Alterations of regular and mature monocytes are distinct,and dependent of intensity and duration of exercise

Circulating monocytes comprise functionally distinct regular (CD14^bright+) and mature (CD141^low+) cells. Cell surface receptors were determined by three colour flow cytometry in 8 healthy control subjects. Compared to regular monocytes, mature monocytes had lower levels of the high affinity Fcy receptor 1 (CD64), complement receptor 3 (CDllb), CD45RO and higher levels for HLA-DR, LFA-1 (CD11a/CD18), interleukin-2 receptor (CD25), CD45RA and the Fc receptor 3 (CD16). Both regular and mature monocytes were measured before and up to three hours after four different types of exercise (Ex) in endurance trained athletes (n=9-16). Immediately after anaerobic exercise of I min with a maximal lactate concentration (la_max) of I2.3 (SD I.4) mmol · l^–1 and exhaustive exercise of 24 (SD 8) min with a maximal lactate concentration (la_max) of 7.4 (SD 2.6) mmol· l^–1 mature monocytes increased more than regular monocytes. Exhaustive endurance exercise of 87 (SD 21) min [la_max 3.7 (SD I.0)] led to a similar increase of regular and mature monocytes. 15–33 min after a 100km run regular monocytes increased significantly, whereas mature monocytes decreased. Up to three hours after the end of all exercises mature monocytes fell below pre-exercise values. In conclusion, duration and intensity of exercise alter distinct maturation stages of monocytes differently. It is probable that the avidity of adhesion molecules like LFA-1 to their endothelial ligands is increased to enable the firm attachment to the endothelium.     

Study of the phagocytic process in neutrophils from elite sportswomen

Summary All the stages of the phagocytic process of blood neutrophils were compared in sedentary young women (no formal exercise during the previous 24 months) and elite sportswomen (basketball players from the Siglo XXI Spain Selection, in the middle of their competitive season) at rest. The sportswomen had performed no exercise since the day before taking the blood samples. Adherence of neutrophils to nylon fibre, which is similar to endothelium adherence, was not different between the two groups [62 (SD 14) and 58 (SD 18) in control and sport groups respectively]. Chemotaxis (studied in a Boyden chamber using a filter with 3 m pore diameter) was found to be stimulated (P<0.001) in the sportswomen [105 (SD 30)] with respect to the controls [39 (SD 9)]. Attachment, ingestion and killing by neutrophils was measured by incubation of the neutrophils with serum and a suspension ofCandida albicans. While no statistical differences were found in attachment ofC. albicans after 15 min incubation [71 (SD 8) in the control group, and 74 (SD 20) in the sport group], the sportswomen showed a higher (P<0.001) ingestion capacity forC. albicans at both 15 min [53 (SD 13) and 111 (SD 32) in control and sportswomen respectively] and 60 min [control 90 (SD 10), and sport group 224 (SD 21)] incubation. The greater phagocytic capacity in sportswomen was correlated with a higher plasma cortisol concentration (P <0.05) and a lower plasma ACTH concentration (P <0.001) in this group. It is concluded that elite women basketball players have a greater phagocytic capacity than sedentary women, possibly mediated by the higher plasma cortisol concentration in the sportswomen.     

Circulating leucocyte and lymphocyte subpopulations before and after intensive endurance exercise to exhaustion

Summary Seventeen healthy cyclists [age 20.8 (SD 4.8) years; body mass 68.3 (SD 7.7) kg; body fat, 11.4 (SD 2.6) %; height, 179.1 (SD 5.9) cm; 60.9 (SD 7.4) ml · kg^–1 · min^–1] conducted intensive endurance exercise to exhaustion (stress test, ST) on a cycle ergometer at 110% of their individual anaerobic threshold [Th_an,individua; exercise intensity, 3.97 (SD 0.6) W · kg^–1 ; duration, 23.9 (SD 8.3) min; maximal lactate concentration, 7.39 (SD 2.59) mmol · 1^–1]. The distribution of leucocyte subpopulations was measured flow cytometrically: before, immediately after (0), 5 (+5), 30 (+30) and 60 (+60) min after ST. The lymphocytes (0 min) and granulocytes (+60 min) were mainly responsible for the increase of leucocytes. Lymphocytes were significantly lower at +30 and + 60 min than before. CD3^–CD16/CD56⁺ (+480%) and CD8⁺-lymphocytes (+211%) increased at 0 min more than the other lymphocyte subpopulations (CD3+-cells, +100%; CD4⁺ cells, +56%; CD19⁺-cells, +64%). CD3^–CD16/CD56⁺-and CD8⁺-cells also were mainly responsible for the decreased values of lymphocytes at +30 min and +60 min compared to before. At 0 min naive CD8⁺ cells (CD45RA⁺, CD45RO^–) increased more than memory CD8⁺-cells (CD45RA^–, CD45RO⁺). Changes of naive and memory CD4⁺-cells did not differ. All lymphocyte subpopulations, in particular CD8⁺- and CD3^–CD16/CD56⁺-cells, decreased rapidly between 0 min and 5 min. We conclude that an intensive endurance exercise to exhaustion causes a mobilisation of lymphocytes, especially of natural killer cells (CD3^–CD16/CD56⁺) and naive, unprimed CD8+ cells (CD45RA⁺, CD45RO^–) which may be transported to injured muscles. The decreased cell numbers of the latter subpopulations are possibly one reason for the susceptibility to infections during the first hours after exercise. Furthermore, an exact definition of the intensity of exercise and times of taking blood is essential for comparing results describing cell parameters during or after exercise.     

