Effects of two injectable anesthetic agents on coagulation assays in the rat.

The rat is commonly used as an animal model to test the efficacy of new anticoagulant and antithrombotic drugs. Many of the protocols require the use of anesthetics for surgical procedures. Three common coagulation assays used to monitor the effects of new anticoagulant and antithrombotic drugs are thrombin time, activated partial thromboplastin time, and prothrombin time. We examined the effects of either urethane- or ketamine/xylazine-induced anesthesia on variables in the three coagulation assays with blood samples obtained from the rats before the induction of anesthesia, compared with samples obtained at 15 or 60 min after anesthesia induction. Statistically significant differences were observed in the ketamine/xylazine group for thrombin time at 60 min (compared with control values) and in the urethane group for activated partial thromboplastin time at 60 min (compared with control values). In both instances a prolongation of times was the effect seen. These results indicate that the choice of anesthetic affects the data in certain coagulation assays in the rat and must be taken into account when one is planning studies and analyzing results.

Superoxide anion production from human neutrophils measured with an improved kinetic and endpoint microassay.

Superoxide dismutase (SOD)-inhibitable reduction of cytochrome c is the basis for a widely used assay to measure superoxide production. We report novel modifications leading to a dual wavelength, high throughput simultaneous kinetic and endpoint microplate assay with high reproducibility. Neutrophils were isolated using a modified elutriation procedure to minimize priming and adherence during isolation. Cytochrome c reduction was measured in a microplate reader using 96-well polystyrene plates with a modified (Plastek A*) surface to prevent the adherence and consequent activation of PMNs. Comparison of Plastek A* treated and untreated plates revealed a statistically significant difference in basal as well as stimulated levels of superoxide production. Absorption measurements were made at both 550 nm, the absorption maximum of reduced cytochrome c, and 557 nm, an isosbestic point. A significant increase in both well-to-well reproducibility and sensitivity (detection limit) was realized by using the normalized 550-557 nm difference values compared to the 550 nm absorbance values alone. These modifications represent an improved method for handling and assessing the function of superoxide production, providing greater experimental reproducibility and lessening the perturbations caused by the microplate.

Fluorescence analysis of the size of a binding pocket of a peptide receptor at natural abundance.

We have studied the topography of interaction of a family of fluorescent formyl peptides containing four (CHO-Met-Leu-Phe-Lys-fluorescein), five (CHO-Met-Leu-Phe-Phe-Lys- fluorescein), and six (CHO-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein and CHO-Met-Leu-Phe-Phe-Phe-Lys- fluorescein) amino acids with their receptor using spectroscopic methods adapted to small sample volumes. Only the fluorescent peptides containing four and five amino acids were quenched upon binding to the receptor, indicating physical contact of the chromophore with the receptor. In contrast, only the hexapeptides were accessible to antibodies to fluorescein. Taken together, these results suggest that the carboxy terminus of the tetrapeptide or the pentapeptide is protected in the receptor binding pocket while the fluorescein on the carboxy terminus of either hexapeptide is exposed and recognized by the antibody to fluorescein. These results indicate that the binding pocket accommodates at least five but no more than six amino acids.

Neutrophils are hyperpolarized after exudation and show an increased depolarization response to formyl-peptide but not to phorbol myristate acetate.

