Sodium stibogluconate (Pentostam) potentiates oxidant production in murine visceral leishmaniasis and in human blood

Sodium stibogluconate (Sbb), a leishmanicidal drug, was studied for its in vivo effect on the formation of reactive oxygen species (ROS), assessed by chemiluminescence (CL) in the whole blood of mice infected with Leishmania infantum. Stimulation of ROS formation induced ex vivo by zymosan particles or the protein kinase C activator phorbol myristate acetate (PMA) was reduced by approximately 25% (P < 0.05) after infection of mice. Treatment of infected mice with Sbb (50 to 400 mg/kg of body weight) enhanced the blood CL induced by zymosan and PMA (47 to 96%, P < 0.01). The drug potentiation effect also occurred in uninfected mice. In vitro treatment of normal human blood with Sbb (1, 10, or 100 microg/ml) for 1 h primed the CL response to PMA (29 to 54%). The priming effect of Sbb was also observed on the production of superoxide by isolated polymorphonuclear leukocytes stimulated either by PMA and zymosan or by the chemoattractants N-formyl-Met-Leu-Phe and platelet-activating factor. These data provide the first evidence of priming of the phagocyte respiratory burst by Sbb. This novel property of Sbb may contribute to the drug's leishmanicidal effect.     

Stimulus- and cumulative dose-dependent inhibition of O2- production by polymorphonuclear leukocytes of patients receiving corticosteroids

Since early in vivo studies in man have remained controversial as to the suppressive effects of glucocorticosteroids on the function of polymorphonuclear leukocytes (PMN), we tried to clarify those effects. The study population involved 19 inpatients on daily and prolonged corticosteroid therapy. Superoxide (O2-) production and chemotaxis were determined as a function of peripheral blood PMN, using various stimuli: concanavalin A (ConA) + cytochalasin D (CD), N-formyl-methionyl-leucyl-phenylalanine (FMLP) and phorbol myristate acetate (PMA), and the chemoattractants FMLP and zymosan activated serum (ZAS). In addition, the relationship between PMN function and corticosteroid dose was also evaluated. There was significant inhibition of PMN O2- production in the patients receiving corticosteroids depending on the stimulus (FMLP or PMA, 51% or 56% of controls; ConA + CD, not inhibited) but no significant inhibition of PMN chemotactic activity. Stimulation with FMLP showed an inverse relationship between O2- production and cumulative prednisolone dose (r = -0.41) in serial determination of O2- production in patients with a negative C-reactive protein (CRP) test. In the serial study of each patient with negative CRP we confirmed the suppression. These results suggest that O2- production by PMN could be inhibited, depending on the cumulative dose of corticosteroids in steroid-treated patients. This may be one possible mechanism of impaired host defences caused by corticosteroid therapy in man.     

Dissociation of human neutrophil activation events by prolonged treatment with amiloride

Human polymorphonuclear leukocytes (PMNs) can be stimulated to release granule contents and to produce superoxide anion. These functional responses are associated with cellular alkalinization and influx of Na+ in exchange for H+. Amiloride is a potassium-sparing diuretic that will inhibit stimulus-induced Na+-H+ exchange and prevent an increase in cell pH. Amiloride has been shown to inhibit a number of protein kinases including the calcium phospholipid-dependent protein kinase. Because PMA, which binds and activates C-kinase, is a potent stimulus of the PMN, this study was undertaken to investigate the effect of prolonged incubation of PMNs with amiloride on PMN stimulation by the chemotactic peptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), the calcium ionophore A23187, opsonized zymosan particles, and the tumor promoter phorbol myristate acetate (PMA). Our results demonstrate that amiloride inhibits superoxide anion production by FMLP, A23187, and opsonized zymosan by causing a slower rate of release and lower maximal release without altering lag time. In contrast, amiloride, despite an inhibition of 22Na+ influx, did not affect superoxide anion production stimulated by PMA. PMN degranulation, phagocytosis, arachidonic acid release, and early influx of calcium were unaffected by preincubation with amiloride. These data suggest that PMN superoxide release induced by FMLP, A23187, and opsonized zymosan is likely modulated by amiloride-sensitive Na+-H+ exchange; and phorbol ester-induced superoxide anion release and degranulation by any stimulant do not appear to be modulated by inhibition of an amiloride-sensitive Na+-H+ exchange.     