Regulation of Con A – dependent cytokine production from CD4+ and CD8+ T lymphocytes by autosecretion of histamine

Objectives: Previously we have shown that both CD4+ T cells and CD8+ T cells produce histamine when activated with Con A. The aim of this study was to examine whether cytokine production by these cells is regulated by autosecretion of histamine.Materials: CD4+ and CD8+ T cells were separated from spleen cells of C57BL/6 mice and mice lacking the H1 receptor (H1R) or H2R, using anti – CD4+ – and anti – CD8+ – coupled magnetic beads, respectively.Results: Depletion of the H1R resulted in decreases in the release of IL-2 and IL-10 from both CD4+ and CD8+ cells and increases in the release of IL-4 from CD4+ T cells and IFN- from CD8+ cells. Mice lacking the H2R showed up – regulation of IFN- secretion from CD8+ cells and of IL-4 from CD4+ and CD8+ T cells. Release of IL-2 and IL-10 from CD4+ as well as CD8+ cells was down – regulated in these mice. Both CD4+ and CD8+ T cell fractions synthesized histamine, which was enhanced in the H1R - deficient CD8+ T cells. Treatment of the cells with -fluoromethyl-histidine, a specific inhibitor of HDC, or histaminase increased IFN- from CD8+ cells, whereas it had no appreciable effect on IL-4 secretion from CD4+ cells.Conclusions: These results suggest that cytokine production by CD4+ and CD8+ T lymphocytes is regulated by autosecretion of histamine.Received 4 July 2003; returned for revision 23 September 2003; returned for final revision 13 October 2003, accepted by M. Parnham 17 October 2003     

Catecholamines,lymphocyte subsets,and cyclic adenosine monophosphate production in mononuclear cells and CD4+ cells in response to submaximal resistance exercise

We examined the effect of 30 min of submaximal resistance exercise on free and sulphoconjugated plasma catecholamine concentrations determined by high performance (-pressure) liquid chromatography separation, the distribution of circulating lymphocytes quantified by flow cytometry, and isoproterenol induced cyclic adenosine monophosphate (cAMP) production in mononuclear cells (MNL) and CD4⁺ cells. Venous blood samples were taken before, immediately after and 45 min after exercise. Resistance exercise increased free plasma adrenaline (A) and noradrenaline (NA) concentrations, whereas sulphoconjugated catecholamine concentrations remained unchanged. Exercise induced leucocytosis and lymphocytosis was predominantly manifested by an increase in the number of total lymphocytes, monocytes, CD3⁺, CD8⁺ cells and CD3^– CD16/CD56^– cells. Redistribution resulted in a decrease in the CD4^–: CD8⁺ ratio. The total number and distribution of lymphocytes returned to baseline after 45-min rest. An exercise-induced increase in the number of CD3^– CD16/CD56⁺ cells was significantly correlated with the increase in plasma NA (r = 0.66;P = 0.035), indicating a NA dependent process of redistribution. The cAMP-production in MNL was significantly elevated after resistance exercise, when cells were stimulated with 1 mol·1^–1 isoproterenol [pre-exercise 16.5 (SD 3.3); postexercise 21.6 (SD 9.8); 45 min postexercise 10.7 (SD 2.8)]. The cAMP production in CD4⁺ cells was not affected by exercise. Therefore, it is discussed whether redistribution is responsible for the exercise induced increase in cAMP production in MNL.     