The migration of polymorphonuclear leucocytes (PMN) to extravascular sites and the interaction with chemotactic substances at such locations is called exudation. Since stimulation of PMN in vitro modifies the characteristics of PMN, we asked the question whether similar modifications take place during in-vivo exudation. We found that resting guinea-pig peritoneal exudate PMN were hyperpolarized in comparison to blood PMN of the same species. Guinea-pig and human exudate PMN responded to lower N-formyl-methionylleucylphenylalanine (fmet-leu-phe) concentrations than blood PMN and exhibited a larger membrane depolarization. Furthermore, experiments with the fluorescence-activated cell sorter revealed increased forward light scatter from resting exudate PMN compared to blood PMN. Experiments with the fluorescence-activated cell sorter using the fluoresceinated ligand fmet-leu-phe-lysin-fluorescein (fmet-leu-phe-lys-F) indicated that the priming of exudate PMN was associated with increased fmet-leu-phe-lys-F binding on the individual cells. In contrast, phorbol myristate acetate (PMA) did not induce an increased membrane depolarization response in human and guinea-pig exudate PMN. With this stimulus, the only sign of priming was the shorter activation time in exudate PMN compared to blood PMN. Thus, in-vivo exudation modifies the characteristics of resting and stimulated PMN. The priming is different for different stimuli. Increased responsiveness to fmet-leu-phe may be due to fmet-leu-phe receptor upregulation. The distinct characteristics of exudate PMN that we describe may allow definition of clinical situations in which PMN are primed in vivo.

Separation and function of neutrophil karyogranuloplasts and comparison with cytoplasts and intact cells.

Since neutrophil cytoplasts lacking nucleus and granules were first prepared by centrifuging neutrophils over a discontinuous Ficoll gradient containing cytochalasin B, several functional deficits have been reported in these cytoplasts. Although these functional deficits have been considered to originate from the absence of organelles, cell damage during preparation could not be excluded. Therefore, in the following experiments the Ficoll gradient was modified to isolate both cytoplasts and karyogranuloplasts, which have a nucleus and granules and represent the cell after loss of the cytoplast. Electron microscopy and analysis of marker proteins and cell volume showed that karyogranuloplasts were distinct from neutrophils. The phorbol myristate acetate (PMA) or N-formylmethionylleucylphenylalanine (FMLP)-induced O2- release, corrected by surface area, was in the following order: neutrophils greater than cytoplasts greater than karyogranuloplasts. Both aggregation and membrane potential depolarization were maximal in neutrophils, intermediate in karyogranuloplasts, and lowest in cytoplasts when either PMA or FMLP was used as a stimulant. Extracellular release of the granule contents (degranulation) was triggered by FMLP in both neutrophils and karyogranuloplasts. Cytochalasin B pretreatment greatly enhanced FMLP-induced O2- release, degranulation, aggregation, and depolarization of membrane potential in neutrophils and karyogranuloplasts, but not in cytoplasts. The ability of cytochalasin B to potentiate FMLP-triggered cell function probably depends on granules or cell organelles which are depleted in cytoplasts. Chemokinesis and chemotaxis were impaired in both karyogranuloplasts and cytoplasts. Specific FML[3H]P binding was greater in karyogranuloplasts than in cytoplasts. Cellular actin content, measured by the DNase I inhibition assay, was abundant in cytoplasts and was extremely low in karyogranuloplasts. Karyogranuloplasts retain various neutrophil functions, except for chemotaxis, and provide an important control when studying the role of cell organelles in cytoplast function.

PMN heterogeneity: long-term stability of fluorescent membrane potential responses to the chemoattractant N-formyl-methionyl-leucyl-phenylalanine in healthy adults and correlation with respiratory burst activity.

