjects, eight of whom had graft vs. host disease. Generation of chemotactic activity by endotoxin activation of serum was reduced in five patients. The results demonstrate a severe defect in neutrophil chemotaxis in some bone marrow transplant patients and suggest that neutrophil dysfunction may predispose to infection in such patients.     

Neutrophil defect associated with hairy cell leukemia

We have investigated the motility, superoxide anion production and tumor cell lysis of neutrophils from five patients affected by hairy cell leukemia. Random locomotion and chemotaxis towards denaturated casein and activated serum was normal as well as the superoxide production by opsonized zymosan. In contrast, chemotaxis and superoxide generation induced by FMLP and TPA were markedly reduced. The low responsiveness of neutrophils to TPA was also observed by evaluating cell lysis, against either non-immunized K562 target cells or antibody-coated tumor targets. In hairy cell leukemia neutrophils showed a selective reduced response to TPA and FMLP that, directly or indirectly, activate PKC, suggesting an impairment in the system of signal transduction.     

A Specific Inhibitor of Complement (C5)-Derived Chemotactic Activity in Serum from Patients with Systemic Lupus Erythematosus

In the course of examining polymorphonuclear leukocyte (PMN) chemotaxis in patients with systemic lupus erythematosus (SLE), we have found a previously undescribed serum inhibitor of complement (C5)-derived chemotactic activity. Serum from a 25-yr-old Black female with untreated SLE, when activated with zymosan, failed completely to attract either her own or normal PMN. Incubation of normal PMN with the patient's serum did not affect their subsequent random motility or chemotactic response toward normal zymosan-treated serum (ZTS). The patient's serum, however, did inhibit the chemotactic activity of normal ZTS and of column-purified C5-derived peptide(s), but had no effect on the chemotactic activity of either the synthetic peptide, N-formylmethionyl leucyl-phenylalanine or a filtrate prepared from a culture of Escherichia coli (bacterial chemotactic factor). The inhibitory activity in the patient's serum resisted heating at 56°C for 30 min and could be separated from C5-derived chemotactic activity in the patient's ZTS (or normal ZTS that had been incubated with the patient's serum) by chromatography on Sephadex G-75. Despite its effect on C5-derived chemotactic activity, the patient's serum did not influence two other C5-derived biologic activities: PMN lysosomal enzyme-releasing activity and PMN-aggregating activity. Chromatography of the patient's serum (65% ammonium sulfate pellet) on Sephadex G-200 yielded three distinct peaks of inhibitory activity. Two were heat labile and exhibited other properties of the previously described chemotactic factor inactivators of normal human serum. The third and most active peak, however, resisted heating at 56°C for 30 min, eluted with an apparent mol wt of 50,000-60,000, and acted specifically on C5-derived chemotactic activity. This uniquely specific, heat-stable inhibitor of C5-derived chemotactic activity has been found thus far in serum from 4 of 11 patients with active SLE and may account, in part, for altered host defenses against infections caused by pyogenic microorganisms.     

Monocyte-chemotactic activity of defensins from human neutrophils.

We investigated the monocyte-chemotactic activity of fractionated extracts of human neutrophil granules. Monocyte-chemotactic activity was found predominantly in the defensin-containing fraction of the neutrophil granules. Purified preparations of each of the three human defensins (HNP-1, HNP-2, HNP-3) were then tested. HNP-1 demonstrated significant chemotactic activity for monocytes: Peak activity was seen at HNP-1 concentrations of 5 X 10(-9) M and was 49 +/- 20% (mean +/- SE, n = 9) of that elicited by 10(-8) M FMLP. HNP-2 (peak activity at 5 X 10(-9) M) was somewhat less active, yielding 19 +/- 10% (n = 11). HNP-3 failed to demonstrate chemotactic activity. Checkerboard analysis of monocyte response to HNP-1 and HNP-2 confirmed that their activity was chemotactic rather than chemokinetic. Neutrophils demonstrated a low level of response to defensins but this reaction was primarily chemokinetic. Defensins may play a role in the recruitment of monocytes by neutrophils into inflammatory sites.     

Adherence of neutrophils to cultured human microvascular endothelial cells. Stimulation by chemotactic peptides and lipid mediators and dependence upon the Mac-1, LFA-1, p150,95 glycoprotein family.