Circulating leucocyte subpopulations in sedentary subjects following graded maximal exercise with hypoxia

Summary Ten healthy sedentary subjects [age, 27.5 (SD 3.5) years; height, 180 (SD 5) cm; mass, 69.3 (SD 6.3) kg] performed two periods of maximal incremental graded cycle ergometer exercise in a supine position. Randomly ordered and using an open spirometric system, one exercise was carried out during normoxia [maximal oxygen consumption ( O_2max)=38.6 (SD 3.5) ml·min^–1·kg^–1; maximal blood lactate concentration, 9.86 (SD 1.85) mmol·l^–1; test duration, 22.6 (SD 2.7) min], the other during hypoxia [ O_2max=33.2 (SD 3.2) ml·min^–1· kg^–1; maximal blood lactate concentration, 10.38 (SD 2.02) mmol·l^–1; test duration, 19.7 (SD 2.8) min]. At rest, immediately (0 p) and 60 min (60 p) after exercise, counts of leucocyte subpopulations (flow cytometry), cortisol and catecholamine concentrations were determined. At 0 p in contrast to normoxia, during hypoxia there was no significant increase of granulocytes. There were no significant differences between normoxia and hypoxia in the increases from rest to 0 p in counts of monocytes, total lymphocytes and lymphocyte subpopulations [clusters of differentiation (CD), CD3⁺, CD4⁺CD45RO^–, CD4⁺CD45RO⁺, CD8⁺CD45RO^–, CD8⁺CD45RO⁺, CD3⁺HLA-DR⁺, CD3^–CD16/CD56⁺, CD3⁺CD16/CD56⁺, CD 19⁺] as well as adrenaline, noradrenaline and cortisol concentrations. The counts of CD3 ^–CD16/CD56⁺-and CD8 ⁺CD45RO⁺-cells increased most. At 60 p, CD3^–CD16/CD56⁺ and CD3⁺CD16/CD56⁺-cell counts were below pre-exercise levels and under hypoxia slightly but significantly lower than under normoxia. We concluded that the exercise-induced mobilization and redistribution of most leucocyte and lymphocyte subpopulations were unimpaired under acute hypoxia at sea level. Reduced increases of granulocyte counts during the study and reduced cell numbers of natural killer cells and cytotoxic, not major histocompatibility complex-restricted T-cells, only indicated marginal effects on the immune system.     

Heterogeneity in phagocytic and oxidative burst activities of neutrophils and neonatal calves

In vitro experiments were conducted to determine the influence of source of neutrophils (individual calves) and calf age on phagocytic and oxidative burst activities of neutrophils isolated from 106 clinically healthy Holstein-Friesian calves. Calves were categorised by age into four groups: group A, 3–7 days; group B, 8–14 days; group C, 15–21 days or group D, 22–31 days. Neutrophils were isolated (purity >90%, viability >97%) from EDTA-anticoagulated blood. Aliquots of 2 x 106 neutrophils were incubated at 37°C for 30 min with fluorescein-labelledEscherichia coli 0111:134 J5,E. coli 0157:117 andSalmonella dublin, and with pooled bovine serum as the opsonin. Percentage phagocytosis and averaged number of intracellular bacteria per neutrophil were evaluated by fluorescence microscopy. Mean channel intensity was determined by flow cytometry using dichlorofluorescein diacetate and phorbol myristate acetate to evaluate 11202 production, a measure of oxidative burst.Significant (p<0.05) variation in neutrophil phagocytic activity for each bacterial type was attributed mainly to the source of neutrophils and partially to the influence of age. In the phagocytic assays, the percentages of variation due to the cell source were 33.3%, 38.7% and 20.9% forE. coli 0111:B4 J5,E. coli 0157:117, andS. dublin, respectively. The respective percentages of variation due to age were 17.4%, 7.7% and 24.5%. Similarly for H₂O₂ production assays, percentages of variation due to cell source and age were 69.2% and 8.1%, respectively.In general, significant variation among the four age-specific groups was found for percentage phagocytic, average number of intracellular bacteria per neutrophil and in H₂O₂ production. Differences were particularly evident between groups A and B, and between groups B and D. These results support the concept of functional heterogeneity of neutrophils among neonatal calves.     

