Changes in neutrophil surface receptor expression, degranulation, and respiratory burst activity after moderate- and high-intensity exercise.

Intense exercise stimulates the systemic release of a variety of factors that alter neutrophil surface receptor expression and functional activity. These alterations may influence resistance to infection after intense exercise. The aim of this study was to examine the influence of exercise intensity on neutrophil receptor expression, degranulation (measured by plasma and intracellular myeloperoxidase concentrations), and respiratory burst activity. Ten well-trained male runners ran on a treadmill for 60 min at 60% [moderate-intensity exercise (MI)] and 85% maximal oxygen consumption [high-intensity exercise (HI)]. Blood was drawn immediately before and after exercise and at 1 h postexercise. Immediately after HI, the expression of the neutrophil receptor CD16 was significantly below preexercise values (P < 0.01), whereas MI significantly reduced CD35 expression below preexercise values (P < 0.05). One hour after exercise at both intensities, there was a significant decline in CD11b expression (P < 0.05) and a further decrease in CD16 expression compared with preexercise values (P < 0.01). CD16 expression was lower 1 h after HI than 1 h after MI (P < 0.01). Immediately after HI, intracellular myeloperoxidase concentration was less than preexercise values (P < 0.01), whereas plasma myeloperoxidase concentration was greater (P < 0.01), indicating that HI stimulated neutrophil degranulation. Plasma myeloperoxidase concentration was higher immediately after HI than after MI (P < 0.01). Neutrophil respiratory burst activity increased after HI (P < 0.01). In summary, both MI and HI reduced neutrophil surface receptor expression. Although CD16 expression was reduced to a greater extent after HI, this reduction did not impair neutrophil degranulation and respiratory burst activity.     

P-selectin Cross-links PSGL-1 and Enhances Neutrophil Adhesion to Fibrinogen and ICAM-1 in a Src Kinase-Dependent,but GPCR-Independent Mechanism

Endothelial and platelet P-selectin (CD62P) and leukocyte integrin αMβ2 (CD11bCD18, Mac-1) are cell adhesion molecules essential for host defense and innate immunity. Upon inflammatory challenges, P-selectin binds to PSGL-1 (P-selectin glycoprotein ligand-1, CD162) to mediate neutrophil rolling, during which integrins become activated by extracellular stimuli for their firm adhesion in a G-protein coupled receptor (GPCR)-dependent mechanism. Here we show that cross-linking of PSGL-1 by dimeric or multimeric forms of platelet P-selectin, P-selectin receptor-globulin, anti-PSGL-1 mAb and its F(ab’)2 induced adhesion of human neutrophils to fibrinogen (Fg) and intercellular cell adhesion molecule-1 (ICAM-1, CD54) and triggered a moderate clustering of αMβ2, but monomeric forms of soluble P-selectin and anti-PSGL-1 Fab did not. Interestingly, P-selectin did not induce a detectable interleukine-8 (IL-8) secretion (&lt;0.1 ng/ml) in 30 minutes, whereas a high concentration of IL-8 (>50 ng/ml) was required to increase neutrophil adhesion to Fg. P-selectin-induced neutrophil adhesion was significantly inhibited by PP2 (a Src kinase inhibitor), but not by Pertussis toxin (PTX; a GPCR inhibitor). Activated platelets also increased neutrophil binding to fibrinogen and triggered tyrosine phosphorylation of cellular proteins. Our results indicate that P-selectin-induced integrin activation (Src kinase-dependent) is distinct from that elicited by cytokines, chemokines, chemoattractants (GPCR-dependent), suggesting that these two signal transduction pathways may cooperate for maximal activation of leukocyte integrins.     

Total serum IL-12 and IL-12p40, but not IL-12p70, are increased in the serum of older subjects; relationship to CD3(+)and NK subsets

