Ontogeny of inflammatory cell responsiveness

Newborn calves, like human infants, are uniquely susceptible to bacterial infections. Part of this increased susceptibility may be related to defects in newborn polymorphonuclear leukocyte (PMN) defensive functions. It remains unclear whether reported deficits in newborn PMN function represent maturational disorders or are manifestations of some form of perinatal suppression phenomenon. We therefore compared the ability of bovine newborn PMNs (less than 24 h old), newborn PMNs (7–10 days of age), fetal PMNs (210–220 days gestational age), and adult PMNs to generate superoxide anion (O ₂ ^– ) as an indicator of respiratory burst activity. Citrated biood was collected, and PMNs were isolated to greater than 95% purity and 98% viability. O ₂ ^– generation was measured as the superoxide dismutase-inhibitable (10 g/ml) reduction of ferricytochrome c (2 mg/ml) after activation of PMNs with phorbol myristate acetate (PMA, 2 g/ml) to directly stimulate protein kinase C. The reaction kinetics were measured (37°C, 550 nm) using a spectrophotometer and chart recorder for continuous monitoring. O ₂ ^– generation was measured for 5 min after the initial lag period and the total nanomoles of O ₂ ^– generated calculated using the extinction coefficient for ferricytochromec. Newborn PMNs (N=10) generated significantly less O ₂ ^– (5.7 ±0.8 nmol O ₂ ^– /10⁶ cells/5 min,P < 0.01) than did adult PMNs (N=14) (9.6 ±2.1 nmol O ₂ ^– /10¹⁰ cells/5 min) or fetal PMNs (N=4) (10.7 ±0.7 nmol O ₂ ^– /10⁶ cells/5 min). PMNs from 7-to 10-day-old calves (N=9) generated almost identical amounts of O ₂ ^– as newborn PMNs (5.7 ±1.6 nmol O ₂ ^– /10⁶ ceils/5 min). There was no difference in measured lag time period between new-born and adult PMNs, but fetal PMNs had significantly reduced (P < 0.01) mean lag time. The data indicated that bovine newborn PMNs have a decreased ability to generate O ₂ ^– in response to PMA stimulation, which persists for at least 7–10 days, and that this functional decrement may be a manifestation of some form of perinatal PMN suppression phenomenon rather than a developmental abnormality since fetal PMNs produced O ₂ ^– as well as adult PMNs.     

Calcium mobilization in C5a-stimulated adult and newborn bovine neutrophils

Calcium (Ca²⁺) is an important second messenger central to many neutrophil (PMN) functional activities. Impaired Ca²⁺ mobilization in newborn PMNs following membrane perturbation could be one of the mechanisms underlying observed abnormalities in neonatal PMN function. To compare Ca²⁺ mobilization in bovine newborn and adult PMNs, cytosolic Ca²⁺ responses after stimulation with recombinant human C5a (rHC5a) were measured. PMNs from normal newborn calves (N=6) and adult cows (N=5) were loaded with fura-2/AM for 60 min at room temperature and the fluorescence changes monitored following stimulation with 0.1, 1, 10, or 50 nM rHC5a in Ca²⁺-containing buffer. Resting levels of Ca²⁺ in both newborn (54.6±1.9 nM) and adult (57.3±1.8 nM) bovine PMNs were comparable. After stimulation with rHC5a, a rapid rise of cytosolic Ca²⁺ was observed, which peaked within 20 sec and was followed by a sustained phase of elevated Ca²⁺ lasting up to 20 min. There were no significant differences (P > 0.05) in peak levels of cytosolic Ca²⁺ obtained by newborn and adult PMNs at 0.1, 10, and 50 nM rHC5a. At 1 nM rHC5a, newborn PMNs reached significantly (P < 0.05) higher levels of cytosolic Ca²⁺ (217.9 ± 21.7 nM) than did adult PMNs (158.7 ± 7.9 nM). At 1 nM rHC5a, newborn PMNs also sustained higher levels of cytosolic Ca²⁺ for 3 min following the peak. At all concentrations of rHC5a tested, the time required to reach the peak and the duration of the peak were comparable in both populations. In the absence of extracellular Ca²⁺ (Ca²⁺-free buffer with 1 mM EGTA), resting levels of cytosolic Ca²⁺ were lower in both newborn (33.3 ± 2.9 nM) and adult PMNs (27.9 ± 2.4 nM) and the magnitude of the peak response to rHC5a was diminished at all concentrations of agonist. Additionally, in the absence of extracellular Ca²⁺, the return to basal cytosolic Ca²⁺ levels occurred rapidly and the sustained phase of increased cytosolic Ca²⁺ seen with rHC5a-stimulated PMNs in Ca²⁺-containing buffer was virtually eliminated. These results indicate that bovine PMNs respond well to rHC5a, that stimulated newborn bovine PMNs can mobilize Ca²⁺ as efficiently as adult PMNs, and that the sustained cytosolic Ca²⁺ response to rHC5a in both age groups requires both release of Ca²⁺ from intracellular stores as well as influx of extracellular Ca²⁺. Such data suggest that observed functional deficits in newborn bovine PMNs are probably not related to improper mobilization of Ca²⁺ following stimulation.     