Differential mobilization of leucocyte and lymphocyte subpopulations into the circulation during endurance exercise

Summary A total of 14 healthy subjects [means (SD): 27.6 (3.8) years; body mass 77.8 (6.6) kg; height 183 (6) cm] performed endurance exercise to exhaustion at 100% of the individual anaerobic threshold (Th_an) on a cycle ergometer (mean workload 207 (55) W; lactate concentrations 3.4 (1.2) mmol · l^–1; duration 83.8 (22.2) min, including 5 min at 50% of individual Th_an). Leucocyte subpopulations were measured by flow cytometry and catecholamines by radioimmunological methods. Blood samples were taken before and several times during exercise. Values were corrected for plasma volume changes and analysed using ANOVA for repeated measures. During the first 10 min of exercise, of all cell subpopulations the natural killer cells (CD3^–CD16/CD56+) increased the most (229%). Also CD3^÷CD16/CD56+ (84%), CD8^÷CD45RO^– (69%) cells, eosinophils (36%) and monocytes (62%) increased rapidly during thattime.CD3⁺, CD3⁺HLA-DR⁺, CD4⁺CD45RO⁺, CD4⁺CD45RO^–, CD8⁺CD45RO^÷ and CD19⁺ cells either did not increase or increased only slightly during exercise. Adrenaline and noradrenaline increased nearly linearly by 36% and 77% respectively at 10 min exercise. The increase of natural killer cells and heart rates between rest and 10 min of exercise correlated significantly (r=0.576,P=0.031). We conclude that natural killer cells, cytotoxic, non-MHC-restricted T-cells, monocytes and eosinophils are mobilized rapidly during the first minutes of endurance exercise. Both catecholamines and increased blood flow are likely to contribute this effect.     

Effects of moderate endurance exercise and training on in vitro lymphocyte proliferation,interleukin-2 (IL-2) production,and IL-2 receptor expression

This study was designed to examine immunological responses to an acute bout of cycle ergometry exercise before and after moderate endurance training. Previously sedentary males were randomly assigned to matched training (n=9) or control (n=6) groups. Training comprised 12 weeks during which supervised cycle ergometer exercise took place [30 min at 65–70% of maximal oxygen intake , 4–5 days · week^–1]. An acute bout of exercise (60 min; 60% was performed initially and after the 12-week interval. Samples of peripheral venous blood were taken at rest, after 30 and 60 min of exercise, and at 30 and 120 min post-exercise. Training improved by an average of 20% (40.6 to 49.2 ml · kg^–1 · min^–1). Relative to baseline and control measures, the resting concentration of (CD3-CD16⁺/CD56⁺) natural killer (NK) cells increased by 22% (P<0.05). The resting count of total CD25⁺ [interleukin-2 receptor (IL-2R) chain] lymphocytes did not change following training, but dual staining analysis showed a 100% increase in the fraction of CD16⁺ CD25⁺ NK cells (P < 0.05). Likewise the resting CD122⁺ (IL-2R chain) lymphocyte count increased 35% after training, the greatest increases (44%) being in CD16⁺ CD122⁺ NK cells (P<0.05). Soluble IL-2R levels also increased 33% (P< 0.05) after training. Following acute exercise at the same relative intensity; trained individuals exhibited a larger increase in the NK cell count, reduced lymphocytopenia, and attenuation of exercise-induced suppression of lymphocyte proliferation and IL-2 production (P<0.05). In addition, smaller increases in CD4 and CD8 counts during exercise were noted, but with faster recovery post-exercise (P<0.05). Addition of recombinant IL-2 (rIL-2) to phytohemagglutinin-stimulated peripheral blood mononuclear cell cultures did not reverse exercise-induced suppression of cell proliferation, either before or after training. However, rIL-2 did augment the spontaneous blastogenesis of exercise and post-training samples relative to baseline (P < 0.05). We conclude that moderate endurance training is associated with sustained alterations in immune function, both at rest and when exercising. Further investigations are necessary to determine the impact on overall health and susceptibility to disease.     

Mobilization of circulating leucocyte and lymphocyte subpopulations during and after short,anaerobic exercise