Interleukin 12 (IL-12), a central cytokine acting on T and natural killer (NK) cells, directs proliferation of activated T lymphocytes towards a Th1 phenotype. The heterodimeric molecule IL-12p70, equates with IL-12 biological activity, while IL-12p40 may antagonize IL-12 and inhibit cytotoxic T lymphocyte (CTL) generation in vitro. This study characterizes age-related changes in serum total IL-12, IL-12p70 and IL-12p40 relating them with CD3(+), NK and related subsets from subjects, aged 30-96 years. Total IL-12, IL-12p40 and the IL-12p40/IL-12p70 ratio, but not IL-12p70, increased significantly with age (P<0.0001). Increases in total IL-12 and IL-12p40 were negatively associated with CD3(+)(P=0.003, P=0.002), CD3(+)CD4(+)(P=0.004, P=0.003), CD3(+)CD8(+)(P=0.04;P=0. 04) and CD4(+)45RA(+)(P=0.0003;P=0.0007) subsets, respectively. Conversely, increases in IL-12p40 showed a non-significant trend for association with increases in NK(P=0.07) and a related CD8(+low)CD57(+)(P=0.07) subset. These findings may have important implications for understanding the functional activity of IL-12 and its p40 and p70 subunits in vivo and with respect to T-or NK-cell activation in aging.     

Systemic administration of IGF-I enhances oxidative status and reduces contraction-induced injury in skeletal muscles of mdx dystrophic mice

The absence of dystrophin and resultant disruption of the dystrophin glycoprotein complex renders skeletal muscles of dystrophic patients and dystrophic mdx mice susceptible to contraction-induced injury. Strategies to reduce contraction-induced injury are of critical importance, because this mode of damage contributes to the etiology of myofiber breakdown in the dystrophic pathology. Transgenic overexpression of insulin-like growth factor-I (IGF-I) causes myofiber hypertrophy, increases force production, and can improve the dystrophic pathology in mdx mice. In contrast, the predominant effect of continuous exogenous administration of IGF-I to mdx mice at a low dose (1.0-1.5 mg.kg(-1).day(-1)) is a shift in muscle phenotype from fast glycolytic toward a more oxidative, fatigue-resistant, slow muscle without alterations in myofiber cross-sectional area, muscle mass, or maximum force-producing capacity. We found that exogenous administration of IGF-I to mdx mice increased myofiber succinate dehydrogenase activity, shifted the overall myosin heavy chain isoform composition toward a slower phenotype, and, most importantly, reduced contraction-induced damage in tibialis anterior muscles. The deficit in force-producing capacity after two damaging lengthening contractions was reduced significantly in tibialis anterior muscles of IGF-I-treated (53 +/- 4%) compared with untreated mdx mice (70 +/- 5%, P < 0.05). The results provide further evidence that IGF-I administration can enhance the functional properties of dystrophic skeletal muscle and, compared with results in transgenic mice or virus-mediated overexpression, highlight the disparities in different models of endocrine factor delivery.     

The effect of inhibition of leukotriene synthesis on the activity of interleukin-8 and granulocyte-macrophage colony-stimulating factor

The cytokines interleukin-8 (IL-8) and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced the extracellular release of arachidonate metabolites from ionophore-stimulated neutrophils by 145 +/- 10% (mean +/- S.E.M., n = 13) and 182 +/- 11% (n = 16), respectively. To determine whether enhanced leukotriene production mediates the effects of these cytokines on neutrophil activity, two different specific arachidonate 5-lipoxygenase (5-LO) inhibitors, piriprost and MK-886, were used to inhibit leukotriene synthesis. Neither inhibitor affected the upregulation of CD11b beta(2)-integrin expression or priming of superoxide generation stimulated by IL-8 and GM-CSF. It is concluded that leukotrienes do not mediate either the direct or priming effects of these cytokines and that these classes of anti-inflammatory drugs are therefore unlikely to inhibit the effects of IL-8 and GM-CSF on neutrophil activation.     

Transmural left ventricular mechanics underlying torsional recoil during relaxation

Early relaxation in the cardiac cycle is characterized by rapid torsional recoil of the left ventricular (LV) wall. To elucidate the contribution of the transmural arrangement of the myofiber to relaxation, we determined the time course of three-dimensional fiber-sheet strains in the anterior wall of five adult mongrel dogs in vivo during early relaxation with biplane cineangiography (125 Hz) of implanted transmural markers. Fiber-sheet strains were found from transmural fiber and sheet orientations directly measured in the heart tissue. The strain time course was determined during early relaxation in the epicardial, midwall, and endocardial layers referenced to the end-diastolic configuration. During early relaxation, significant circumferential stretch, wall thinning, and in-plane and transverse shear were observed (P < 0.05). We also observed significant stretch along myofibers in the epicardial layers and sheet shortening and shear in the endocardial layers (P < 0.01). Importantly, predominant epicardial stretch along the fiber direction and endocardial sheet shortening occurred during isovolumic relaxation (P < 0.05). We conclude that the LV mechanics during early relaxation involves substantial deformation of fiber and sheet structures with significant transmural heterogeneity. Predominant epicardial stretch along myofibers during isovolumic relaxation appears to drive global torsional recoil to aid early diastolic filling.     