Neutrophil migration,oxidative metabolism,and adhesion in elderly and young subjects

Objective. To evaluate neutrophil functions in the elderly.Methods. We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects.Results. No difference was found in PMN migration in vivo (62.9±21.3×10⁶ and 65.5±9.1×10⁶ PMN/cm²/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6±2.7 and 9.3±3.3 nMOLES O ₂ ^– /10⁶ PMN respectively, p<0.0001) and by exudate PMNs (13.6±4.3 and 19.4±6 nMOLES O _2– ^{10 6} PMNs respectively, p<0.005).Conclusion. Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Patients with adult respiratory distress syndrome (ARDS) demonstrate in vivo neutrophil activation associated with diminished binding of neutrophil-specific monoclonal antibody 31D8

Neutrophils (PMNs) from patients with adult respiratory distress syndrome (ARDS) were assessed for light scattering, membrane potential, and phagocytic responses using fluorescent probes and flow cytometry to evaluate individual cells. Qualitative and quantitative oxidant responses were measured by nitroblue tetrazolium (NBT) and cytochromec reduction assays, respectively. The results were correlated with the proportion of cells binding the PMN subset-specific monoclonal antibody 31D8. Despite an increased forward scatter signal (4.3±1.6 vs. 1.3±1.1 ARDS vs. control,P=0.041) and spontaneous NBT test (12.6±4.7% vs. 2.5 ±0.8% positive, ARDS vs. control,P=0.033) indicating in vivo priming of ARDS PMNs, there were no significant differences between ARDS and control PMNs in assays of stimulated membrane potential, NBT, and O·₂ ^– production or phagocytosis. However, positive correlations between the degree of prestimulus forward light scatter and subsequent O·₂ ^– production to FMLP (r=0.673,P=0.006) and between the percentage of bands and the O·₂ ^– response to PMA (r=0.660,P =0.003), suggest that the great variability of the ARDS PMN functional responses may relate to varying degrees of in vivo cell priming and/or deactivation. ARDS PMNs demonstrated a significantly lower percentage of 31D8 positive cells (73.4 ±7.5% vs. 94.5±1.6%,P=0.012) and a lower level of 31D8 staining when compared to normals (60.1±10.4% of control level,P=0.001). The lower 31D8 expression did not directly correlate with any functional parameter tested or with the proportion of immature cells. However, patients receiving an intravenous PGE₂1-infusion demonstrated a significant increase in 31D8 staining relative to controls and inhibition of PMA-stimulated O·₂ ^– production. The data suggest that the function of PMNs from ARDS patients varies widely and reflects great in vivo variation in cell priming. While the mechanism responsible for the lowered expression of the 31D8 antigen and its apparent modulation by PGE₁ is unknown, 31D8 may be an indirect marker for in vivo stress factors that regulate the preferential release of a structurally distinct PMN subset from the bone marrow.This work was supported in part by NIH grant P30-DK35747, a University of California, Davis, Dean's Research Grant, and The Upjohn Company.     

Polyl-histidine

Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O ₂ ^– ) and hydrogen peroxide (H₂O₂) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O ₂ ^– took place with 4–5 ×10^–6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/10⁷ PMNs/min, suggesting that this polycation is one of the most potent stimulators of O ₂ ^– generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O ₂ ^– by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O ₂ ^– and H₂O₂ by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O ₂ ^– and H₂O₂ following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O ₂ ^– which were inhibited by CYB. Generation of O ₂ ^– and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H₂O₂ by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.Supported by a research grant from Dr. S. M. Robbins of Cleveland, Ohio.     