Summary A group of 11 healthy athletes [age, 27.4 (SD 6.7) years; body mass, 75.3 (SD 9.2) kg; height, 182 (SD 8) cm; maximal oxygen uptake, 58.0 (SD 9.9) ml · kg^–1 · min^–1] conducted maximal exercise of 60-s duration on a cycle ergometer [mean exercise intensity, 520 (SD 72) W; maximal lactate concentration, 12.26 (SD 1.35) mmol · l^–1]. Adrenaline and noradrenaline, and leucocyte subpopulations were measured flow cytometrically at rest, after 5-min warming up at 50% of each individual's anaerobic threshold (followed by 5-min rest), immediately after (0 min), 15 min, 30 min, and 1, 2, 4 and 24 h after exercise. Granulocytes showed two increases, the first at 15 min and, after return to pre-exercise values, the second more than 2 h after exercise. Eosinophils also increased at 15 min but decreased below pre-exercise values 2 h after exercise. Total lymphocytes and monocytes had their maximal increases at 0 min. Out of all lymphocyte subpopulations CD3^–CD16/CD56⁺- and CD8S⁺ CD45RO^–-cells increased most and had their maximal cell counts at 0 min. The CD3⁺-, CD4⁺CD45RO⁺-, CD8⁺ CD45RO⁺-, and CD19⁺- increased at 0 min, but had their maximum at 15 min. During the hours after exercise CD3^– CD16/CD56⁺-, CD3⁺CD16/CD56⁺-, CD8⁺CD45RO⁺- and CD8⁺ CD45RO^–-cells were responsible for the lymphocytopenia. The CD3⁺- and CD3^– CD16/CD56⁺-cells were lower 24h after exercise than before exercise. Adrenaline and noradrenaline increased during exercise. In conclusion, short anaerobic exercise led to a sequential mobilization of leucocyte subpopulations. The rapid increase of natural killer cells and monocytes may have been due to increased blood flow and catecholamine concentrations. We interpreted from these results that those cells forming the first line of defence can be mobilized faster and disappear out of circulation more rapidly than all other cell populations.     

The effect of maximal exercise on the activity of neutrophil granulocytes in highly trained athletes in a moderate training period

Summary Leucocyte cell counts and the phagocytic and chemotactic activities of neutrophil granulocytes were investigated in highly endurance-trained long-distance runners (n = 10) and triathletes (n = 10) during a moderate training period and compared with untrained subjects (n= 0) before and up to 24 h after a graded exercise to exhaustion on a treadmill. After exercise a leucocytosis was noted with a significant increase in lymphocyte (P0.01) and neutrophil (P0.01) counts in all groups. In neutrophils the number of ingested inert latex beads was significantly increased (P 0.01) from 0.21 (SD 0.09) to 0.45 (SD 0.22) in controls, from 0.20 (SD 0.12) to 0.56 (SD 0.16) in long-distance runners and from 0.25 (SD 0.08) to 1.03 (SD 0.42) particles per cell in triathletes 24 h after exercise, compared with resting values. The capability of neutrophils to produce microbicidal reactive oxygen species fell (P:_ 0.05) immediately after exercise in all subjects and then increased by 36 (SD 8) %, 31 (SD 6) % and 19 (SD 9) % in controls, runners and triathletes respectively up to 24 h after exercise (P 0.05) compared with pre-start values. With respect to the absolute number of neutrophils, ingestion capacity, production of superoxide anions and chemotactic activity, no significant differences were found between athletes and control subjects at rest and after exercise. These data indicate, on the one hand, no impairment of the granulocyte system during a moderate training period in long-distance runners and triathletes but, on the other, that the prolonged activation of the phagocytosis reaction after exercise might impair the granulocyte system in periods of intensive training with high training frequency.     

Modulation of phagocytic and oxidative burst activities of bovine neutrophils by human recombinant TNF-α, IL-1-α, IFN-γ, G-CSF, GM-CSF

In vitro experiments were conducted to determine the effects of recombinant cytokines on phagocytic and oxidative burst activities of bovine neutrophils. Neutrophils were isolated (purity >91%, viability >97%) from EDTA-anticoagulated blood from healthy Holstein-Friesian heifers. Aliquots of neutrophils (10 × 10⁶ cells/ml) were incubated for 1 h at 37 °C with equal volumes of recombinant human cytokines, namely, tumour necrosis factor-alpha (rhTNF-, 0.5–1000 ng/ml), interleukin-1-alpha (rhIL-1-, 0.001–10 ng/ml), interferon-gamma (rhIFN-, 0.01–100 ng/ml), granulocyte colony-stimulating factor (rhG-CSF, 25 ng/ml), and granulocyte-macrophage colony-stimulating factor (rhGM-CSF, 10 ng/ml). Then, the percentage phagocytosis and average number of intracellular bacteria per cell were evaluated by flow cytometry and/or fluorescent microscopy using FITC-labelled opsonised bacteria (Escherichia coli 0111:B4). Unlabelled opsonised bacteria and dichlorofluorescin diacetate were used to evaluate H₂O₂ production, a measure of oxidative burst, by flow cytometry. The results showed that all five cytokines significantly (p <0.05) increased percentage phagocytosis (52.4–86.1%), number of intracellular bacteria per cell (24.9–47.9%), and H₂O₂ production (31.3–58.2%) when compared to untreated neutrophils. A gradual increase in mean channel fluorescence but not in percentage phagocytosis was consistently seen with increasing concentrations of rhTNF-, rhIL-1-, and rhIFN-, thereby indicating a concentration-dependent stimulation of phagocytic capacity by these three cytokines.     