Function of the lectin domain of Mac-1/complement receptor type 3 (CD11b/CD18) in regulating neutrophil adhesion

A lectin function within CD11b mediates both cytotoxic priming of Mac-1/complement receptor type 3 (CR3) by beta-glucan and the formation of transmembrane signaling complexes with GPI-anchored glycoproteins such as CD16b (FcgammaRIIIb). A requirement for GPI-anchored urokinase plasminogen activator receptor (uPAR; CD87) in neutrophil adhesion and diapedesis has been demonstrated with uPAR-knockout mice. In this study, neutrophil activation conditions generating high-affinity (H-AFN) or low-affinity (L-AFN) beta(2) integrin adhesion were explored. A role for the Mac-1/CR3 lectin domain and uPAR in mediating H-AFN or L-AFN adhesion was suggested by the inhibition of Mac-1/CR3-dependent adhesion to ICAM-1 or fibrinogen by beta-glucan or anti-uPAR. The formation of uPAR complexes with Mac-1/CR3 activated for L-AFN adhesion was demonstrated by fluorescence resonance energy transfer. Conversely, Jurkat cell LFA-1 H-AFN-adhesion to ICAM-1 was not associated with uPAR/LFA-1 complexes, any requirement for GPI-anchored glycoproteins, or inhibition by beta-glucan. A single CD11b lectin site for beta-glucan and uPAR was suggested because the binding of either beta-glucan or uPAR to Mac-1/CR3 selectively masked two CD11b epitopes adjacent to the transmembrane domain. Moreover, treatment with phosphatidylinositol-specific phospholipase C that removed GPI-anchored proteins increased CD11b-specific binding of (125)I-labeled beta-glucan by 3-fold and this was reversed with soluble recombinant uPAR. Conversely, neutrophil activation for generation of Mac-1/CR3/uPAR complexes inhibited CD11b-dependent binding of (125)I-labeled beta-glucan by 75%. These data indicate that the same lectin domain within CD11b regulates both the cytotoxic and adhesion functions of Mac-1/CR3.     

Polymorphonuclear leucocyte (PMN) inhibitory factor prevents PMN-dependent endothelial cell injury by an anti-adhesive mechanism

Neutrophil inhibitory factor (NIF), a 41-kD glycoprotein isolated from the canine hookworm, inhibits CD11b/CD18-dependent neutrophil adhesion by binding to CD11b. We studied the effects of NIF on neutrophil-dependent endothelial cell injury using bovine pulmonary microvessel endothelial cells grown on microporous filters. Endothelial injury was determined as an increase in the transendothelial ¹²⁵I-albumin clearance rate (a measure of transendothelial permeability). Layering of neutrophils on the endothelial cell monolayer (ratio of 10 neutrophils: 1 endothelial cell) followed by activation of neutrophils with 500 nM of phorbol 12-myristate 13-acetate (PMA) increased transendothelial permeability of albumin by 3- to 4-fold over control monolayers. Pretreatment of neutrophils with NIF at concentrations of 100 nM and above prevented the increased permeability. Pretreatment of neutrophils with the anti-CD18 monoclonal antibody (mAb) IB4 similarly prevented the increase of permeability. Pretreatment of neutrophils with OKM-1, a control isotype-matched mAb directed against an irrelevant epitope on CD11b mAb, did not affect the neutrophil-dependent increase in permeability. NIF reduced the adhesion of neutrophils at concentrations of ≥100 nM and this effect was abolished by an anti-NIF polyclonal Ab. However, NIF did not prevent the generation of superoxide anions following PMA-induced activation of neutrophils layered on endothelial cell. These findings indicate that NIF inhibits the neutrophil-dependent endothelial injury by preventing CD11b/CD18-mediated neutrophil adhesion, but without altering the oxidant generating capacity of neutrophils interacting with the endothelial cell monolayer. J. Cell. Physiol. 171:212–216, 1997. © 1997 Wiley-Liss, Inc.     