Functional effects of monoclonal antibodies (mAbs) against human polymorphonuclear (PMN) adherence antigens

Two mouse monoclonal antibodies (mABs), 25.31 raised against an subunit epitope of LFA1 antigen and Mol against an epitope of the complement receptor type 3 (CR3) were used for investigating their effects on human polymorphonuclear (PMN) functions. The two mABs have an inhibitory effect on PMN adherence. Furthermore, the PMN adherence strength depends upon the support and the adherence induces the capping process of these antigens. Other PMN functions dependent upon adherence were also altered by these two mAbs: random locomotion and that directed by formyl-methionyl-leucyl-phenylalanine (FMLP) or by activated serum, degranulation induced by opsonized or non opsonized zymosan but not by phorbol myristate acetate (PMA), iodination, K562 cell cytotoxicity. Luminol enhanced chemiluminescence of PMN was diminished by both mAbs when PMN were stimulated either by opsonized zymosan or by PMA. Our results confirm other workers' findings, and they are consistent with PMN functional abnormalities observed in children with congenital LFA1, Mol antigens defect.     

Microtiter plate assay for phagocyte-derived taurine-chloramines.

Despite their potential importance, the role of phagocyte-derived chloramines ("long-lived oxidants") has not yet been investigated in inflammatory or infectious diseases. We have developed a sensitive spectrophotometric microtiter plate assay for chloramines based on their capacity to oxidize potassium iodide (KI). Consistent levels of endogenous chloramines were detected in normal human polymorphonuclear neutrophil (PMN) supernatants after stimulation by phorbol myristate acetate (PMA) or opsonized zymosan. Exogenous taurine strongly enhanced chloramine secretion and was used to quantify the chlorinating potential of PMN. Taurine-chloramines were also detectable in monocyte supernatants, although in smaller amounts. The specificity of the KI assay was assessed both in terms of effect of compounds inhibiting (KBr) or interacting with (sodium azide and catalase) chloramine formation and by showing that PMN from patients with chronic granulomatous disease, due to a hereditary lack of oxidative response capacity, were unable to produce chloramines. Taurine-chloramine levels secreted by PMA (but not zymosan)-stimulated PMN were closely related to the cellular luminol-amplified chemiluminescence (CL) responses although the CL assay failed to detect chloramines in PMN supernatants. We consider that this KI assay should be of use in studying the role of long-lived phagocyte-derived oxidants in clinical medicine.     

Re-evaluation of the phagocytic respiratory burst in the physiological or inflammatory state and in aging

We studied the differences in oxygen metabolite generation, using a chemiluminescence (CL) assay, in peripheral blood phagocytic cells from various donors including healthy young volunteers, patients with acute or chronic inflammation, pregnant women, and elderly persons. The CL response of polymorphonuclear leukocytes (PMN) after stimulation with serum-opsonized zymosan was increased in patients with acute inflammation due to infection and in pregnant women as compared with that in controls. Monocytes from those patients also showed a slight increase of the CL response. In contrast, CL of monocytes from patients with chronic inflammation (Crohn's disease patients) and elderly persons was significantly enhanced, whereas that of their PMN remained in the range of control values. The significance of these results was discussed.     