Polymorphonuclear neutrophils (PMNs) were isolated from 24 healthy adults 20 to 61 years of age and the proportion of cells that demonstrated depolarization responses to the synthetic chemotaxin N-formyl-methionyl-leucyl-phenylalanine (FMLP) were enumerated using the lipophilic fluorescent cyanine dye 3,3'-di-pentyl-oxacarbocyanine [di-O-C(5)(3)] and flow cytometry. The membrane potential responses were correlated to the cells' respiratory burst capabilities as measured by nitroblue tetrazolium (NBT) reduction and/or superoxide production in response to FMLP; 40.2% +/- 15.1% (mean +/- SD) of cells depolarized to FMLP. The mean SE for duplicate determinations 1 hour apart was 3.8% (range 0.4% to 13.6%, n = 15). There was no correlation between the percentage of depolarizing PMNs and the yield of cells, the percentage of immature cells, or the circulating WBC count. There was no difference in the average age of men (34.9 +/- 9.9 years, n = 11) v women (33.8 +/- 8.5, n = 13) (mean +/- SD) studied, or in the percentage of depolarizing PMNs when men and women were compared (42.2 +/- 10.6% v 43.1 +/- 13.3%, respectively). However, there was a significant increase in the percentage of depolarizing PMNs with increasing age of women (r = .61, P less than .025) but not men (r = .03, P greater than .05). Analysis of variance revealed significantly greater person-to-person variability in the membrane potential response than in day-to-day variability in the same person (P less than .0005). The percentage of depolarizing PMNs in response to FMLP was significantly correlated with the percentage of NBT-positive cells from both purified PMNs and from whole blood (r = .849, P less than .0005, r = .857, P less than .05, respectively), and with the amount of superoxide produced, expressed as a percentage of that amount produced by cytochalasin B (cyto-B)-pretreated cells (r = .565, P less than .01). The data indicate that PMNs from healthy adults demonstrate a heterogeneous membrane potential response to the chemotaxin FMLP that correlates with the cells' oxidative responsiveness and that intersubject differences can be detected. In addition, the proportion of responsive PMNs increases with increasing age in women.

A neutrophil membrane marker reveals two groups of chronic myelogenous leukemia and its absence may be a marker of disease progression.

An IgG1 monoclonal antibody, 31D8, that recognizes normal neutrophil (PMN) membranes, was used to study PMN from patients with chronic myelogenous leukemia (CML). Nineteen patients with Philadelphia chromosome positive CML were followed over a ten-month period and compared with 23 normals, six patients with leukemoid reactions, and eight patients with phagocytic cell defects. The percentage of PMN binding of 31D8 among normal subjects was variable about a normal distribution with an average of 95 +/- 2% of cells binding 31D8. In contrast, there were two groups of CML patients: in 14 patients 88 +/- 3% PMN bound 31D8 while in the remaining five patients only 6 +/- 6% PMN bound 31D8. PMN 31D8 binding was normal in the control patient groups. Control antibodies 7C3 (binds to PMN precursors) and OKM1 (binds to the CR3 (iC3b) receptor) bound normally to CML neutrophils. Functionally, CML cells had normal chemotaxis to several stimuli and normal superoxide generation to phorbol myristate acetate. However, superoxide production in response to fmet-leu-phe was significantly less in 31D8 negative CML PMN than both 31D8 positive CML PMN and normal PMN which contained 85% 31D8 positive and 15% 31D8 negative PMN. Clinically, 2 of 14 CML patients with 31D8 positive PMN were in blast crisis (one extramedullary) at the time of study and the other 12 patients remained clinically stable in the chronic phase during the ten months of study. In contrast, one of five patients with 31D8 negative PMN was in blast crisis at the time of study and all four of the remaining patients progressed to either the accelerated phase or blast crisis. Three of these patients died of their disease eight to ten months after their initial study. Thus, failure of CML cells to bind 31D8 may be useful for predicting which patients are likely to progress to the accelerated phase or blast crisis.

Voltage-sensitive cyanine dye fluorescence signals in lymphocytes: plasma membrane and mitochondrial components.

The origin of the cyanine dye fluorescence signal in murine and human peripheral blood leukocytes was investigated using the oxa- and indo-carbocyanines di-O-C5(3) and di-I-C5(3). Fluorescence signals from individual cells suspended with nanomolar concentrations of the dyes were measured in a flow cytometer modified to permit simultaneous four-parameter analysis (including two-color fluorescence or fluorescence polarization measurements). The contributions of mitochondrial membrane potential (psi m) and plasma membrane potential (psi pm) to the total voltage-sensitive fluorescence signal were found to depend on the equilibrium extracellular dye concentration, manipulated in these experiments by varying the ratio of dye to cell density. Hence, conditions could be chosen that amplified either the psi m or the psi pm component. Selective depolarization of lymphocytes or polymorphonuclear leukocytes (PMN) in mixed cell suspensions demonstrated that defining the partition of dye between cells and medium is requisite to assessing the heterogeneity of cell responses by cyanine dye fluorescence. At extracellular dye concentrations exceeding 5 nM in equilibrated cell suspensions, both mitochondrial and plasma membrane dye toxicity were observed. In murine splenic lymphocytes, plasma membrane toxicity (dye-induced depolarization) was selective for the B lymphocytes. Certain problems in calibration of psi pm with valinomycin at low dye concentrations and perturbations of psi pm by mitochondrial inhibitors are presented. These findings address the current controversy concerning psi m and psi pm measurement in intact cells by cyanine dye fluorescence. The finding of selective toxicity at low cyanine dye concentrations suggest that purported differences in resting psi m among cells or changes in psi pm with cell activation may reflect variable susceptibility to dye toxicity rather than intrinsic cell properties.