Effect of sodium azide on hydrogen peroxide production by zymosan-activated human neutrophils

Stimulated neutrophils (PMNs) produce large quantities of superoxide anion, which is the precursor for hydrogen peroxide (H₂O₂). We developed a new fluorimetric assay to measure the H₂O₂ released by zymosan A-activated PMNs utilizing the oxidation ofp-hydroxyphenylacetic acid by H₂O₂ to its fluorescent dimer in the presence of horseradish peroxidase. Zymosan-activated PMNs isolated from nine healthy volunteers and 20 patients with acute hypoxemic respiratory failure (AHRF) released after 90 min 2.3±0.3 and 2.4±1.3 nmol H₂O₂/10⁶ PMNs, respectively. Inhibition of the heme enzymes by 1.0 mM sodium azide (NaN₃) increased the H₂O₂ production to 21.6±4.4 nmol H₂O₂/10⁶ PMNs in the control group (P<0.001), and to 22.5±14.7 nmol H₂O₂/10⁶ PMNs in patients with AHRF (P<0.001). Incubation temperature, room temperature or 37C, did not change the total amount of H₂O₂ produced after 90 min by zymosan-activated PMN. Addition of NaN₃ improved both the sensitivity and reproducibility of the measurement of H₂O₂ and allowed detection of H₂O₂ released by PMNs with coefficients of variation of less than 5% at PMN concentrations as low as 0.1×10⁶ cells/ml. The amount of H₂O₂ released by activated PMNs did not distinguish healthy controls from patients with AHRF.     

Human polymorphonuclear leukocytes release leukotriene B4 during phagocytosis ofStaphylococcus aureus

We studied release of leukotriene B₄ (LTB₄) by human polymorphonuclear leukocytes (PMNs) during phagocytosis of staphylococci in the presence or absence of arachidonic acid. The 12×10⁷ PMNs incubated with 3×10⁹ opsonizedS. aureus and 50M arachidonic acid released 1.45±0.42 nmol LTB₄. No LTB₄ was detected after stimulation of PMNs withS. aureus or arachidonic acid by themselves. However, by increasing the concentration of arachidonic acid to 200 or 400M, 1.22±0.45 and 1.98±0.49 nmol LTB₄, respectively, was released by PMNs. The effect of different bacteria-PMN ratios on LTB₄ production was also studied. LTB₄ varied from 0.3 to 2.0 nmol when bacteria/PMN ratios increased from 5 to 50 (respectively) in the presence of 50 M arachidonic acid. Thus, phagocytizing PMNs produce LTB₄ in the presence of arachidonic acid, and its production is dependent on the number of bacteria phagocytized.     

Stimulus-dependent actin polymerization in bovine neutrophils

Polymorphonuclear leukocytes (PMNs) are responsible for much of the first wave of leukocyte-mediated host defense against microbial pathogens. In order to migrate through the endothelium of vessel walls, undergo chemotaxis, and phagocytize microbes, PMNs must modulate their cytoskeletal elements and undergo change of cellular shape. We have used fluorescence flow cytometric analysis and cellular microscopic observations to demonstrate actin polymerization in bovine PMNs and to examine the kinetics of PMN actin polymerization utilizing different PMN stimuli. In addition, we compared temporal relationships between cellular shape and actin polymerization. Actin polymerization occurred rapidly, and the kinetics of actin polymerization were similar for each of the three PMN agonists used, ZAS (10%), PAF (10^–6 M), and rhC5a (10^–7 M). Actin polymerization was near-maximal by 10 sec poststimulation (95.4% of maximal F-actin content attained by 10 sec poststimulation with ZAS stimulation), and reached peak values by 30 sec. The maximal increase in F-actin content of agonist-stimulated cells as compared to resting cells was 2.8-fold with ZAS; 2.3-fold with PAF; and 2.3-fold with rhC5a. PMN shape change (pseudopodia, membrane raffles) was not as rapid, with only 22.4% of cells attaining visible membrane deformation by 10 sec and requiring 120 sec to reach peak shape-change values. After attaining peak values, the two events also differed. Whereas the percent of shape-changed PMNs remained plateaued up to 5 min poststimulation, the F-actin content gradually decreased after 30 sec, approaching F-actin values of unstimulated PMNs.     

Occupancy of adenosine receptors on human neutrophils inhibits respiratory burst stimulated by ingestion of complement-coated particles and occupancy of chemoattractant but not Fc receptors