Glycogen metabolism in stored granulocytes

Optimal function of transfused granulocytes (PMNs) requires adequate glycogen metabolism. Previous studies in our laboratory suggested that stored PMNs had decreased glycogen. We report here the glycogen content and chemotaxis of stored PMNs, and the ability of fresh and stored PMNs to use glycogen as the fuel source for chemotaxis. PMNs were prepared from 8 fresh units of blood drawn into citrate-phosphate-dextrose-adenine, suspended at 2 or 8 X 10(7) PMN per ml in autologous plasma with or without 15 mM sodium bicarbonate, and stored at 22 to 24 degrees C in transfer packs for 48 hours. Glycogen was measured on resting PMNs, and after challenge with opsonized zymosan and F-Met-Leu-Phe (FMLP). The chemotaxis of fresh and stored PMNs was measured in the presence or absence of extracellular glucose. Fresh PMNs contained 10.3 +/- 0.5 (mean +/- SEM) micrograms of glycogen per 10(6) PMN. Glycogen decreased by 4.2 +/- 0.9 micrograms per 10(6) PMN after challenge with opsonized zymosan and by 1.1 +/- 0.6 micrograms per 10(6) PMN after FMLP. After 48 hours of storage, neutrophil glycogen increased by 18 percent, except in units stored at a concentration of PMN of 8 X 10(7) per ml without sodium bicarbonate. In PMNs from these units stored without bicarbonate, glycogen decreased by 9 percent (p less than .05), and there was a 19 and 55 percent decrease in the ability of PMN from these units to metabolize glycogen after exposure to opsonized zymosan and FMLP, respectively (p less than 0.05). In addition, in PMNs from units stored at a concentration of PMN of 8 X 10(7) per ml without bicarbonate, there was a 47 and 70 percent decrease in chemotaxis at 24 and 48 hours, respectively (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)     

Abnormal granulocyte functions in autoimmune disorders demonstrated by microcalorimetry

Heat production by polymorphonuclear granulocytes (PMN) was assayed in 14 patients suffering from diseases known to be associated with circulating immune complexes (CIC) and in 10 healthy blood donors. The patients' PMN produced significantly more heat than the normal PMN when the cells were suspended in autologous plasma (23.8 +/- 3.9 pW/cell vs. 18.1 +/- 1.9 pW/cell, p less than 0.0005), but not when the cells were suspended in tissue culture medium (5.6 +/- 1.2 pW/cell vs. 5.6 +/- 0.8 pW/cell, p greater than 0.05). After mixing with IgG-coated latex particles the heat production by patients' PMN were significantly less stimulated than that of normal PMN when the cells were suspended in autologous plasma (42 +/- 13% vs. 75 +/- 22%, p less than 0.0005), but not when the cells were suspended in tissue culture medium (82 +/- 24% vs. 83 +/- 22%, p greater than 0.05). The increase of heat production by normal PMN after stimulation with IgG-coated latex particles was significantly lower when the cells were preincubated with heat aggregated human gammaglobulin compared to that obtained when the cells were preincubated in saline (11 +/- 10% vs. 51 +/- 11%, p less than 0.001). This finding shows that the increased PMN heat production which occurs after binding of the IgG-coated particles is mediated via Fc receptors. A plasma factor(s), possibly circulating immune complexes, can explain the abnormal PMN functions in the patients.     

Effects of adenosine on functions of polymorphonuclear leukocytes from patients with septic shock

Inasmuch as polymorphonuclear leukocytes (PMNs) play a major role in antibacterial defense but can also cause substantial tissue injury, drugs are needed which are able to attenuate tissue-toxic PMN reactions without inhibiting bactericidal mechanisms. Adenosine as a retaliatory metabolite is produced in response to metabolically unfavorable conditions like inflammation. However, it is not known whether adenosine can selectively downregulate adverse PMN reactions in sepsis. In this prospective clinical study, we characterized the effects of adenosine ex vivo on PMN functions in patients with septic shock ([SS] n = 33) and healthy volunteers ([HV] n = 33). The PMNs were primed by tumor necrosis factor-alpha (TNF-alpha) and subsequently stimulated with N-formyl methionyl-leucyl-phenylalanine (fMLP) to test for the formation of hydrogen peroxide (H2O2) in response to soluble inflammatory stimuli. The PMNs were also challenged by opsonized zymosan particles to assess adhesion, phagocytosis, and the associated H2O2 production.As compared with HV, PMNs from SS patients showed strongly enhanced tissue-toxic H2O2 production elicited by TNF-alpha/fMLP. Increasing concentrations of adenosine dose-dependently reduced this tissue-toxic H2O2 production in both groups with a half-maximal inhibitory concentration of 25 nmol/L and 114 nmol/L in HV and SS patients, respectively. This 4.6-fold decrease in the adenosine-mediated inhibition of PMNs from patients with septic shock was compensated by a 3-fold increase in the plasma concentrations of the nucleoside (HV, 42.5 +/- 2.9 nmol/L vs. SS, 125.6 +/- 18.2 nmol/L; mean +/- SEM). When the effects of adenosine were tested at a very high A2A receptor saturating concentration of 10 mol/L, neither adhesion, phagocytosis, nor the associated H2O2 production induced by opsonized zymosan was affected in both groups. These results were confirmed by the highly selective A2A agonist, CGS21680.Thus, adenosine or A2A agonists may be useful to selectively inhibit the potentially tissue-toxic H2O2 production elicited by soluble inflammatory mediators in patients with septic shock.     

