Leukocyte arrest during cytokine-dependent inflammation in vivo

Leukocyte rolling along the walls of inflamed venules precedes their adhesion during inflammation. Rolling leukocytes are thought to arrest by engaging beta2 integrins following cellular activation. In vitro studies suggest that chemoattractants may instantaneously activate and arrest rolling leukocytes. However, how leukocytes stop rolling and become adherent in inflamed venules in vivo has remained rather mysterious. In this paper we use a novel method of tracking individual leukocytes through the microcirculation to show that rolling neutrophils become progressively activated while rolling down the venular tree. On average, leukocytes in wild-type mice roll for 86 s (and cover 270 microm) before becoming adherent with an efficiency around 90%. These rolling leukocytes exhibit a gradual beta2 integrin-dependent decrease in rolling velocity that correlates with an increase in intracellular free calcium concentration before arrest. Similar tracking analyses in gene-targeted mice demonstrate that the arrest of rolling leukocytes is very rare when beta2 integrins are absent or blocked by a mAb. Arrest is approximately 50% less efficient in the absence of E-selectin. These data suggest a model of leukocyte recruitment in which beta2 integrins play a critical role in stabilizing leukocyte rolling during a protracted cellular activation period before arrest and firm adhesion.     

Leukocyte adhesion molecule expression during intense resistance exercise

We hypothesized that expression of L-selectinand very late antigen-4 (VLA-4) integrin adhesionmolecules would influence cell type-specific redistribution duringexercise. Women subjects performed six sets of 10-repetition maximumsquats. L-selectin and VLA-4 integrin were measured by using flowcytometry pre- and postexercise on peripheral blood neutrophils andlymphocytes (n = 29 subjects) andlymphocyte subsets (n = 70 subjects),respectively. Neutrophil concentration increased 41.8%(P < 0.001), whereas the percentexpressing L-selectin was unchanged (79%). Lymphocyte concentrationincreased 61.8% (P < 0.001). Thepercent of T cells expressing L-selectin decreased from 73.5 ± 8.9 to 68.2 ± 11.4% (P < 0.001); the combined population of natural killer and B cells expressing L-selectin decreased from 80.4 ± 22.5 to 62.7 ± 25.8% (P < 0.001).VLA-4 integrin was expressed by nearly all lymphocytes both pre- andpostexercise. The proportional decrease in L-selectin positive cellscould have resulted from 1) sheddingof L-selectin, 2) selective entry ofL-selectin-negative subsets, or 3)selective removal of L-selectin-positive subsets.

     

Leukocyte chemotactic cytokine and leukocyte subset responses during ultra-marathon running

PurposeThe aim of this study was to assess leukocyte chemotactic cytokine and leukocyte subset responses during ultra-marathon running. Leukocyte chemokines such as interleukin (IL)-8, interferon gamma-induced protein-10 (IP-10), regulated upon activation, normal T-cell expressed and secreted (RANTES), and eotaxin are involved in leukocyte recruitment.MethodsAmong 60 male amateur endurance runner volunteers, 18 finished the course (a 308 km continuous race from Kanghwado to Kangneung, South Korea). Their average age, height, and body mass were 52.8 ± 5.0 years, 167.6 ± 5.2 cm, and 64.5 ± 1.2 kg, respectively. Blood samples were collected at 0, 100, 200, and 308 km during the race for analysis of white blood cells and serum concentrations of IL-8, IP-10, RANTES, eotaxin, IL-6, creatine kinase (CK), and C-reactive protein (CRP).ResultsMuscle and liver damage indicators (IL-6, CK, and CRP) were maximally elevated as a result of marathon running. Total leukocytes, neutrophils, and monocytes increased significantly during the event (leukocytosis, neutrophilia, and monocytosis, respectively). However, lymphocytes and eosinophils decreased significantly during the event (lymphopenia and eosinopenia, respectively). Serum levels of the neutrophil chemokine IL-8 increased maximally at 100 km and were maintained. Monocyte-lymphocyte chemokine IP-10 concentration decreased during the latter part of the race. The eosinophil chemokine eotaxin decreased gradually during the race, and no difference was observed in eosinophil chemokine RANTES levels.ConclusionThese observations indicate that prolonged endurance ultra-marathon running was associated with significant systemic inflammation and perturbation in leukocyte subsets. Leukocyte chemotactic cytokines such as IL-8, IP-10, eotaxin showed similar patterns of responses in related leukocyte subsets, but RANTES did not.     

Leukocyte elastase induces epithelial apoptosis: role of mitochondial permeability changes and Akt

During acute inflammation, neutrophil-mediated injury to epithelium may lead to disruption of epithelial function, including the induction of epithelial apoptosis. Herein, we report the effects of neutrophil transmigration and of purified leukocyte elastase on epithelial cell survival. Neutrophil transmigration induced apoptosis of epithelial cells [control monolayers: 5 +/- 1 cells/25 high-power fields (HPF) vs. neutrophil-treated monolayers: 29 +/- 10 cells/HPF, P < 0.05, n = 3 as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling assay] as did low concentrations (0.1 U/ml) of purified leukocyte elastase (control monolayers: 6.4 +/- 2.5% apoptotic vs. elastase: 26.2 +/- 2.9% apoptotic, P < 0.05, as determined by cytokeratin 18 cleavage). Treatment with elastase resulted in decreased mitochondrial membrane potential, release of cytochrome c to the cytosol, and cleavage of caspases-9 and -3 as determined by Western blot analysis, implicating altered mitochondrial membrane permeability as a primary mechanism for elastase-induced apoptosis. Additionally, incubation of epithelial cells with leukocyte elastase resulted in an early increase followed by a decrease in the phosphorylation of epithelial Akt, a serine/threonine kinase important in cell survival. Inhibition of epithelial Akt before elastase treatment potentiated epithelial cell apoptosis, suggesting that the initial activation of Akt represents a protective response by the epithelial cells to the proapoptotic effects of leukocyte elastase. Taken together, these observations suggest that epithelial cells exhibit a dual response to cellular stress imposed by leukocyte elastase with a proapoptotic response mediated via early alterations in mitochondrial membrane permeability countered by activation of the survival pathway involving Akt.     