Chemoattractants are generated at inflammatory loci that not only induce neutrophils (PMNs) to leave the vasculature but also stimulate PMNs to release potentially toxic agents (e.g., H₂O₂, O ₂ ^– or OH). We have recently demonstrated that endothelium releases adenosine which, when bound to a specific receptor on the PMN surface, inhibits release of toxic oxygen metabolites from stimulated PMN. To determine whether occupancy of adenosine receptors modulates generation and release of oxygen metabolites, we have studied the effect of 2-chloroadenosine on O ₂ ^– generation and O₂ consumption in response to opsonized zymosan particles (STZ) and immune complexes (IC). 2-Chloroadenosine inhibits, in a dose-dependent fashion, Of generation by neutrophils that have been exposed to C3b-coated particles (STZ). Inhibition of Of generation is similar in the presence or absence of cytochalasin B (IC₅₀=53 ±19 and 16 ±5nM, respectively,P=NS). Since occupancy of adenosine receptors might inhibit only externalization but not generation of oxygen metabolites, we studied the effect of 2-chloroadenosine on oxygen consumption by activated neutrophils. 2-Chloroadenosine inhibited O₂ consumption stimulated by STZ and the surrogate bacterial chemoattractant FMLP; however, inhibition of O₂ consumption varied with the presence or absence of cytochalasin B. In contrast, when neutrophils were stimulated by immune complexes, 2-chloroadenosine only minimally inhibited O ₂ ^– release and O₂ consumption (10 ± 5 and 5 ± 4% inhibition, respectively). Thus, occupancy of adenosine receptors inhibits O₂ consumption in parallel with inhibition of O ₂ ^– release. These results support the hypothesis that ingestion of complement-opsonized particles stimulates the respiratory burst by a mechanism different from that by which the respiratory burst is stimulated after occupancy of Fc receptors. Moreover, these observations suggest that endothelium, by releasing adenosine, prevent activated neutrophils from damaging the microvasculature at inflammatory loci. In contrast, deposition of immune complexes in vessel walls leads to vascular damage because endothelial cells are incapable of preventing attack by immune complex-stimulated neutrophils.This research was supported by grants from the U.S. Public Health Service (AI-10343 and HL29034) and the Veterans Administration.Dr. Cronstein is the recipient of a Clinical Investigator Award (K11-AR-01490) and was a fellow of the Arthritis Foundation. Dr. Broekman was an Established Investigator of the American Heart Association.     

Neutrophil responses to intravascular pneumococcal sonicate

Leukopenia and pulmonary leukostasis are prominent features in patients succumbing to pneumococcal (PNC) infections. We examined mechanisms involved in recruitment of polymorphonuclear neutrophils (PMNs) into pulmonary capillaries and alveolae after PNC sonicate injection. We showed that by 15 min postinjection, PMN chemotactic activity was found in bronchoalveolar lavage (BAL) fluids and increased with time until the end point of the study at 90 min. Accompanying the increased chemotactic activity in BAL fluids was a decrease in circulating PMNs more pronounced in the femoral artery (FA) then the pulmonary artery (PA). Super-oxide anion (O ₂ ^– ) production by peripheral PMNs was depressed following PNC sonicate injection, and comparison of FA and PA showed that FA PMNs produced less O ₂ ^– than PA PMNs. PA PMNs also showed enhanced random migration when compared to the depressed random migration of FA PMNs. This study demonstrated that an intravascular challenge of PNC sonicate was associated with increased chemotactic activity for PMNs in BAL fluid. Fewer PMNs and altered PMN function resulted from passage through the pulmonary microvasculature after PNC sonicate injection.Supported in part by USDHHS grant CA 20819 from the National Cancer Institute and from the Veterans Administration Research Service.     

Granulocyte chemotaxis in the canine trachea: Inhibition by lipid mediator antagonists and systemic inhibitors

Inflammation of the airways contributes to the multicomponent disease known as asthma. The primary cells that infiltrate the airways in response to antigen exposure are PMNs and eosinophils, cells that can release cellular components, and damage the airways. We adapted a double-ballon endotracheal tube to study the cellular response to threede novo synthesized lipid mediators (LTB₄, PAF-acether and 15 HETE) found in respiratory fluids following antigen exposure.In random repeat challenges in groups of 7 dogs using mongrel dogs at 240 min following exposure to 10^–6 M agonists, the PMN content of the perfused fluid was 870±240, 1632±883, 515±395, and 1575±214 cells/ml/5 high power fields for vehicle, LTB₄, PAF, and 15 HETE respectively. Eosinophils that infiltrated the lumen at 240 min were 162±23, 608±287, 502±23, 115±14 cells/ml/5 HPF for vehicle, LTB₄, PAF, and 15 HETE respectively. Thus LTB₄ and PAF-acether significantly (p<0.05) increased eosinophils, and LTB₄ and 15 HETE increased PMNs (p<0.05). After determining the agonist response for the 3 agonists we included 2 specific antagonists in the perfusate. The LTB₄ antagonist U-75,302 10^–5 M, and the PAF antagonist L 652,731 10^–5 M in chambers containing LTB₄ and PAF-acether respectively blocked significantly the influx of PMNs and eosinophils compared to vehicle (p<0.01). Methylprednisolone 5 mg/kg i.m.-18 hrs blocked eosinophilia to PAF and LTB₄. Oral U-78,517F a Trolox amine lazaroid, active as an inhibitor of lipid peroxidation, 30 mg/kg-18 hrs significantly blocked eosinophilia to PAF-acether and LTB₄ directed chemotaxis compared to vehicle (p<0.05) but not 15 HETE. Specificity was shown for each antagonist since the PAF and LTB₄ antagonists did not block the opposite agonist. Use of this novelin vivo chemotaxis model allows the additional advantage of studying chemotaxis in living tissue.     

Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic

Net transepithelial transport (and cellular accumulation) of the dipeptide glycylsarcosine (Gly-Sar), across the apical membrane of human intestinal Caco-2 epithelia, is driven by a proton gradient (Na⁺-free conditions) and displays saturation kinetics (K_m 17.4±5.1 mM, V_max of 92.8±15.6 nmol.cm^–2.h^–1). Net Gly-Sar transport is associated with the stimulation of an inward short-circuit current (I_sc). This dipeptide-stimulated I_sc is observed in both Na⁺-containing and Na⁺-free conditions, is stimulated by apical acidity, and displays saturation kinetics (in Na⁺-free media at apical pH 6.0, K_m of 13.6±4.5 mM and a V_max of 284.1±39.3 nmol.cm^–2.h^–1). The maximal capacities of Gly-Sar transport and I_sc suggest a dipeptide/proton stoichiometry greater than unity (13).     

Neutrophils from asthmatics exhibit diminished responsiveness to 2-chloroadenosine which is reversed by theophylline

We have shown previously that neutrophils (PMNs) from patients with asthma have a more potent stimulated respiratory burst than normals and that their respiratory burst is significantly less suppressed with exposure to 2-chloroadenosine (2-CADO). The present studies investigated the basis of this defect in responsiveness to 2-CADO. PMNs obtained from asthmatics either not on theophylline (minus theophylline) or taking theophylline (plus theophylline) generated significantly more superoxide in response to 2×10^–8 M FMLP (2.08±0.36 nmol/5×105 PMNs (minus theophylline) (P<0.01 compared to controls) vs. 2.16±0.44 (plus theophylline) (P<0.01) as compared to controls (1.05±0.17 nmol). In the presence of FMLP (2×10^–8 M), PMNs from the minus theophylline cohort had less 2-CADO (10^–6 M) -mediated suppression of superoxide generation as compared to controls (38.3±3.8% vs. 67±3.8%; (P<0.001). The plus theophylline group exhibited suppression values similar to controls (64.5±7.2%). Theophylline, in the presence of a physiological concentration of 2-CADO (0.1M) accentuated the suppression of the respiratory burnt in normals (74.1±5.9%, 80.1±4.9% (P<0.02) and 84.7±3.8% (P<0.02) at 0, 10, and 100M, respectively). PMNs from asthmatics not taking theophylline demonstrated suppression values of 46.2±6%, 53.8±6.6% (P=NS), and 63.2±7.1% (P<0.01), respectively. Resting PMNs from normal controls generated 0.97±0.20 pmol cAMP/10⁷ cells compared to 2.83±0.75 pmol in the pressnce of 0.1M 2-CADO. The combination of 2-CADO and theophylline (10–100M) produced cAMP concentrations not significantly different from that observed with 2-CADO alone. These findings support the existence of a novel cAMP-independent adenosine receptor in PMNs. The specific binding of 10^–8 M³H-labeled 2-CADO (in cpm) was 10,358 ± 1502 (P < 0.001 compared to controls), 5468 ± 843 (NS compared to controls), and 3751 ± 477 in the plus theophylline group, minus theophylline group, and controls, respectively. Such up-regulation of specific binding may represent the effects of theophylline as shown by the specific binding of [³H]2-CADO in PMNs from normal controls exposed to 10 M theophylline for 30 min (6013 ± 969) compared to unexposed PMNs (3768 ± 656; P < 0.05). These data support an antiinflammatory mechanism of action for theophylline in the therapy of asthma and suggest that this may occur through potentiation of the antiinflammatory mediator adenosine.This research was supported by NIH grants AI24843 and AI24848.     