Neutrophil activation on biological surfaces. Massive secretion of hydrogen peroxide in response to products of macrophages and lymphocytes.

Recombinant tumor necrosis factor alpha (rTNF alpha) and beta (rTNF beta) did not trigger H2O2 release from PMN in suspension. However, when PMN were plated on polystyrene surfaces coated with serum, fibronectin, vitronectin, laminin, or human umbilical vein endothelial cells (HUVEC), rTNFs induced a massive, prolonged secretory response, similar to that elicited by phorbol myristate acetate (PMA) or bacteria. On serum-coated plates, the maximum sustained rate of H2O2 release in response to rTNF alpha was 2.6 +/- 0.2 nmol/min per 10(6) PMN, the same as that with PMA; release continued for 73 +/- 4 min. On laminin-coated surfaces or HUVEC, release of H2O2 in response to rTNFs was slower, but lasted approximately 3.5 h, reaching the same total (greater than 100 nmol/10(6) PMN). Not only was this response far longer and larger than for other soluble stimuli of the respiratory burst studied with PMN in suspension, but the concentration necessary to elicit a half-maximal response (EC50) for rTNF alpha was orders of magnitude lower (55 pM). Responses were similar with FMLP, but ranged from zero to small with recombinant IFN alpha, recombinant IFN beta, recombinant IFN gamma, platelet-derived growth factor, recombinant IL-1 beta, or bacterial lipopolysaccharide. Adherent monocytes did not secrete H2O2 in response to rTNFs. H2O2 secretion by adherent PMN was first detectable 15-90 min after addition of rTNFs or FMLP. This lag period was unaffected by prior exposure of PMN to rTNF alpha in suspension, by allowing PMN to adhere before adding rTNF alpha, or by incubating adherent PMN in medium conditioned by rTNF alpha-treated PMN. Cytochalasins abolished H2O2 secretion in response to rTNFs, but not FMLP, if added during, but not after, the lag period. Thus, H2O2 secretion from rTNF alpha-treated PMN appears to be a direct but delayed response that requires assembly of microfilaments during exposure to the cytokine. These results suggest that PMN adherent to intra- or extravascular surfaces may undergo a massive, prolonged respiratory burst at the command of macrophages and lymphocytes reacting to microbial products and antigens.     

Phagocytic cell chemiluminescence using different zymosan preparations

This study compared the effectiveness of opsonized and unopsonized zymosan prepared in our laboratories with a commercially available opsonized preparation used for induction of luminol-dependent oxidative burst in phagocytic cells. The production of chemiluminescence (CL) by human whole blood, isolated human neutrophils, normal BALB c mouse splenocytes, and an immortal BALB c mouse macrophage cell line (J774A.1) was tested in an automated luminometer. Recombinant murine or human interferon-gamma (IFN-gamma) and lipopolysaccharide (LPS) were used as priming agents in some of the experiments. With human leukocytes and normal mouse spleen cells the laboratory-prepared zymosans (regardless of opsonization) induced equal or significantly greater CL than did the commercially prepared zymosan. In addition, greatly increased CL was evident with IFN-gamma- and LPS-primed neutrophils tested with our zymosans compared with the commercial preparation. These results suggest that effective zymosans capable of inducing strong, reproducible CL responses from several different phagocytic cell populations can be readily made in the laboratory.     