Selective elimination of lymphocyte subpopulations by monoclonal antibody-enzyme conjugates.

A novel method for the selective depletion of lymphocyte subpopulations has been developed. Conjugates of glucose oxidase (GOx) and phospholipase-C (PL-C) coupled to a monoclonal mouse anti-rat IgG (MAR) were shown to be selectively cytotoxic for targeted lymphocyte subsets in the presence of various rat monoclonal antibodies directed toward murine cell surface antigens. The ability of both conjugates to bind specifically to rat monoclonal antibodies was demonstrated by flow cytometry. The PL-C-MAR conjugate was more stable than the GOx-MAR conjugate. The PL-C conjugate, in conjunction with primary rat anti-mouse monoclonal antibodies, produced selective killing of T or B cells as detected by a loss of proliferative capacity in response to mitogens and by specific cell depletion demonstrated by flow cytometry. Normal mouse serum protected against the cytotoxicity of free enzymes but had no protective effect on enzyme conjugates. Because the substrates of these enzymes are abundant in vivo and serum did not interfere with their cytotoxicity, these enzyme-antibody conjugates may be valuable for selective lymphocyte depletion in vivo.

Evidence for distinct intracellular pools of receptors for C3b and C3bi in human neutrophils.

In this report, the modulation and localization of complement receptors CR1 and CR3 in neutrophils were examined with the use of monoclonal antibodies (mab) directed against these membrane proteins. We first studied complement receptor modulation in a patient with neutrophil-specific granule deficiency. With flow cytometric analysis, we determined that, while N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) (10(-6) M) caused an increase in the binding of both anti-CR1 and anti-CR3 mab to normal neutrophils, the fmet-leu-phe-stimulated neutrophils from our patient increased anti-CR1 binding but decreased anti-CR3 binding. This suggested that CR3, but not CR1, might be associated with specific granules. We next studied receptor modulation in organelle-depleted neutrophil cytoplasts obtained from normal donors. Unlike the specific granule-deficient neutrophils, the normal cytoplasts failed to augment expression of either receptor after stimulation. Immunofluorescence studies of permeabilized polymorphonuclear leukocytes (PMN) revealed considerable internal binding of both anti-CR1 and anti-CR3. In additional studies, phorbol myristate acetate (PMA) was used as a stimulus for receptor modulation in normal neutrophils. Unlike fmet-leu-phe and C5a, PMA elicited a biphasic dose-response curve. High doses of PMA (greater than 0.5 ng/ml) caused a reduction in the magnitude of membrane expression of both CR1 and CR3. In studies designed to localize the internal pool of receptors, we evaluated the binding of 125I-anti-receptor mab to plasma membrane-, specific granule, and azurophilic granule-enriched fractions obtained from sucrose gradient fractionation of disrupted neutrophils. 125I-anti-CR1 mab bound to the membrane-enriched fraction but bound little to either granule-enriched fraction. In contrast, 125I-anti-CR3 mab bound more to the specific granule-enriched fraction than to the plasma membrane-enriched fraction. Azurophilic granules showed no increased anti-CR3 binding. Immunoprecipitation of radiolabeled solubilized subcellular fractions with anti-receptor mab confirmed these findings. CR3 was present in the plasma membrane-, and specific granule-enriched fraction but not in the azurophilic granule-enriched fraction. CR1, however, was present only in the plasma membrane-enriched fraction. These data indicate that there are intracellular pools for both the CR1 and CR3, but the intracellular locations for these pools are distinct. The pool for CR3 co-sediments with specific granules, while the pool for CR1 does not. Nonetheless, a variety of stimulatory agents increase and decrease the membrane expression of both receptors in parallel.