Effects of combination of tumor necrosis factor alpha and chemotactic peptide,f-met-leu-phe,on phagocytosis of opsonized microspheres by human neutrophils

Pretreatment of normal, human neutrophils with 8 units/ml of TNF- followed by treatment with 10^–8 M FMLP resulted in a synergistic effect of the combination of the two mediators on the enhancement of the phagocytic capacity of the cells. This enhancement of phagocytosis occurred without an additional increase in the upregulation of C3b receptors (CR1) beyond that caused by each mediator alone. Pretreatment of the cells with 8 units/ml of TNF- followed by 10^–6 M FMLP resulted in an additive effect of the mediators on neutrophil phagocytosis, again without an additional up-regulation of CR1. This additive effect resulted in an increase in phagocytic capacity of the neutrophils greater than that obtained by treatment of the cells with 10^–6 M FMLP alone, which heretofore has resulted in the greatest enhancement of phagocytic capacity obtained by any pretreatment condition. These synergistic and additive effects of the combination of mediators could be of great importance in host defense against bacterial infections and have important implications regarding the mechanisms of receptor upregulation and phagocytosis.Parts of this study were supported by NIH grant AI 12936 and by the Shriners of North America.     

Effects of gold compounds on function of phagocytic cells

The effect of sodium aurothiomalate and auranofin on the generation of superoxide anions (O ₂ ^– ) by polymorphonuclear leukocytes (PMNLs) and adherent mononuclear phagocytic cells (AMNCs) has been investigated. Sodium aurothiomalate at final concentrations of 1, 10, and 100 g Au/ml and auranofin ranging from 0.1 to 2.0 g Au/ml were used in the reactions involving all celt types. Results have been compared between cells drawn from normal controls and patients with active rheumatoid disease. The effect of gold compounds on both cell types was assessed following activation by phorbyl myristate acetate (1×10^–8 M) and N-formyl-methionylleucyl-phenylalanine (1×10^–4 M) using a cytochromec reduction method. Sodium aurothiomalate at the maximum concentration modestly inhibited O ₂ ^– generation by PMNLs but not AMNCs. Auranofin inhibits O ₂ ^– generation by both cell types. Inhibition of cells from patients with rheumatoid arthritis was greater than that seen with cells from normal controls.     

Assessment of bacterial exposure on phagocytic capability and surface marker expression of sputum macrophages and neutrophils in COPD patients

Defective phagocytosis has been shown in chronic obstructive pulmonary disease (COPD) bronchoalveolar lavage and blood monocyte‐derived macrophages. Phagocytic capabilities of sputum macrophages and neutrophils in COPD are unknown. We investigated phagocytosis in these cells from COPD patients and controls. Phagocytosis of Streptococcus pneumoniae or fluorescently labelled non‐typeable Haemophilus influenzae (NTHi) by sputum macrophages and neutrophils was determined by gentamycin protection assay (COPD; n = 5) or flow cytometry in 14 COPD patients, 8 healthy smokers (HS) and 9 healthy never‐smokers (HNS). Sputum macrophages and neutrophils were differentiated by adherence for the gentamycin protection assay or receptor expression (CD206 and CD66b, respectively), by flow cytometry. The effects of NTHi on macrophage expression of CD206 and CD14 and neutrophil expression of CD16 were determined by flow cytometry. There was greater uptake of S. pneumoniae [~10‐fold more colony‐forming units (CFU)/ml] by sputum neutrophils compared to macrophages in COPD patients. Flow cytometry showed greater NTHi uptake by neutrophils compared to macrophages in COPD (67 versus 38%, respectively) and HS (61 versus 31%, respectively). NTHi uptake by macrophages was lower in HS (31%, p = 0.019) and COPD patients (38%, p = 0.069) compared to HNS (57%). NTHi uptake by neutrophils was similar between groups. NTHi exposure reduced CD206 and CD14 expression on macrophages and CD16 expression on neutrophils. Sputum neutrophils showed more phagocytic activity than macrophages. There was some evidence that bacterial phagocytosis was impaired in HS sputum macrophages, but no impairment of neutrophils was observed in HS or COPD patients. These results highlight the relative contributions of neutrophils and macrophages to bacterial clearance in COPD.     