Macrophage depletion by clodronate liposome attenuates muscle injury and inflammation following exhaustive exercise

Exhaustive exercise promotes muscle injury, including myofiber lesions; however, its exact mechanism has not yet been elucidated. In this study, we tested the hypothesis that macrophage depletion by pretreatment with clodronate liposomes alters muscle injury and inflammation following exhaustive exercise. Male C57BL/6J mice were divided into four groups: rest plus control liposome (n=8), rest plus clodronate liposome (n=8), exhaustive exercise plus control liposome (n=8), and exhaustive exercise plus clodronate liposome (n=8). Mice were treated with clodronate liposome or control liposome for 48 h before undergoing exhaustive exercise on a treadmill. Twenty-four hours after exhaustive exercise, the gastrocnemius muscles were removed for histological and PCR analyses. Exhaustive exercise increased the number of macrophages in the muscle; however, clodronate liposome treatment reduced this infiltration. Although exhaustive exercise resulted in an increase in injured myofibers, clodronate liposome treatment following exhaustive exercise reduced the injured myofibers. Clodronate liposome treatment also decreased the mRNA expression levels of inflammatory cytokines (TNF-α, IL-1β, and IL-6) in the skeletal muscle after exhaustive exercise. These results suggest that macrophages play a critical role in increasing muscle injury by regulating inflammation.     

Supramaximal exercise mobilizes hematopoietic progenitors and reticulocytes in athletes

Marathon runners show increased circulating CD34+ cell counts and postexercise release of interleukin-6 (IL-6), granulocyte-colony stimulating factor (G-CSF) and flt3-ligand (Bonsignore MR, Morici G, Santoro A, Pegano M, Cascio L, Bonnano A, Abate P, Mirabella F, Profita M, Insalaco G, Gioia M, Vignola AM, Majolino I, Testa U, and Hogg JC. J Appl Physiol 93: 1691-1697, 2002). In the present study we hypothesized that supramaximal ("all-out") exercise may acutely affect circulating progenitors and reticulocytes and investigated possible mechanisms involved. Progenitor release was measured by flow cytometry (n = 20) and clonogenic assays (n = 6) in 20 young competitive rowers (13 M, 7 F, age +/- SD: 17.1 +/- 2.1 yr, peak O2 consumption: 56.5 +/- 11.4 ml.min(-1).kg(-1)) at rest and shortly after 1,000 m "all-out." Release of reticulocytes, cortisol, muscle enzymes, neutrophil elastase, and several cytokines/growth factors was measured. Supramaximal exercise doubled circulating CD34+ cells (rest: 7.6 +/- 3.0, all-out: 16.3 +/- 9.1 cells/mul, P < 0.001), and increased immature reticulocyte fractions; AC133+ cells doubled, suggesting release of angiogenetic precursors. Erythrocyte burst forming units and colony forming units for granulocytes-monocytes and all blood series increased postexercise by 3.4-, 5.5-, and 4.8-fold, respectively (P < 0.01 for all). All-out rowing acutely increased plasma cortisol, neutrophil elastase, flt3-ligand, hepatocyte growth factor, VEGF, and transforming growth factor-beta1, and decreased erythropoietin; K-ligand, stromal-derived factor-1, IL-6, and G-CSF were unchanged. Therefore, all-out exercise is a physiological stimulus for progenitor release in athletes. Release of reticulocytes and proangiogenetic cells and mediators suggests tissue hypoxia as possibly involved in progenitor mobilization.     

Low degree of activation of circulating neutrophils determined by flow cytometry during cardiac surgery with cardiopulmonary bypass

BACKGROUND: Enhanced expression of adhesion molecules LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18) following cardiac surgery with cardiopulmonary bypass (CPB) is held responsible for postoperative complications. Surface expression of these molecules, intracellular pH (pH(i)), and oxidative burst capacity was analyzed to test for neutrophil activation during pediatric cardiac surgery. METHODS: Blood samples were drawn from 36 patients (age: 3--16 years) 24 h preoperatively, after onset of anesthesia, after connection to CPB (CPB1, before and after passing CPB, n = 15), at reperfusion (CPB2), and up to 7 days postoperatively. Cells adhering to CPB filters were isolated (n = 11). Antigen expression, pH(i), and oxidative burst capacity on neutrophils was analyzed by flow cytometry. RESULTS: During surgery, oxidative burst capacity was at low level with a mild increase only 1 day after surgery. pH(i) was decreased throughout the surgery. Surgery induced more than 36% decrease of LFA-1 and Mac-1 expression (P < 0.03). Up to postoperative day 7, no increase of antigen expression above baseline was found. Neutrophils isolated from filters of the CPB had increased LFA-1 and Mac-1 expression (all P < 0.05). Integrin expression on neutrophils passing the CPB at CPB1 was decreased (P < 0.05). CONCLUSION: Reduced adhesion molecule expression on neutrophils may be due to selective filtration of highly adhesive cells. This, in combination with low-level oxidative burst capacity, induced by immunosuppressive cytokines (e.g., interleukin-10), reduced the neutrophil activity. Our data indicate that increased activity of circulating neutrophils cannot exclusively be held responsible for postoperative complications after surgery with CPB.     