Monitoring human neutrophil granule secretion by flow cytometry: secretion and membrane potential changes assessed by light scatter and a fluorescent probe of membrane potential.

Purified human peripheral blood polymorphonuclear neutrophils (PMN) were incubated at 37 degrees C with the fluorescent membrane potential sensitive cyanine dye di-O-C(5)(3) and exposed to a number of stimulatory agents (N-formylmethionylleucylphenylalanine (FMLP), cytochalasin B (cyto B) + FMLP, phorbol myristate acetate (PMA). Flow cytometry was utilized to measure changes in forward light scatter (FS), orthogonal light scatter (90 degrees-SC), and fluorescence intensity of individual cells over time. A saturating (10(-6) M) dose of FMLP lead to a significant increase in the cells' FS without a change in 90 degrees-SC as well as a heterogeneous loss of di-O-C(5)(3) fluorescence. PMA (100 ng/ml) also caused an increase in FS but a uniform loss of dye fluorescence by all cells (apparent depolarization). Cyto B + FMLP produced an increase in FS, a marked loss of 90 degrees-SC, and a uniform loss of fluorescence. Secretion experiments under identical incubation conditions indicated a significantly positive relationship between loss of enzyme markers or cell granularity and orthogonal light scatter (r = 0.959, 0.998, and 0.989 for loss of 90 degrees-SC vs lysozyme, beta-glucuronidase, and granularity index, respectively). Sequential exposure of PMN to PMA and then cyto B + FMLP produced a stepwise shift in scatter parameters (increased FS then loss of 90 degrees-SC). Normalization of membrane potential dye fluorescence changes for the changes in light scatter did not abrogate the heterogeneous fluorescence response of cells to stimulus, indicating that stimulus-induced scatter changes were not responsible for such fluorescence shifts. The data demonstrate that loss of 90 degrees-SC relates closely to secretion of primary granules while changes in FS reflect alterations in cell shape and/or surface/volume ratios that accompany cell activation. Flow cytometric light scatter measurements may yield important information on the extent of prior cell degranulation or activation.

Cytochrome b translocation to human neutrophil plasma membranes and superoxide release. Differential effects of N-formylmethionylleucylphenylalanine, phorbol myristate acetate, and A23187.

The role of specific granules and cytochrome b in superoxide (O(2)) release was studied by comparing the effects of three different stimuli on normal human neutrophils, neutrophils congenitally deficient in specific granules, and granule-free normal neutrophil cytoplasts. Phorbol myristate acetate (PMA) stimulated normal neutrophils to release more O(2) than did N-formylmethionylleucylphenylalanine (fMet-Leu-Phe), which stimulated greater release than the calcium ionophore A23187. Neutrophils lacking specific granules produced variable amounts of O(2) in response to all stimuli. Stimulation with PMA, fMet-Leu-Phe, and A23187 produced maximal rates of O(2) release that were 32, 55, and 21% of that by normal cells. Likewise, granule-free neutrophil cytoplasts released 24, 20, and 0% of the O(2) released by intact cells. These data suggest that the stimuli require different mechanisms for activation. Three subcellular fractions (azurophil granule rich, specific granule rich, and plasma membrane rich) were separated by Percoll gradients from normal resting and stimulated neutrophils. In resting neutrophils, the cytochrome b content in the plasma membrane was 31% of the total, with the rest in the specific granule-rich fraction. Ten minutes after stimulation, PMA, fMet-Leu-Phe, and A23187 induced translocation of 27, 8, and 49%, respectively, of the cytochrome b from the specific granule-rich fraction to the plasma membrane. Although our data support a role for specific granule factors in A23187-induced O(2) release, there is no correlation between the amount of cytochrome b incorporated into the plasma membrane and the extent of O(2) production activated by the different stimuli.