Shedding of tumor necrosis factor receptors by activated human neutrophils

The capacity of human neutrophils (PMN) to bind tumor necrosis factor (TNF) was rapidly lost when the cells were incubated in suspension with agents that can stimulate their migratory and secretory responses. Both physiological (poly)peptides (FMLP, C5a, CSF-GM) and pharmacologic agonists (PMN, calcium ionophore A23187) induced the loss of TNF receptors (TNF-R) from the cell surface. Half-maximal loss in TNF-R ensued after only approximately 2 min with 10(-7) M FMLP at 37 degrees C, and required only 10(-9) M FMLP during a 30-min exposure. However, there were no such changes even with prolonged exposure of PMN to FMLP at 4 degrees or 16 degrees C. Scatchard analysis revealed loss of TNF-binding sites without change in their affinity (Kd approximately 0.4 nM) as measured at incompletely modulating concentrations of FMLP, C5a, PMA, or A23187. The binding of anti-TNF-R mAbs to PMN decreased in parallel, providing independent evidence for the loss of TNF-R from the cell surface. At the same time, soluble TNF-R appeared in the medium of stimulated PMN. This inference was based on the PMN- and FMLP-dependent generation of a nonsedimentable activity that could inhibit the binding of TNF to fresh human PMN or to mouse macrophages, and the ability of mAbs specific for human TNF-R to abolish inhibition by PMN-conditioned medium of binding of TNF to mouse macrophages. Soluble TNF-R activity was associated with a protein of Mr approximately 28,000 by ligand blot analysis of cell-free supernatants of FMLP-treated PMN. Thus, some portion of the FMLP-induced loss of TNF-R from human PMN is due to shedding of TNF-R. Shedding was unaffected by inhibitors of serine and thiol proteases and could not be induced with phosphatidylinositol-specific phospholipase C. Loss of TNF-R from PMN first stimulated by other agents may decrease their responsiveness to TNF. TNF-R shed by PMN may be one source of the TNF-binding proteins found in body fluids, and may blunt the actions of the cytokine on other cells.     

Comparison of superoxide generation and luminol-dependent chemiluminescence with eosinophils and neutrophils from normal individuals

Although eosinophilia is found in many allergic and hypersensitivity diseases, the function of the eosinophil is not clearly established. To evaluate and characterize this function, anticoagulated blood from normal subjects was separated into purified populations of both eosinophils and neutrophils by a modified method for Percoll gradients. With this separation procedure, highly purified populations of eosinophils (95.0% +/- 2.1%) and neutrophils (97.2% +/- 0.4%) were obtained. Functional response of these two isolated granulocyte cell types was measured by luminol-dependent chemiluminescence (CL) and superoxide generation to opsonized zymosan and phorbol 12-myristate 13-acetate (PMA). Both the eosinophil and neutrophil peak CL response and superoxide generation to zymosan (1 mg), in the presence of autologous serum (10%), were identical. In contrast, when PMA (10(-4) to 10(0) micrograms/ml) was the stimulant, eosinophil CL was at least twofold greater than the neutrophil light emission (1,595,741 +/- 122,435 cpm/5 X 10(5) cells vs. 765,448 +/- 24,171 cpm/5 X 10(5) cells; n = 6). This same differential in responsiveness was seen in superoxide generation. Thus, under certain conditions the eosinophil's respiratory burst may be greater than that of the neutrophil, and this differential in metabolic activity may contribute directly to the eosinophil's inflammatory potential.     

Hydroxyl radical generation by polymorphonuclear leukocytes measured by electron spin resonance spectroscopy.