The process of neutrophil adhesion to and migration through the microvascular endothelium, an early event in the induction of the acute inflammatory response, has been attributed to the generation of extravascular chemoattractants. Although both chemotactic peptides and lipid mediators enhance neutrophil adherence in vitro and in vivo, the mechanism(s) involved in the interaction between circulating neutrophils and microvascular endothelial cells is still not completely understood. In a microtiter well adherence assay, the chemotactic peptides, FMLP and C5a, and the lipid mediators, leukotriene B4 (LTB4) and platelet activating factor (PAF), enhanced human neutrophil adherence to cultured human microvascular endothelial cells as well as to human umbilical vein endothelial cells in a dose-dependent manner with a rapid time course. This stimulated adhesive interaction between neutrophils and cultured human endothelial cells was dependent on the expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface since neutrophils from patients with leukocyte adhesion deficiency, lacking surface expression of the adhesive glycoproteins, exhibited markedly diminished adherence to human endothelial cells in response to stimulation with chemotactic factors compared to normal control neutrophils. All four mediators enhanced expression of the glycoprotein family on the surface of normal neutrophils as determined by flow cytofluorimetry using a monoclonal antibody (TS1/18) to the glycoprotein common beta subunit. In addition, TS1/18 inhibited up to 100% the adherence of normal neutrophils to endothelial cells stimulated by maximal concentrations of FMLP, C5a, LTB4, or PAF. Moreover, HL-60 cells, human promyelocytic leukemia cells, neither increased glycoprotein surface expression nor adherence in response to stimulation. Thus, peptide and lipid mediators of the acute inflammatory response appear to enhance adherence of circulating neutrophils to the microvascular endothelium by a mechanism dependent on expression of the Mac-1, LFA-1, p150,95 glycoprotein family on the neutrophil surface.     

Recombinant human tumor necrosis factor-alpha. Regulation of N-formylmethionylleucylphenylalanine receptor affinity and function on human neutrophils.

Preincubation of neutrophils with recombinant human tumor necrosis factor-alpha (rH TNF-alpha) enhanced the subsequent release of superoxide anion in response to various concentrations of N-formylmethionylleucylphenylalanine (FMLP). Enhanced superoxide anion production was evident by 5 min and had reached a plateau by 15 min. Not only was the total amount of superoxide anion released greater, but the rate of release was also enhanced threefold by rH TNF-alpha. In contrast, rH TNF-alpha reduced or abolished neutrophil locomotion under agarose in response to a gradient of FMLP. Binding studies of f-Met-Leu-[3H]Phe to purified human neutrophils revealed a heterogeneous binding to unstimulated cells. The high affinity component consisted of approximately 2,000 sites per cell and had an average Kd of 2 +/- 0.7 nM (n = 4). The low affinity component consisted of approximately 40,000 sites per cell and had an average Kd of 180 +/- 50 nM (n = 4). rH TNF-alpha caused conversion to a linear Scatchard plot showing no significant change in total binding sites but a single Kd of 40 +/- 10 nM (n = 4). These data indicate that rH TNF-alpha may influence neutrophil responses to FMLP by regulating the affinity of FMLP receptors.     

Investigation of the in-vitro uptake, intraphagocytic biological activity and effects on neutrophil superoxide generation of dirithromycin compared with erythromycin.

The cellular uptake by human neutrophils and the intraphagocytic biological activity of the new macrolide antimicrobial agent dirithromycin (0.01-2 mg/L) compared with erythromycin was investigated in vitro. Staphylococcus aureus, Listeria monocytogenes and Legionella pneumophila were used as the test intracellular microbial pathogens. After coincubation (45 min at 37 degrees C) of neutrophils with a fixed concentration of 2 mg/L of each antibiotic the respective intracellular/extracellular ratios for erythromycin and dirithromycin were 6.1 +/- 2.5 and 10.6 +/- 2 respectively (P < 0.005). Using a combination of techniques (colony counting, radiometry and fluorescence microscopy) both erythromycin and dirithromycin at concentrations of 0.01 and 0.5 mg/L and higher, respectively, were found to possess dose-related intraphagocytic bacteristatic activity for each of the test microbial pathogens. The effects of dirithromycin and erythromycin (1-20 mg/L) on neutrophil chemotaxis and generation of reactive oxidants by these cells were also investigated in vitro. Both antimicrobial agents caused a dose-related stimulation of neutrophil migration which was associated with inhibition of leucoattractant-activated generation of superoxide and activity of the myeloperoxidase/H2O2/halide system. However, superoxide generation by neutrophils activited with opsonized zymosan or phorbol myristate acetate was unaffected by the macrolides. These findings demonstrate that dirithromycin accumulates in human neutrophils, is biologically active intracellularly and modulates leucoattractant-activated superoxide generation and chemotaxis.     

Miconazole and amphotericin B alter polymorphonuclear leukocyte functions and membrane fluidity in similar fashions.

The influence of miconazole on polymorphonuclear leukocytes (PMN) was investigated and compared with that of amphotericin B (AmB). Human PMN were preincubated in vitro with miconazole or AmB at therapeutically attainable concentrations in plasma, and their chemotactic functions were assessed with the synthetic chemotactic peptide N-formylmethionyl-leucyl-phenylalanine (FMLP). Changes in membrane fluidity of PMN were examined by an excimer-forming lipid technique. Adherence of PMN was measured by a nylon fiber column method. Miconazole and AmB pretreatment irreversibly depressed PMN random migration and chemotaxis under agarose but did not influence superoxide anion production. Both miconazole and AmB increased PMN adherence. Miconazole and AmB lowered the binding affinity of FMLP receptors on PMN and decreased the membrane fluidity in a similar manner. These results demonstrate that miconazole and AmB alter selected in vitro membrane properties of human PMN.     