Pulmonary immune cells and inflammatory cytokine dysregulation are associated with mortality of IL-1R1-/-mice infected with influenza virus (H1 N1)

Respirovirus infection can cause viral pneumonia and acute lung injury (ALI).The interleukin-1 (IL-1) family consists of proinflammatory cytokines that play essential roles in regulating immune and inflammatory responses in vivo.IL-1 signaling is associated with protection against respiratory influenza virus infection by mediation of the pulmonary anti-viral immune response and inflammation.We analyzed the infiltration lung immune leukocytes and cytokines that contribute to inflammatory lung pathology and mortality of fatal H1N1 virus-infected IL-1 receptor 1 (IL-1R1) deficient mice.Results showed that early innate immune cells and cytokine/chemokine dysregulation were observed with significantly decreased neutrophil infiltration and IL-6,TNF-α,G-CSF,KC,and MIP-2 cytokine levels in the bronchoalveolar lavage fluid of infected IL-1R1-/-mice in comparison with that of wild type infected mice.The adaptive immune response against the H1N1 virus in IL-1R1-/-mice was impaired with downregulated anti-viral Th1 cell,CD8+ cell,and antibody functions,which contributes to attenuated viral clearance.Histological analysis revealed reduced lung inflammation during early infection but severe lung pathology in late infection in IL-1R1-/-mice compared with that in WT infected mice.Moreover,the infected IL-1R1-/-mice showed markedly reduced neutrophil generation in bone marrow and neutrophil recruitment to the inflamed lung.Together,these results suggest that IL-1 signaling is associated with pulmonary anti-influenza immune response and inflammatory lung injury,particularly via the influence on neutrophil mobilization and inflammatory cytokine/chemokine production.     

Reactive oxygen species formation during tetanic contractions in single isolated Xenopus myofibers

Contracting skeletal muscle produces reactive oxygen species (ROS) that have been shown to affect muscle function and adaptation. However, real-time measurement of ROS in contracting myofibers has proven to be difficult. We used amphibian (Xenopus laevis) muscle to test the hypothesis that ROS are formed during contractile activity in isolated single skeletal muscle fibers and that this contraction-induced ROS formation affects fatigue development. Single myofibers were loaded with 5 μM dihydrofluorescein-DA (Hfluor-DA), a fluorescent probe that reacts with ROS and results in the formation of fluorescein (Fluor) to precisely monitor ROS generation within single myofibers in real time using confocal miscroscopy. Three identical periods of maximal tetanic contractions (1 contraction/3 s for 2 min, separated by 60 min of rest) were conducted by each myofiber (n = 6) at 20°C. Ebselen (an antioxidant) was present in the perfusate (10 μM) during the second contractile period. Force was reduced by ～30% during each of the three contraction periods, with no significant difference in fatigue development among the three periods. The Fluor signal, indicative of ROS generation, increased significantly above baseline in both the first (42 ± 14%) and third periods (39 ± 10%), with no significant difference in the increase in fluorescence between the first and third periods. There was no increase of Fluor in the presence of ebselen during the second contractile period. These results demonstrated that, in isolated intact Xenopus myofibers, 1) ROS can be measured in real time during tetanic contractions, 2) contractile activity induced a significant increase above resting levels of ROS production, and 3) ebselen treatment reduced ROS generation to baseline levels but had no effect on myofiber contractility and fatigue development.     

Bronchoalveolar lavage fluids of ventilated patients with acute lung injury activate NF-kappaB in alveolar epithelial cell line: role of reactive oxygen/nitrogen species and cytokines.