Electron spin resonance spectroscopy using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) was employed to detect the formation of hydroxyl radicals (OH.) by phagocytosing polymorphonuclear leukocytes (PMN). An electron spin resonance signal with the identical g value and splitting characteristics of the DMPO/OH). adduct was detected on incubation of normal PMN with opsonized zymosan. Adduct formation was strongly inhibited by superoxide dismutase and by the OH. scavenger mannitol, but catalase had little or no effect. (DMPO/OH). was not formed by PMN from a patient with chronic granulomatous disease; in contrast, adduct formation by PMN which lack myeloperoxidase was greater than normal. These findings are discussed in relation to the formation of OH. by PMN.     

Defective polymorphonuclear leukocyte formyl peptide receptor(s) in juvenile periodontitis.

Juvenile periodontitis (JP) is a disease characterized by severe gingival infections. PMN from some JP patients exhibit abnormal chemotactic responsiveness when challenged with the synthetic formyl peptide, FMLP. While investigating PMN function in JP, we found a patient in whom abnormal PMN chemotactic responses to FMLP were associated with a defective population of PMN formyl peptide receptor(s) (FPR). JP PMN failed to respond chemotactically when challenged with FMLP, but exhibited normal chemotactic responses upon exposure to purified human C5a. Furthermore, JP PMN were capable of degranulating and generating superoxide anion radicals as well as normal PMN upon exposure to FMLP. Binding studies demonstrated that JP PMN had a diminution in the number of high-affinity FPR. Studies in which FPR was radiolabeled by chemical cross-linking demonstrated that JP PMN FPR exhibited the same molecular weight and N-linked glycosylation as normal PMN FPR. JP PMN FPR, however, was more resistant to papain cleavage than normal PMN FPR. Autoradiograms obtained from 2D-PAGE of normal and JP PMN FPR demonstrated decreased amounts of FPR isoforms in JP PMN.     

Inhibited neutrophil functions in patients treated with nifedipine but not with verapamil or diltiazem

Neutrophil functions were studied in patients receiving calcium channel blockers: nifedipine, diltiazem or verapamil. Neutrophils from patients treated with nifedipine showed a significantly lower superoxide generation stimulated by phorbol myristate acetate (PMA) (50 ng mL⁻¹), opsonized zymosan (1 mg mL⁻¹) or formyl-methionyl-leucyl-phenylalanine (FMLP) (10⁻⁷  m ), whereas superoxide generation by neutrophils of patients receiving diltiazem or verapamil showed only a slight and insignificant reduction compared with controls. Similarly, chemotaxis towards 10⁻⁷  m FMLP and phagocytosis were significantly lower in patients receiving nifedipine compared with controls and were only slightly reduced in patients receiving diltiazem or verapamil. Nifedipine was the most efficient drug in inhibiting the rise in intracellular calcium ion concentration ([Ca²⁺]_i) when added in vitro and in neutrophils of patients receiving this drug, whereas verapamil had no significant effect. The correlation between the inhibitory effect of nifedipine on neutrophil function and the elevation of [Ca²⁺]_i suggests that nifedipine inhibits neutrophil functions through its effect on [Ca²⁺]_i. However, it is not the sole mechanism as superoxide generation induced by PMA, an agent that does not induce a rise in [Ca²⁺]_i, is also inhibited. The unique effect of nifedipine in reducing neutrophil functions in vivo suggests its clinical implications concerning response to acute ischaemic myocardial events.     

Regulation of monocyte oxidative metabolism: chemotactic factor enhancement of superoxide release, hydroxyl radical generation, and chemiluminescence

Human peripheral blood mononuclear cells exposed to the synthetic chemotactic factor n-formyl-methionyl-leucyl-phenylalanine (FMLP) were enhanced in their ability to generate superoxide anion (O-2), hydroxyl radical (OH.), and chemiluminescence when later exposed to phorbol myristate acetate (PMA). When compared to oxidative responses of cells treated with PMA alone, the degree of enhancement by pretreatment with FMLP was 1.85-fold for O-2 generation, 1.73-fold for OH. production, and 1.34-fold for chemiluminescence. Similarly, pretreatment of mononuclear leukocytes with 5% zymosan-activated serum also enhanced subsequent oxidative responses of cells exposed to PMA. FMLP did not enhance subsequent O-2 release or chemiluminescence by mononuclear leukocytes stimulated by opsonized zymosan or 20 mM sodium fluoride (F-), demonstrating that the O-2 generating system of monocytes stimulated by phagocytosis or F- is not susceptible to chemotactic factor regulation in a manner similar to the system stimulated by PMA. The latter system, like that of neutrophils, is susceptible to regulation by cellular processes activated during an initial encounter with chemoattractants. These processes may provide a mechanism to amplify oxidative responses at sites of infection or inflammation, leading to enhanced efficiency of microbicidal activity or increased tissue damage in vivo.     