Characteristics of aggregated immunoglobulin G as an immunologic phagocytic stimulus for granule enzyme release from human neutrophils

Aggregated immunoglobulin G (AggIgG) induced a time- and concentration-dependent phagocytic release of granule-associated lysozyme and myeloperoxidase (MPO) from human neutrophils. Degranulation was significantly enhanced in the presence of calcium or magnesium, and maximum granule exocytosis was observed when both divalent cations were present. AggIgG-stimulated enzyme release was inhibited with the intracellular calcium antagonist, TMB-8[8-(N, N-diethyl-amino)-octyl-(3,4,5-trimethoxy)benzoate] in the absence of extracellular calcium, DIDS (4,4-diisothiocyano-2,2-disulfonic acid stilbene), a permeant anion channel blocker, also suppressed AggIgG-induced degranulation. Cycloheximide, an inhibitor of protein synthesis, enhanced granule exocytosis from AggIgG-treated neutrophils. Two inhibitors of transmethylation reactions, 3-deazaadenosine (3-DZA) and homocysteine thiolactone (HCTL) in combination, suppressed AggIgG-elicited granule enzyme release. These data indicate that AggIgG is a useful probe for investigating the requirements for phagocytic enzyme release from human neutrophils.     

Reduced iC3b-mediated phagocytotic capacity of pulmonary neutrophils in cystic fibrosis

Cystic fibrosis (CF) is characterized by a neutrophil-dominated chronic inflammation of the airways with persistent infections. In order to investigate whether neutrophils contribute to an inadequacy in the pulmonary defence mechanism, the phagocytic activity of pulmonary and peripheral blood neutrophils from CF and non-CF respiratory patients were compared. Neutrophils were isolated from both the blood and bronchoalveolar lavage fluid of 21 patients with CF (12 male, 9 female; mean age 7.5 years, range 0.25-16.4 years) and 17 non-CF subjects (9 male, 8 female; mean age 5.4 years, range 0.2-13.1 years). The ex vivo phagocytic rate of normal pulmonary neutrophils to internalize zymosan particles opsonized with iC3b was faster than that of circulating neutrophils (P < 0.05), but the maximum capacity (9 particles/cell) was similar. In contrast, pulmonary neutrophils from patients with CF had a lower phagocytic capacity than circulating neutrophils either from the same patients or from normal subjects. This deficiency could not be attributed to (i) the cell surface density of CR3 (CD18/CD11b) receptors, which were not significantly different between the other groups (ii) the signalling ability of the CR3 receptors, using cytosolic free Ca(2+) signalling as the receptor activity read-out or (iii) a decrease in cellular ATP concentration. As CFTR was not detectable on neutrophils from any source by either histochemistry or Western blotting, it was concluded that the reduced phagocytic capacity was not the direct result of a CFTR mutation, but was attributed to a failure of neutrophil phagocytic priming during translocation into the CF lung.     

Functional regulation of human neutrophil Fc γ receptors

Interaction between Fc receptors expressed on phagocytic cells and antibodies play a critical role in innate immune response. Interestingly, immune cells such as neutrophils, monocytes, and dendritic cells (DCs) express multiple Fc receptors for IgG (FγR) with overlapping ligand specificity. These receptors compete for the same ligand on the target and are known to transduce positive and negative signals to the same cell, depending on presence of type of signaling motif in their cytoplasmic domain. Neutrophils, the first line of defense against bacterial infection and the major phagocytic cell in the blood, express two types of FcγRs depending on the species. In humans, the neutrophils co-express immunoreceptor tyrosine-based activation motif (ITAM) containing CD32A and glycosylphosphatidyl inositol (GPI)-anchored CD16B, which is in contrast to co-expression of ITAM containing CD16A and ITIM containing CD32B in mouse neutrophils. Recent studies in gene knockout mice have demonstrated that the negative signaling by CD32B plays a critical role in preventing immune complex (IC)-mediated autoimmune diseases by regulating the activation signal delivered by CD16A. However, it is not known how the function of ITAM signaling CD32A is regulated in human neutrophils. Recent observations from our laboratory suggest that in human neutrophils, the CD32A receptor is regulated at the ligand-binding stage. Using a CD16B-deficient donor, we found that the CD32A expressed on resting neutrophils is unable to bind ligand; however, once neutrophils are activated with fMLP, a bacterial chemotactic peptide, the CD32A is functionally active in binding ligand. We also observed that this regulation is neutrophil-specific phenomenon. These observations suggest that FcγR can be regulated by distinct mechanisms and factors such as membrane-anchoring, cell-specific signaling, and avidity modulation that may be coordinately involved in regulating the function of human FγR. Because neutrophils may be activated during infectious and inflammatory diseases, the knowledge of functional regulation of Fc γR will be useful in designing therapies for many autoimmune diseases.     