In human alveolar epithelial cell line, we investigated the binding activity of NF-kappaB induced by the bronchoalveolar lavage fluids (BALs) from ventilated patients with acute lung injury (ALI), in correlation with the concentrations of inflammatory cytokines, RNOS, and the severity of the ALI. In BALs obtained in 67 patients (16 bronchopneumonia, 14 infected ARDS, 20 ARDS, and 17 ALI patients without bronchopneumonia and no ARDS), we measured endotoxin, IL-1beta, IL-8, and nitrated proteins (NTP), the activity of myeloperoxidase, and the capacity to activate the NF-kappaB in alveolar A549 cells by electrophoretic mobility shift and supershift assays. The neutrophil counts and mean IL-1beta, IL-8, myeloperoxidase, and NTP values were increased in bronchopneumonia and infected ARDS groups compared to ARDS and ALI without bronchopneumonia and no ARDS groups (P<0.001). The number of neutrophils was correlated to those of IL-1beta, IL-8, myeloperoxidase, NTP, and endotoxin in all groups (P<0.0001). NF-kappaB activity was induced in alveolar like cells by BALs in all groups, was higher in bronchopneumonia and infected ARDS groups (P<0.02), and was correlated to IL-1beta (P=0.0002), IL-8 (P=0.02), NTP (P=0.014), myeloperoxidase (P=0.016), and neutrophil counts (P=0.003). BALs of bronchopneumonia and infected ARDS patients had increased inflammatory mediators (compared to ARDS and ALI without bronchopneumonia and no ARDS patients) that correlated to neutrophil counts and to the NF-kappaB-binding activity. These mediators and NF-kappaB activation may induce an amplification of inflammatory phenomena. By in vitro studies, we confirmed that NO-derived species (10(-6) to 10(-5)M peroxynitrite and 10(-5)M nitrites) and myeloperoxidase (at concentration equivalent to that found in BALs) can participate in the NF-kappaB activation.     

Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice.     

Hindlimb suspension reduces muscle regeneration

Exposure of juvenile skeletal muscle to a weightless environment reduces growth and satellite cell mitotic activity. However, the effect of a weightless environment on the satellite cell population during muscle repair remains unknown. Muscle injury was induced in rat soleus muscles using the myotoxic snake venom, notexin. Rats were placed into hindlimb-suspended or weightbearing groups for 10 days following injury. Cellular proliferation during regeneration was evaluated using 5-bromo-2′-deoxyuridine (BrdU) immunohistochemistry and image analysis. Hindlimb suspension reduced (P<0.05) regenerated muscle mass, regenerated myofiber diameter, uninjured muscle mass, and uninjured myofiber diameter compared to weightbearing rats. Hindlimb suspension reduced (P<0.05) BrdU labeling in uninjured soleus muscles compared to weightbearing muscles. However, hindlimb suspension did not abolish muscle regeneration because myofibers formed in the injured soleus muscles of hindlimb-suspended rats, and BrdU labeling was equivalent (P>0.10) on myofiber segments isolated from the soleus muscles of hindlimb-suspended and weightbearing rats following injury. Thus, hindlimb suspension (weightlessness) does not suppress satellite cell mitotic activity in regenerating muscles before myofiber formation, but reduces growth of the newly formed myofibers. Accepted: 11 December 1997     

E. coli pneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability

We examined the relationship between neutrophil [polymorphonuclear leukocyte (PMN)] influx and lung vascular injury in response to Escherichia coli pneumonia. We assessed lung tissue PMN uptake by measuring myeloperoxidase and transvascular PMN migration by determining PMN counts in lung interstitium and bronchoalveolar lavage fluid (BALF) in mice challenged intratracheally with E. coli. Lung vascular injury was quantified by determining microvessel filtration coefficient (Kf,c), a measure of vascular permeability. We addressed the role of CD18 integrin in the mechanism of PMN migration and lung vascular injury by inducing the expression of neutrophil inhibitory factor, a CD11/CD18 antagonist. In control animals, we observed a time-dependent sixfold increase in PMN uptake, a fivefold increase in airway PMN migration, and a 20-fold increase in interstitial PMN uptake at 6 h after challenge. Interestingly, Kf,c increased minimally during this period of PMN extravasation. CD11/CD18 blockade reduced lung tissue PMN uptake consistent with the role of CD18 in mediating PMN adhesion to the endothelium but failed to alter PMN migration in the tissue. Moreover, CD11/CD18 blockade did not affect Kf,c. Analysis of BALF leukocytes demonstrated diminished oxidative burst compared with leukocytes from bacteremic mice, suggesting a basis for lack of vascular injury. The massive CD11/CD18-independent airway PMN influx occurring in the absence of lung vascular injury is indicative of an efficient host-defense response elicited by E. coli pneumonia.     