Mechanism of NaCl secretion in rectal gland tubules of spiny dogfish (Squalus acanthias)

Segments of rectal gland tubules (RGT) the spiny dogfish (Squalus acanthias) were perfused in vitro to study the cellular mechanism by which NaCl secretion is stimulated. Transepithelial PD (PD_te), transepithelial resistance (R_te), the PD across the basolateral membrane (PD_bl), the fractional resistance of the lumen membrane (FR₁), and the cellular activities for Cl^–, Na⁺, and K⁺ (a _x ^cell ) were measured. In series 1 the effects of stimulation (S) (dbcAMP 10^–4, adenosine 10^–4, and forskolin 10^–6 mol · l^–1) on these parameters were recorded and compared to nonstimulated state (NS). PD_te increased from –1.9±0.2 mV to –11.0±0.9 mV (n=51). PD_bI depolarized from –86±1 to –74±1.4 mV (n=52). R_te fell from 29±2.8 to 21±2 cm² (n=23), and FR₁ fell from 0.96±0.005 to 0.79±0.04 (n=9).a _K+ ^cell was constant (123±13 versus 128±17 mmol · 1^–1) (n=6), buta _Cl– ^cell -fell significantly from 48±4 to 41±3 mmol · l^–1 (n=7).a _Na+ ^cell increased from 11±2.1 to 29.5±6.6 mmol · l^–1 (n=4). In series 2 the conductivity properties were examined by rapid K⁺, and Cl^– concentration steps on the basolateral and luminal cell side respectively in NS and S states. In NS-segments reduction of bath K⁺ led to a hyperpolarization of PD_bI with a mean slope of 28±1.3 mV/decade (n=9) (as compared to 19 mV/decade for S-state). Reduction of lumen Cl^– led to very little depolarization of the lumen membrane PD in NS-state: 6.5±2.3 mV/decade (n=4) (as compared to 13 mV/decade for S-state). In series 3 the effects of furosemide (7 · 10^–5 mol l^–1, bath) were examined in NS and S tubules. In NS RGT segments furosemide had no effect on PD_bI or PD_te;a _Cl– ^cell fell slowly after furosemide with an initial rate of 0.33 mmol · l^–1 s^–1, as compared to 1.5 mmol · l^–1 · s^–1 for S-state. The increase ina _Cl– ^cell after removal of furosemide from NS to S-states was examined in the presence of furosemide. Despite the presence of furosemide stimulation was accompanied by a fall in R_te, FR₁, anda _Cl– ^cell . From these data we conclude that (a) stimulation by cyclic AMP increases the Cl^–-conductance of the apical cell membrane at least by a factor of 10, that (b) in the NS-state the Na⁺2Cl^–K⁺ carrier can be triggered to work at rates similar to the S state by loweringa _Cl– ^cell , and that (c) the increase in apical Cl^–-conductance is the primary event in cyclic AMP mediated stimulation of NaCl secretion.Supported by Deutsche Forschungsgemeinschaft Gr 480/8-1, and by NIH Grant AM 34208     

Decreased production of nitric oxide by human neutrophils during septic multiple organ dysfunction syndrome

The objective of this study was to determine nitric oxide (NO) and superoxide anion release (O ₂ ^– ) by neutrophils (PMNs) in the septic multiple organ dysfunction syndrome (MODS) and to compare them with the response of normal cells to lipopolysaccharide (LPS) and cytokines. NO production was measured by the release of nitrites in the medium, its maximal production rate by a modified oxyhemoglobin assay and O ₂ ^– by standard methods. Normal cells were incubated with LPS, gamma interferon (IFN-), or tumor necrosis factor (TNF-) alone or in combination. Results showed that PMN release of both NO and O ₂ ^– was reduced in septic samples; in contrast, an association of LPS, IFN-, and TNF- promoted maximal NO release by normal cells (40–50%). We conclude that while interaction of normal PMNs with cytokines increases NO and O ₂ ^– release, progression of sepsis to a multiple organ dysfunction impairs these responses in both functions.     

Role of myeloperoxidase in respiratory burst of human polymorphonuclear leukocytes