Metabolic adaptation to daily exercise of moderate intensity to exhaustion in the rat

Summary The rats were made to run daily to exhaustion, for 28 days at a speed of 1,200 m·h^–1 on a treadmill set at a gradient of + 10°. The training increased the time of running to exhaustion [184 (SD 49) and 308 (SD 28) min on the 1st and 28th day, respectively; P<0.001]. The body mass was reduced by training [257 (SD 21) g before and 221 (SD 20) g after; P<0.001] whereas the food intake increased [9 (SD 1) g·100 g^– body mass before and 14 (SD 2) g after; P0.001]. The heart mass was not affected by training. Training increased the resting glycogen concentration in muscles composed of different fibre types (soleus, white and red vastus muscles) and in the liver, but had no effect on its concentration in the heart and diaphragm. During exercise lasting for 30 min glycogen mobilization in the red vastus and soleus muscles and the liver was more pronounced after than before training. A sparing effect of training on the skeletal muscles and liver glycogen was markedly apparent only after exericse to exhaustion. The trained rats, contrary to the untrained, did not develop hypoglycaemia during exercise to exhaustion. An increase in the plasma free fatty acid concentration during exercise after training was delayed and attenuated compared to that before training. The 24-h excretion of urea after exercise to exhaustion on the 28th day of training was higher than on the 1st day by 39% (P<0.001). It is concluded that metabolic adaptation to training consisting of daily bouts of exercise to exhaustion differs in many aspects from that so far described for other endurance training protocols.     

Cell numbers and in vitro responses of leucocytes and lymphocyte subpopulations following maximal exercise and interval training sessions of different intensities

Summary In vitro lymphocyte function and the mobilisation of peripheral blood leucocytes was examined in eight trained subjects who undertook an incremental exercise test to exhaustion and a series of interval training sessions. Venous blood samples were obtained before the incremental test, immediately after, and 30, 60, and 120 min after the test. Interval training sessions were undertaken on separate days and the exercise intensities for each of the different sessions were 30%, 60%, 90% and 120% of their maximal work capacity respectively, as determined from the incremental exercise test. There were 15 exercise periods of 1-min duration separated by recovery intervals of 2 min in each session. Venous blood samples were obtained immediately after each training session. Significant increases in lymphocyte subpopulations (CD3⁺, CD4⁺, CD8⁺, CD20⁺, and CD56⁺) occurred following both maximal and supramaximal exercise. This was accompanied by a significant decrease in the response of cultures of peripheral blood lymphocytes to Concanavalin A (ConA), a T-cell mitogen. The state of lymphocyte activation in vivo as measured by CD25⁺ surface antigen was not, however, affected by acute exercise. The total number of lymphocytes, distribution of lymphocyte subpopulations and in vitro lymphocyte response to ConA had returned to pre-exercise levels within half an hour of termination of exercise but serum cortisol concentrations had not begun to fall at this time. There was a significant decrease in the CD4⁺:CD8⁺ cell ratio following exercise; this was more the result of increases in CD3^–CD8⁺ cells (CD8⁺ natural killer cells) than to CD3⁺CD8⁺ cells (CD8⁺ T-lymphocytes). Decreased responsiveness of T-cells to T-cell mitogens, postexercise, may have been the result of decreases in the percentage of T-cells in postexercise mixed lymphocyte cultures rather than depressed cell function. The cause of this was an increase in the percentage of natural killer cells which did not respond to the T-cell mitogen. The results indicated that while a substantial immediate in vitro immunomodulation occurred with acute exercise, this did not reflect an immunosuppression but was rather the result of changes in the proportions of reactive cells in mononuclear cell cultures. We have also demonstrated that the degree of the change in distribution of lymphocyte subpopulation numbers and responsiveness of peripheral blood mononuclear cells in in vitro mitogen reactions increased with increasing exercise intensity. Plasma volume changes may have contributed to some of the changes seen in leucocyte population and subpopulation numbers during and following exercise.