CD8+ T cells negatively regulate IL-4-dependent, IgG1-dominant posttransplant alloantibody production

We have previously reported that CD8(+) T cells significantly influence Ab production based on the observation that posttransplant alloantibody levels in CD8-deficient murine hepatocyte transplant recipients are markedly enhanced. However, the precise mechanisms contributing to enhanced alloantibody production in the absence of CD8(+) T cells is not understood. We hypothesized that alloactivated CD8(+) T cells inhibit Ab production by skewing toward a proinflammatory cytokine profile, whereas when these cells are absent, an anti-inflammatory cytokine profile shifts the alloimmune response toward alloantibody production. To investigate this possibility, alloantibody isotype profiles were examined in CD8-deficient and wild-type hepatocyte recipients. We found that IgG1 (IL-4-dependent isotype) was the dominant alloantibody isotype in wild-type recipients as well as in CD8-deficient recipients, although the amount of alloantibody in the latter group was substantially higher. Utilizing real-time PCR we found that CD4(+) T cells from wild-type recipients significantly upregulated IFN-γ but not IL-4 mRNA. In contrast, in the absence of CD8(+) T cells, CD4(+) T cells switched to significantly upregulate IL-4 mRNA, while IFN-γ was downregulated. IL-4 knockout mice do not produce any posttransplant alloantibody. However, adoptive transfer of wild-type CD4(+) T cells into CD8-depleted IL-4 knockout mice restores high alloantibody levels observed in CD8-depleted wild-type recipients. This suggests that IL-4-producing CD4(+) T cells are critical for posttransplant alloantibody production. Additionally, this CD8-mediated regulation of posttransplant alloantibody production is IFN-γ-dependent. Further elucidation of the mechanisms by which CD8(+) T cells influence Ab production will significantly contribute to development of therapies to manipulate humoral responses to Ag.     

Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells.

Interleukin-8 (IL-8) participates in the generation of dense neutrophil accumulations in bronchopulmonary infections caused by Pseudomonas aeruginosa (P. aeruginosa). We have recently reported that nitrite reductase, a bifunctional enzyme located in the periplasmic space of P. aeruginosa, induces IL-8 generation in bronchial epithelial cells (K. Oishi et al. Infect. Immun. 65: 2648-2655, 1997). We examined whether or not Pseudomonas nitrite reductase (PNR) could also stimulate human alveolar macrophages (AM) and pulmonary type II epithelial-like cells (A549) to induce IL-8 production and mRNA expression as well as the production of TNF alpha and IL-1beta. We demonstrated a time- and dose-dependent IL-8 protein synthesis and IL-8 mRNA expression, but no TNF alpha or IL-1beta production, by A549 cells in response to PNR. New protein translation was not required for PNR-mediated IL-8 mRNA expression in the same cells. Furthermore, simultaneous stimulation of PNR with serial doses of TNF alpha or IL-1beta resulted in additive IL-8 production in A549 cells. In adherent AM, PNR enhanced IL-8 protein synthesis and IL-8 mRNA expression in a time-dependent fashion. PNR similarly induced a time-dependent production of TNF alpha and IL-1beta by human adherent AM. Neutralization of TNF alpha or IL-1beta did not influence the levels of IL-8 production in adherent AM culture. We also evaluated whether the culture supernatants of the A549 cells or AM stimulated with PNR could similarly mediate neutrophil migration in vitro. When anti-human IL-8 immunoglobulin G was used for neutralizing neutrophil chemotactic factor (NCF) activities in the culture supernatants of these cells stimulated with 5 microg/ml of PNR, the mean percent reduction of NCF activities were 49-59% in A549 cells and 24-34% in AM. Our present data support that PNR directly stimulates AM and pulmonary epithelial cells to produce IL-8. PNR also mediates neutrophil migration, in part, through IL-8 production from AM and pulmonary epithelial cells. These data suggest the contribution of PNR to the pathogenesis of bronchopulmonary infections due to P. aeruginosa.