Leukocyte biophysics

The biophysical properties of leukocytes in the passive and active state are discussed. In the passive unstressed state, leukocytes are spherical with numerous membrane folds. Passive leukocytes exhibit viscoelastic properties, and the stress is carried largely by the cell cytoplasm and the nucleus. The membrane is highly deformable in shearing and bending, but resists area expansion. Membrane tension can usually be neglected but plays a role in cases of large deformation when the membrane becomes unfolded. The constant membrane area constraint is a determinant of phagocytic capacity, spreading of cells, and passage through narrow pores. In the active state, leukocytes undergo large internal cytoplasmic deformation, pseudopod projection, and granule redistribution. Several different measurements for assessment of biophysical properties and the internal cytoplasmic deformation in form of strain and strain rate tensors are presented. The current theoretical models for active cytoplasmic motion in leukocytes are discussed in terms of specific macromolecular reactions.     

Leukocyte reactivity as an objective means of quantifying mental loading during ergonomic evaluation

Psychological stress evokes rapid changes to the cardiovascular and neuroendocrine systems, responses that can become habituated following repeated exposure. This study, comprising of two phases, suggests that the immune system follows a similar trend. Phase 1: 15 healthy subjects (aged between 26 and 56 years) provided capillary blood samples before and after completing three basic tasks using, in turn, two automotive touch screen interfaces (Interface 1—antecedent version, Interface 2—improved version). Using a chemiluminescent technique termed leukocyte coping capacity (LCC), the ability of leukocytes to produce reactive oxygen species in vitro was assessed. Significant differences in leukocyte activity were shown between treatment groups, where the greatest post-test decrease occurred after using Interface 1. Phase 2: a randomly selected sub-group (n = 4) underwent weekly repeat testing using both interfaces. Significant differences in post-test leukocyte reactivity were exhibited between test weeks for each interface—the magnitude of response decreasing with successive exposure.     

Real-Time Imaging Reveals the Dynamics of Leukocyte Behaviour during Experimental Cerebral Malaria Pathogenesis

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C^hi monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8⁺ T lymphocytes. A critical number of CD8⁺ T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8⁺ T lymphocytes in regulating vascular pathology in this disease.     

Ribosome changes during translation

Studies on the quantitative binding of [³H]anisomycin are useful in determining conformational and/or structural changes on eukaryotic ribosomes. We have shown that yeast ribosomes have different structures depending on their functional states during the ribosome cycle as defined by their affinity for [³H]anisomycin.Free ribosomes, either in vivo run-off ribosomes (1 mm-sodium azide treatment or 8 °C incubation of spheroplasts) or puromycin-dependent released ribosomes, have an affinity defined by K_d = 3.3 to 3.6 μm.Ribosomes forming polysomes engaged in protein synthesis have at least two new different conformations (defined by K_d,H = 0.81 μm and K_d,L = 12 μm). These conformations have been ascribed to the pre and post-translocated steps of the elongation cycle in protein synthesis by blocking the polysomes with specific inhibitors of translation. Pre-translocated polysomes (polysomes blocked with cycloheximide) have an affinity of K_d^CHX = 12 μm and post-translocated polysomes (polysomes blocked with doxycycline) have an affinity of K_d^DC = 0.82 μm. These dissociation constants are identical to K_d,L and K_d,H obtained with control untreated polysomes, respectively.Moreover, a new ribosome conformation defined by K_d^DT = 1.5 μm and K_d^FA = 1.8 μm was found, by blocking the polysomes with the elongation factor, EF-2, bound by using either diphtheria toxin or fusidic acid.We also present evidence of the previously reported heterogeneity of standard preparations of eukaryotic ribosomes (Barbacid & Vazquez, 1974a) being a direct consequence of the high-salt washing treatment of ribosomes.     

Leukocyte subpopulations in the uteri of leukemia inhibitory factor knockout mice during early pregnancy

Leukemia inhibitory factor (LIF) is transiently expressed on Day (D) 1 of pregnancy by the uterine epithelium and on D4 specifically by the glandular epithelium. The Lif knockout female mice are infertile because of uterine defects that affect embryo implantation, but pregnancy can be rescued in these mice by injections of LIF on D4 of pregnancy. Many of the specific actions of LIF in the uterus are unknown, especially with regard to uterine cell biology. Leukocytes, such as macrophages, natural killer (NK) cells, and eosinophils, are present in the pregnant uterus and are thought to be beneficial, because alterations in their proportions can adversely affect pregnancy. Immunocytochemistry and cell counting were used to compare the distributions and dynamics of leukocyte subpopulations in wild-type and Lif knockout mice. The percentage of macrophages was reduced by more than half in the Lif knockout mice on D3 of pregnancy, and their distribution was disrupted, suggesting that LIF is a chemokine for these cells. The NK cells were detected as early as D3 of pregnancy, but the Lif knockout mice had double the percentage of NK cells compared to wild-type mice at this time, indicating that LIF restricts the migration of NK cells to the uterus. The Lif knockout mice also had significantly higher percentages of eosinophils in the outer stroma on D3, and in the midstroma on D4, of pregnancy, suggesting that LIF also may restrict eosinophil migration to the uterus. These alterations of the uterine leukocyte subpopulations in Lif knockout mice may disrupt pregnancy and contribute to failure of implantation.