Mobilization and adaptation of human neutrophil chemoattractant fMet-Leu-Phe receptors.

Neutrophil responsiveness to N-formylmethionylleucylphenylalanine (fMet-Leu-Phe) appears to involve either receptor recycling and/or a storage pool of receptors that provide a mechanism for replenishment of receptors at the cell surface. Asymmetric distribution of the fMet-Leu-Phe receptor has been shown by others, with increased receptor density reported at the front of the cell. This receptor asymmetry may result from receptor capping in the front, receptor internalization or shedding in the rear, or addition of new receptors at the front of the cell. In addition to receptor asymmetry, affinity adaptation of the receptor through heterogeneity and/or negative cooperative interaction of the receptors is probably important. It has been suggested that a high-affinity state of the receptor is associated with transduction of chemotaxis and a low-affinity state with transduction of superoxide production and degranulation. Thus, the fMet-Leu-Phe receptor is an important model to study how neutrophils recognize and integrate signals for their complex functions.

Auranofin affects early events in human polymorphonuclear neutrophil activation by receptor-mediated stimuli.

Auranofin, a new oral antirheumatic gold compound, in concentrations achieved therapeutically, inhibits neutrophil phagocytosis, chemotaxis, chemiluminescence, reduction of cytochrome c, and release of lysosomal enzymes. To further characterize the mechanism by which auranofin affects neutrophils, we studied the effects of auranofin on unstimulated properties and functions of neutrophils as well as on rapidly stimulated functions. When examined by electron microscopy, 4 micrograms/ml of auranofin significantly decreased the number of visualized centriole-associated microtubules in resting cells. Furthermore, auranofin inhibited neutrophil spreading on glass and caused a decrease in negative surface charge (electrophoretic mobility). In addition, auranofin inhibited several fmet-leu-phe-stimulated responses such as shape change, increases in centriole-associated microtubules, decreases in surface charge, and elicited membrane potential changes (di-O-C5(3) dye response). Auranofin (1 micrograms/ml) inhibited fmet-leu-phe-stimulated superoxide and hydrogen peroxide production by 80% (p less than 0.05), and also increased the affinity of receptors for fmet-leu-phe (from Ka 0.035 to Ka 0.48, p less than 0.001). Auranofin also affected neutrophil responses to phorbol myristic acetate (PMA). The total amount of PMA-stimulated superoxide production was suppressed by as little as 0.4 micrograms/ml of auranofin, but the lag time for activation was shortened by low concentrations of auranofin (0.5 to 1 microgram/ml). Four micrograms per milliliter of auranofin suppressed the decrease in surface charge induced by PMA. However, auranofin did not influence superoxide production elicited by the ionophore A23187. The results indicate that auranofin affects the earliest detected responses in neutrophil activation by certain receptor-mediated stimuli.

NIH conference. Recent advances in chronic granulomatous disease.

Chronic granulomatous disease represents a group of disorders of phagocytic cell oxidative metabolism involving recurrent infections with catalase-positive microorganisms and chronic inflammation. Genetic heterogeneity and phagocyte abnormalities, including enzyme deficiencies, abnormal elicited membrane potential changes, abnormal acidification of the phagocytic vacuole, and deficiencies of an electron transport cascade, have been associated with its pathogenesis. In addition, recently we have shown abnormal neutrophil C3b-receptor expression, antibody-dependent cellular cytotoxicity, and abnormal microtubule metabolism (tyrosinolation of the alpha-chain of tubulin). Fourteen patients with the disease who were followed at the National Institutes of Health had life-threatening infections, on average, once every 9.6 months. In most of the 119 febrile episodes seen in these patients, no infectious agent was found. Retrospective studies indicated that prophylactic antibiotic therapy, particularly with trimethoprim-sulfamethoxazole, significantly prolonged disease-free intervals to greater than 40 months (p less than 0.05). In serious, life-threatening infections, leukocyte transfusions have been used in therapy. Transfused leukocytes localize and persist at infectious sites, and the clinical efficacy of leukocyte transfusions has been suggested.