A comparative study of the respiratory burst [monitored as superoxide (O₂ ^–) production] of normal and myeloperoxidase (MPO) -deficient poiymorphonuclear leukocytes (PMNs) was carried out on 11 MPO-deficient subjects that represent the largest sample of this kind ever studied. The rate of O₂ ^– production by isolated PMNs and whole blood from normal and MPO-deficient subjects was comparable during the initial 30–40 min of incubation with serum-treated zymosan (STZ). Afterwards, the amount of O₂ ^– produced became progressively higher in MPO-deficient cells at least until 120 min incubation with STZ. On the contrary the rate of O₂ ^– production by both cell types in response to 4--phorbol-12-myristate-13-acetate (PMA) was the same. The PMNs of four MPO-deficient subjects were tested for their ingestion ability by counting the number of ingested particles on toluidine blue-stained sections of epoxy-embedded PMN suspensions. Both cell types ingested STZ particles at a comparable rate at early postphagocytic times, whereas on prolonged incubation MPO-deflcient PMNs ingested more STZ particles than normal PMNs. These results suggest that the ingestion capacity of normal cells may undergo a more rapid deterioration than that of MPO-deficient cells during incubation with STZ. Evidence for a higher deterioration of normal PMNs with respect to MPO-deficient PMNs was obtained also from studies on the effect of storage on O₂ ^– generation. After standing at melting ice temperature for 3 h, normal PMNs produced less O₂ ^– than MPO-deficient PMNs in response to PMA, and the difference in O₂ ^– production by the two cell types in response to STZ was evident at earlier postphagocytic periods than with freshly isolated cells. Taken all together these results suggest that normal PMNs and MPO-deficient PMNs do not intrinsically differ in O₂ ^– generating potential and that the difference in the respiratory burst observed during phagocytosis may be accounted for by a more marked deterioration, in normal PMNs, of one or more functions related to the respiratory burst.     

Influence of neutrophil cationic proteins on generation of superoxide by human polymorphonuclear cells during phagocytosis

The cationic proteins from neutrophyl lysosomes have been shown to modulate phagocytic activity of granulocytes. The present study reports the effects of the cationic protein fractions on the generation of O ₂ ^– by human PMNs during phagocytosis. Human PMNs were reacted win different phagocytic stimuli in the presence and absence of lysosomal cationic proteins and the amount of O ₂ ^– generated was determined by superoxide dismutase inhibitable reduction of cytochromec. Total cationic protein extract from neutrophil lysosomes enhanced O ₂ ^– generated by PMNs during the phagocytosis of IgG-coated latex beads and opsonized zymosan particles. The analysis of the fractions of cationic proteins obtained from a Sephadex G-75 column showed that the O ₂ ^– generation-enhancing activity was associated with the proteins eluted in fractions III and IV. A protein fraction mainly eluted in void volume inhibited the cytochromec reduction by O ₂ ^– formed during phagocytosis. This was due to the presence of superoxide dismutase-like activity since O ₂ ^– generated by the xanthine-xanthine oxidase system was also inhibited by this fraction. The cationic protein fractions III and IV from the Sephadex G-75 column were further subfractionated. Although the O ₂ ^– -enhancing activity was eluted in the same fractions as chymotrypsin activity, there was no quantitative correlation between the amount of O ₂ ^– generation and chymotrypsin activity. Moreover, commercial chymotrypsin did not enhance O ₂ ^– generation. Electrophoretic analysis of the isolated protein fractions suggests that O ₂ ^– generation enhancing protein (SGEP) is different from lysozyme or chymotrypsin and probably represents previously undescribed protein.     

Effects of dietary supplementation with eicosapentaenoic acid or gamma-linolenic acid on neutrophil phospholipid fatty acid composition and activation responses

Previous data that alimentation with fish oil rich in eicosapentaenoic acid (EPA; 20: 5n-3) or vegetable oil rich in gamma-linolenic acid (GLA; 18: 3n-6) can reduce symptoms of inflammatory skin disorders lead us to determine the effects of dietary supplements of oils rich in EPA or GLA on guinea pig (GP) neutrophil (PMN) membrane potential (), secretion, and Superoxide (O ₂ ^– ) responses. Weanling GPs were initially fed diets supplemented with olive oil (<0.1% EPA; <0.1% GLA) for 2 weeks, followed by a crossover by two sets of animals to diets supplemented with fish oil (19% EPA) or borage oil (25% GLA). At 4-week intervals, 12% sterile casein-elicited peritoneal neutrophils (PMN) were assessed for membrane polyunsaturated fatty acid (PUFA) profiles and FMLP-, LTB ₄ ^s- , and PMA-stimulated changes, changes in flow cytometrically measured forward scatter (FWD-SC) (shape change), 90 scatter (90-SC) in cytochalasin B-pretreated-PMN (secretion response), and Superoxide responses. GP incorporated EPA and GLA (as the elongation product, dihomo-GLA or DGLA) into their PMN phospholipids by 4 weeks. The peritoneal PMN of all groups demonstrated broad resting FWD-SC and poor activation-related FWD-SC increases, suggesting in vivo activation. While secretion was comparable in the three groups in response to FMLP, there was a trend toward inhibition of LTB₄-stimulated 90-SC loss in both fish and borage oil groups. This was significant only with borage oil (21.7±2.1 vs 15.3±1.2% loss of baseline 90-SC, olive vs borage;P=0.03). PMN from borage- and fish oil-fed GPs showed a progressively lower O ₂ ^– response to FMLP than the olive oil group (73.9±3.9 and 42.9±6.8% of olive oil response for borage and fish oils, respectively;P< 0.005 andP<0.01, respectively, at 12 weeks), while PMA-stimulated O ₂ ^– was inhibited only in the fish oil-fed group and only at 12 weeks (62.0±2.7% of control;P<0.025). We conclude that dietary supplementation with oils rich in PUFAs can modify PMN activation responses.This work was supported in part by Research Grants AM-30679, AR 39040, and P30-AM 35747-02 from the U.S. Public Health Service.     