Enhanced phagocytic cell respiratory burst induced by spinal manipulation: potential role of substance P.

The effect of spinal manipulation on the respiratory burst of polymorphonuclear neutrophils (PMN) and monocytes from treated adults was measured by zymosan-stimulated chemiluminescence (CL). Peripheral blood was collected 15 min before and 15 min after treatment (sham manipulation, thoracic spine manipulation, or soft tissue manipulation), the cells were isolated, challenged with a standardized, opsonized luminol-containing suspension of zymosan, and monitored for CL. Plasma from two subsets of subjects was radioimmunoassayed for Substance P (SP). PMN were also preincubated with SP in vitro over the dose range 5 x 10(-12) M to 5 x 10(-8) M and the CL response monitored. The CL responses of both PMN and monocytes from subjects who received spinal manipulation were significantly higher after than before treatment, and significantly higher than the response in sham or soft-tissue treated subjects. Measurement of the force applied by sham and spinal manipulation suggested a force threshold for the enhancement of the CL response. Plasma levels of SP before and after treatment in sham treated subjects did not differ significantly; however, elevated plasma SP was observed in subjects after spinal manipulation. Preincubation of PMN with 1 x 10(-11) M, 5 x 10(-11) M or 1 x 10(-10) M SP in vitro primed PMN for an enhanced respiratory burst when the cells were subsequently challenged.     

The influence of phorbol myristate acetate on the metabolism of neutrophils from carriers of sex-linked chronic granulomatous disease.

The present investigation has compared the influences of phorbol myristate acetate (PMA) and heat-killed bacteria (HKB) on oxygen consumption and glucose oxidation by polymorphonuclear leukocytes (PMN) from carriers of sex-linked chronic granulomatous disease (CGD). PMA or HKB caused neutrophils from CGD carriers, considered as a group, to consume oxygen and oxidize glucose-1-14C at rates that were statistically distinguishable from rates of normal controls and affected CGD hemizygotes. PMA at a final concentration of 1.0 micrograms per milliliter wass more effective and reproducible than a ratio of 50 HKB: 1 PMN in discriminating the partial abnormality of carrier PMN from normal PMN. Moreover, a deficiency in glucose oxidation by the PMN of one individual carrier was detectable using PMA stimulation when no defect was apparent with HKB. Results of the present investigation confirm and extend previous observations which have demonstrated the similarity in responses of PMA-treated normal and CGD PMN to the reactions produced by particulates under similar conditions.     

Functional capacity of neutrophil granulocytes in deep-sea divers

Neutrophil granulocytes (PMN) are main defenders against invading microbes. We evaluated the adaptive response of PMN from divers exposed for weeks to high total and oxygen pressures. Under these conditions PMN could be primed to give a heightened respiratory burst upon stimulation with the bacterial peptide analogue, formyl-methionyl-leucyl-phenylalanine (FMLP): blood PMN sampled both shortly after operational saturation dives offshore and during an onshore test-dive gave larger responses than control pre- or post-dive PMN from the same subjects and PMN from laboratory personnel. The assays used measured oxygen consumption, intracellular H2O2 availability, and chemiluminescence. The submaximal responses provoked by the non-metabolizable diacylglycerol analogue phorbol myristate acetate (PMA) were less and less often increased. Such enhanced PMN responsiveness may possibly decrease resistance to skin and other infections that are encountered in divers, if PMN thereby failed to localize correctly to inflamed tissues.