Comparison of indirect probes of membrane potential utilized in studies of human neutrophils.

Four indirect probes of membrane potential, triphenylmethylphosphonium ion (TPMP+), 3,3'dipentyloxacarbocyanine [di-O-C5(3)], 3,3'dipentylindocarbocyanine [di-I-C5(3)], and 3,3'dipropylthiodicarbocyanine [di-S-C3(5)] have been used to study neutrophil (PMN) activation. The data extend previous studies indicating that the cyanine dye di-S-C3(5) not only exhibits a different fluorescence response mechanism from di-O-C5(3) [and di-I-C5(3)] but also that the fluorescence of di-S-C3(5) is destroyed by reactive oxygen products produced by neutrophils following stimulation. When these aspects of the probes are taken into account, the interpretations of the results using all three cyanine dyes are identical. Studies with the isotope TPMP+ indicate that long incubations are necessary for PMN to fully equilibrate during which time the PMNs depolarize. Use of TPB-, to shorten the TPMP+ equilibration time, produces results identical with those obtained using the cyanine dyes. The cyanine dyes and TPMP+/TPB- are toxic to neutrophil functions although they do not cause cell death. Toxicity can be avoided by using low concentrations of di-O-C5(3) and di-I-C5(3) but cannot be avoided with di-S-C3(5) or TPMP+/TPB-. Using di-O-C5(3) with the fluorescence-activated cell sorter, we demonstrate that heterogeneity of neutrophil responsiveness confuses the interpretation of studies characterizing the ionic basis of the fluorescence responses stimulated by certain stimuli. We conclude that some of the discrepancies currently reported in the literature using these probes are not due to inherent differences in the ability of the different probes to monitor the same event (i.e., PMN membrane potential) but instead are due to failure to correct for probe-specific problems or response heterogeneity.

Histamine modulation of human neutrophil oxidative metabolism, locomotion, degranulation, and membrane potential changes.

Previous reports have suggested that histamine modulates neutrophil chemotaxis, but this has not been observed by all laboratories. We have re-addressed this controversial point and demonstrate that histamine and H1- and H2-receptor-specific agonists cause limited inhibition of chemotaxis while stimulating chemokinesis. Furthermore, using the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (f-met-leu-phe) as a stimulus, we demonstrate that histamine and H1/H2 agonists inhibit f-met-leu-phe-stimulated changes in membrane potential (monitored with the cyanine dye dipentyloxacarbocyanine), superoxide anion production (cytochrome c reduction), hydrogen peroxide formation (scopoletin fluorescence), and degranulation of granule contents (lysozyme and beta-glucuronidase) in a dose-dependent manner but have no effect on neutrophil functions stimulated by the secretagogues phorbol myristate acetate or A23187. All inhibitory effects of histamine and the H1/H2 agonists are reversed in a competitive manner by the H2 antagonist cimetidine. In addition, structure-activity studies using H1 and H2 receptor agonists and antagonists indicate that a single site with specificity for both H1 and H2 analogue structures modulates the various f-met-leu-phe-stimulated functions studied. Kinetic studies demonstrate that the inhibitory effects of histamine on neutrophil function are only observed when histamine is added before f-met-leu-phe and that inhibition occurs within 10 to 20 sec of histamine addition, does not persist after its removal, and is reversed by addition of cimetidine 10 to 20 sec before stimulation with f-met-leu-phe. Although the inhibitory effects of histamine are exerted early in the sequence of PMN activation by f-met-leu-phe, histamine does not affect the binding or internalization of f-met-leu-[3H]phe. The ability of histamine to modify the variety of neutrophil responses demonstrated in this report suggests an important and direct role for histamine in the regulation of inflammatory reactions in acute allergic settings or other disease states in which histamine release may occur.