Functional heterogeneity in the hamster medullary thick ascending limb of Henle's loop

Cellular heterogeneity was examined in the hamster medullary thick ascending limb (MAL) perfused in vitro by electrophysiological measurements with an intracellular microelectrode. Random measurements of fractional resistance of basolateral membrane (Rf _B) revealed two cell populations, high basolateral conductance (HBC) cells havingRf _B of 0.05±0.01 (n=24) and low basolateral conductance (LBC) cells havingRf _B of 0.80±0.03 (n=32). Basolateral membrane potentials (V _B) were not different between HBC cells and LBC cells (–72.6±1.2,n=43 vs. –70.0±1.2,n=35). Addition of 2 mmol/l Ba²⁺ to the bath depolarized the basolateral membrane in the HBC cells from –70.4±3.2 to –20.9±5.9 mV (n=8) but not in the LBC cells (from –74.4±1.9 to –72.0±2.1 mV). Increasing K⁺ or decreasing Cl^– in the bathing solution caused marked positive deflection ofV _B in the HBC cells but little or no change inV _B in the LBC cells. Elimination of Cl^– from the lumen or addition of furosemide to the lumen enhanced the potential response of the HBC cells to basolateral application of Ba²⁺. Accordingly, with Ba²⁺ present in the bath, the potential response of the HBC cells to a decrease in bath Cl^– concentration was enhanced. These observations suggest that a K⁺ conductance exists in the basolateral membrane of HBC cells in paralled with a Cl^– conductance. The basolateral cell membrane of LBC cells also contains a Cl^– conductance. In these cells, but not in HBC cells, the potential response to decreasing bath Cl^– concentration increased when bath pH was decreased from 7.4 to 6.0 Apparent K⁺ transference numbers of the luminal membrane were higher in LBC cells (0.74±0.05,n=7) than in HBC cells (0.20±0.02,n=5). From these data, we conclude: (1) there are two distinct cell types in the hamster medullary thick ascending limb; (2) there is a low Cl^– conductance in basolateral membrane of LBC cells which is stimulated by low pH.     

Interleukin-18 Primes the Oxidative Burst of Neutrophils in Response to Formyl-Peptides: Role of Cytochrome b558 Translocation and N-Formyl Peptide Receptor Endocytosis

Using flow cytometry, we observed that interleukin-18 (IL-18) primed human neutrophils (PMNs) in whole blood to produce superoxide anion (O₂°⁻) in response to N-formyl peptide (fMLP) stimulation, whereas IL-18 alone had no significant effect. In contrast to tumor necrosis factor alpha (TNF-α), which is a cytokine known to strongly prime O₂°⁻ production, IL-18 did not induce either p47^phox phosphorylation or its translocation from the cytosol to the plasma membrane. However, IL-18 increased PMN degranulation, as shown by increased levels of cytochrome b558 and CD11b expression at the PMN surface. Moreover, addition of IL-18 to whole blood for 45 min reduced the ability of PMNs to bind to fMLP, suggesting endocytosis of fMLP receptors, as visualized by confocal microscopy. 2,3-Butanedione 2-monoxime, which inhibits endosomal recycling of plasma membrane components back to the cell surface, concomitantly accentuated the diminution of fMLP binding at the PMN surface and increased IL-18 priming of O₂°⁻ production by PMNs in response to fMLP. This suggests that fMLP receptor endocytosis could account, at least in part, for the priming of O₂°⁻ production. In addition, genistein, a tyrosine kinase inhibitor, and SB203580, a p38 mitogen-activated protein kinase (p38MAPK) inhibitor, completely reversed the decreased level of fMLP binding and increased the level of CD11b expression after IL-18 treatment. Flow cytometric analysis of intact PMNs in whole blood showed that IL-18 increased p38MAPK phosphorylation and tyrosine phosphorylation. In particular, IL-18 induced phosphorylation of focal adhesion kinase (p125^FAK), which has been implicated in cytoskeleton reorganization. Taken together, our findings suggest several mechanisms that are likely to regulate cytokine-induced priming of the oxidative burst in PMNs in their blood environment.