Rapid changes in light scattering from human polymorphonuclear leukocytes exposed to chemoattractants. Discrete responses correlated with chemotactic and secretory functions

A platelet aggregometer was adapted for the simultaneous measurement of perpendicular light scattering in addition to light transmission. The addition of chemoattractants to polymorphonuclear leukocyte suspensions evoked a single wave of increased light transmission, whereas the perpendicular scattering measurement demonstrated a previously unrecognized biphasic response. The first perpendicular scattering response had no detectable latency and peaked at 10 +/- 1 s, then decayed rapidly. The second response peaked at 40 +/- 5 s, and decayed over several minutes. The dose-response curve of chemoattractants for inducing the rapid (10 +/- 1 s) perpendicular scattering peak corresponded to that which initiated chemotaxis. Initiation of the slow (40 +/- 5 s) peak required 10-fold higher amounts of chemoattractants, and the dose-response curve correlated with the induction of lysosomal enzyme secretion and superoxide anion production. Low doses of aliphatic alcohols, which have been shown to enhance chemotaxis but to inhibit secretion and superoxide anion production, abolished the slow perpendicular light-scattering response but left the fast response intact. Stimulants of secretion induced only slow and prolonged responses that were best observed in transmission measurements. In an attempt to resolve the origin of the light-scattering responses, the morphological changes of polymorphonuclear leukocytes were examined microscopically. Neither aggregation nor morphological whole cell polarization could be correlated with changes in light transmission or perpendicular scattering, which suggested that the source of scattering is of subcellular dimensions. The rapid perpendicular light-scattering response of polymorphonuclear leukocytes to chemoattractants appears to record an initial event in the stimulus-response coupling, and its measurement should provide a useful new tool for the study of leukocyte function. The biphasic nature of the light-scattering responses to chemoattractants, moreover, correlates with the dual regulation of the chemotactic and secretory responses of leukocytes.     

Modulation of human neutrophil function by endogenous opiate enkephalins

On the basis of recent evidence, the natural opiate enkephalins, which previously were believed to be confined to the central nervous system, are now known, in fact, to be released from the adrenal glands by sympathetic activation or trauma. To determine if enkephalins (EKs) affect peripheral function, the influence of synthetic leucine and methionine enkephalin (leuEK and metEK) on several relevant functions of human polymorphonuclear leukocytes was evaluated. Initial attempts to detect interaction of leuEK and metEK with neutrophils yielded inconsistent results. Further studies were done using protease-resistant methionine enkephalin-amide (metEKamide). MetEKamide was able to induce degranulation when present at 10(-3) and 10(-4) mmol/L as determined by release of beta-glucuronidase and lysozyme. Using the under-agarose chemotaxis method, treatment with metEKamide resulted in no change of the neutrophil's chemotactic response to an optimal concentration of the chemotactic peptide formyl-methionyl-leucyl-phenyl-alanine (FMLP). However, responsiveness to low levels of FMLP increased in cells treated with 10(-3)-10(-5) mmol/L metaEKamide. This appeared to be a result of increased chemokinesis of the treated cells. Scanning electron microscopic studies of cells exposed to metEKamide revealed that treatment resulted in changes in neutrophil morphology. When metEKamide itself was tested as a potential chemotactic agent, 10(-2) mmol/L metEKamide in an opposing well served to induce chemotaxis. Our results, along with those of recent studies of EKs as immunomodulators of T cell function, suggest that neurohormones can function as regulators of the immune response.     

The adenosine/neutrophil paradox resolved: human neutrophils possess both A1 and A2 receptors that promote chemotaxis and inhibit O2 generation, respectively

Occupancy of specific receptors on neutrophils by adenosine or its analogues diminishes the stimulated release of toxic oxygen metabolites from neutrophils, while paradoxically promoting chemotaxis. We now report evidence that two distinct adenosine receptors are found on neutrophils (presumably the A1 and A2 receptors of other cell types). These adenosine receptors modulate chemotaxis and O2- generation, respectively. N6-Cyclopentyladenosine (CPA), a selective A1 agonist, promoted neutrophil chemotaxis to the chemoattractant FMLP as well as or better than 5'N-ethylcarboxamidoadenosine (NECA). In contrast, CPA did not inhibit O2- generation stimulated by FMLP. Pertussis toxin completely abolished promotion of chemotaxis by CPA but enhanced inhibition by NECA of O2- generation. Disruption of microtubules by colchicine or vinblastine also abrogated the enhancement by NECA of chemotaxis whereas these agents did not markedly interfere with inhibition by NECA of O2- generation. FMLP receptors, once they have bound ligand, shift to a high affinity state and become associated with the cytoskeleton. NECA significantly increased association of [3H]FMLP with cytoskeletal preparations as it inhibited O2-. Disruption of microtubules did not prevent NECA from increasing association of [3H]FMLP with cytoskeletal preparations. Additionally, CPA (A1 agonist) did not increase binding of [3H]FMLP to the cytoskeleton as well as NECA (A2 agonist). These studies indicate that occupancy of one class of adenosine receptors (A1) promotes chemotaxis by a mechanism requiring intact microtubules and G proteins whereas engagement of a second class of receptors (A2) inhibits O2- generation. Signalling via A2 receptors is independent of microtubules, insensitive to pertussis toxin and is associated with binding of [3H]FMLP to cytoskeletal preparations.