Dynamics of human neutrophil receptors for the chemoattractant fmet-leu-phe.

Neutrophils contain an intracellular pool of binding sites for the chemoattractant fmet-leu-phe with the same density as specific granules. This pool of receptors appears to be translocated to the plasma membrane during degranulation and may be important for receptor renewal during chemotaxis. Scatchard analyses of fmet-leu-phe binding to neutrophils and isolated plasma membranes are nonlinear and calculated Hill coefficients of binding to these fractions were about 0.67, suggesting negative cooperativity and/or heterogeneity of receptors. Some of the features of the nonlinear Scatchard curves may reflect heterogeneity of binding among cells since binding of fmet-leu-phe-lys-fluorescein to cells is heterogeneous when simultaneously monitored in the fluorescence activated cell sorter with a fluorescent probe of membrane potential. Following addition of a chemoattractant cells showing a depolarization have more chemoattractant associated with them than cells showing no change of membrane potential or a hyperpolarization. The cell associated chemoattractant is largely in a displaceable pool on the cells showing depolarization while it is in a nondisplaceable pool in the cells that either do not respond or show a hyperpolarization. Studies of the adaptation of human neutrophils responsiveness to fmet-leu-phe elicited membrane potential changes indicate that fmet-leu-phe stimulated decreases in receptor affinity accompany such adaptation. This decreased affinity appears to involve heterogeneity and/or negative cooperative interaction of receptors. The dynamics of chemoattractant receptors that have been reviewed provide further insights for studying the concepts behind the spatial and temporal mechanisms by which cells respond and adapt to a gradient of chemoattractant.

Human neutrophil-specific granule deficiency: a model to assess the role of neutrophil-specific granules in the evolution of the inflammatory response.

It has been suggested that neutrophil (PMN) specific granules are important in cell aggregation, locomotion, hydroxyl radical formation, and in extracellular functions such as the generation of complement-related inflammatory mediators (C5a) and the feedback regulation of myelopoiesis. In the current studies, a 9-yr-old boy with a history of recurrent infections and specific granule deficiency (absent lactoferrin, B-12 binding proteins, and characteristic specific granules on sucrose gradient centrifugation of cell homogenates) was studied to assess some of these concepts. In vivo, the patient had decreased PMN and monocyte accumulation into Rebuck skin windows but an expected febrile episode with an associated neutropenia (PMN margination) and neutrophilia (mobilization of marrow reserves) in response to intravenous endotoxin. In vitro, the patient's resting PMN showed increased ruffling, increased surface-to-volume ratio, and increased numbers of centriole-associated microtubules. His PMN showed a significant decrease in cell negative surface charge (which may relate to aggregation) in response to several stimuli and adhered better than normally to plastic. In addition, his PMN aggregated normally in response to the chemoattractant f-met-leu-phe, although the subsequent disaggregation normally seen with PMN did not occur with the patient's cells. Chemotaxis of the patient's PMN to several stimuli was abnormal, and specific saturable and displaceable binding of the chemoattractant f-met-leu-[3H]phe was decreased. Similarly, following incubation with secretagogues, there was a less than normal increase in f-met-leu-[3H]phe binding and an absence of the normal increases in PMN surface area. The patient's PMN bactericidal activity, stimulated oxygen metabolism (cytochrome-c reduction, chemiluminescence, and NBT reduction), and elicited changes in membrane potential were also abnormal. Studies assessing the mechanism for the abnormal monocyte accumulation into skin windows indicated the patient's monocyte chemotaxis was better than normal in vitro. However, the patient's PMN homogenates lacked a stimulus of monocyte locomotion and did not generate chemotactic activity normally from serum. Thus, the data indicate that specific granule constituents are not required for neutrophil margination in vivo or aggregation in vitro. However, the data support the concept that PMN-specific granules are important for PMN locomotion and oxidative metabolism. In addition, extracellular release of specific granule constituents appears to be important for amplification of the initial and subsequent phases of the inflammatory response.