The marginated pool

The pulmonary circulation harbors a large intravascular reservoir of leukocytes refered to as the Marginated Pool. This marginated pool is balanced by propeling and retaining forces acting on leukocytes during their passage through the pulmonary circulation. The present paper discusses these factors and their underlying mechanisms. Copyright (C) 2002 S. Karger AG, Basel

PECAM-1 engagement counteracts ICAM-1-induced signaling in brain vascular endothelial cells

Interactions between leukocytes and vascular endothelial cells are mediated by a complex set of membrane adhesion molecules which transduce bi-directional signals in both cell types. Endothelium of the cerebral blood vessels, which constitute the blood–brain barrier, strictly controls adhesion and trafficking of leukocytes into the brain. Investigating signaling pathways triggered by the engagement of adhesion molecules expressed on brain endothelial cells, we previously documented the role of ICAM-1 in activation of the tyrosine phosphorylation of several actin-binding proteins and subsequent rearrangements of the actin cytoskeleton. In the present study, we show that, whereas PECAM-1 is known to control positively the trans-endothelial migration of leukocytes via homophilic interactions between leukocytes and endothelial cells, PECAM-1 engagement on brain endothelial surface unexpectedly counteracts the ICAM-1-induced tyrosine phosphorylation of cortactin and rearrangements of the actin cytoskeleton. We present evidence that the PECAM-1-associated tyrosine phosphatase SHP-2 is required for ICAM-1 signaling, suggesting that its activity might crucially contribute to the regulation of ICAM-1 signaling by PECAM-1. Our findings reveal a novel activity for PECAM-1 which, by counteracting ICAM-1-induced activation, could directly contribute to limit activation and maintain integrity of brain vascular endothelium

Leukocyte sequestration in pulmonary microvessels and lung injury following systemic complement activation in rabbits

Inflammatory reactions are associated with sequestration of leukocytes in the lung. Complement activation leads to accumulation of leukocytes in alveolar septa and alveoli, to lung edema and hemorrhage. Although in organs other than the lung leukocytes interact with the vascular endothelium only in postcapillary venules, alveolar capillaries are considered to be the site of leukocyte sequestration in the lung. However, pulmonary venules and arterioles have not been investigated systematically after complement activation so far, A closed thoracic window was implanted in anesthetized rabbits; leukocytes and red blood cells were stained, and the movement of these cells was measured in superficial pulmonary arterioles, venules and alveolar capillaries using fluorescence video microscopy before and 30 and 60 min after infusion of cobra venom factor (CVF). Erythrocyte velocity and macrohemodynamic conditions did not change after CVF infusion and were not different from the sham-treated controls. The number of sticking leukocytes increased significantly compared to baseline and control: by 150% in arterioles and in venules and by 740% in alveolar capillaries within 60 min after CVF infusion. The width of alveolar septa in vivo was significantly enlarged after CVF infusion, indicating interstitial pulmonary edema. At the end of the experiments, myeloperoxidase activity was higher in the CVF group, showing leukocyte sequestration in the whole organ. It is concluded that complement activation by CVF induces leukocyte sequestration in lung arterioles, venules and alveolar capillaries and leads to mild lung injury

Differential expression and biochemical activity of the immune receptor Tim-3 in healthy and malignant human myeloid cells

The T cell immunoglobulin and mucin domain 3 (Tim-3) is a plasma membrane-associated receptor which is involved in a variety of biological responses in human immune cells. It is highly expressed in most acute myeloid leukaemia (AML) cells and therefore may serve as a possible target for AML therapy. However, its biochemical activities in primary human AML cells remain unclear. We therefore analysed the total expression and surface presence of the Tim-3 receptor in primary human AML blasts and healthy primary human leukocytes isolated from human blood. We found that Tim-3 expression was significantly higher in primary AML cells compared to primary healthy leukocytes. Tim-3 receptor molecules were distributed largely on the surface of primary AML cells, whereas in healthy leukocytes Tim-3 protein was mainly expressed intracellularly. In primary human AML blasts, both Tim-3 agonistic antibody and galectin-9 (a Tim-3 natural ligand) significantly upregulated mTOR pathway activity. This was in line with increased accumulation of hypoxia-inducible factor 1 alpha (HIF-1α) and secretion of VEGF and TNF-α. Similar results were obtained in primary human healthy leukocytes. Importantly, in both types of primary cells, Tim-3-mediated effects were compared with those induced by lipopolysaccharide (LPS) and stem cell factor (SCF). Tim-3 induced comparatively moderate responses in both AML cells and healthy leukocytes. However, Tim-3, like LPS, mediated the release of both TNF-α and VEGF, while SCF induced mostly VEGF secretion and did not upregulate TNF-α release

Leukocyte margination in alveolar capillaries: Interrelationship with functional capillary geometry and microhemodynamics

The pulmonary capillary microvasculature harbors a large pool of intravascularly marginated leukocytes. In this study, we investigated the interrelationship of leukocyte margination with characteristics of functional capillary geometry and microhemodynamics in alveolar capillary networks. In 22 anesthetized rabbits we assessed functional capillary density, average capillary length, red blood cell velocity and leukocyte kinetics in alveolar capillary networks in vivo by intravital fluorescence microscopy. In alveolar wall areas of 12,800 +/- 1,800 mu m(2), we detected 3.6 +/- 0.5 sticking leukocytes and 21.0 +/- 1.9 functional capillary segments with an average capillary length of 35.7 +/- 2.1 mu m. We calculated that approximately 15% of functional capillary segments are blocked by marginated leukocytes. Leukocyte margination was predominantly observed in capillary networks characterized by a high functional capillary density, short capillary segments and low red blood cell velocities. The multitude of interconnected capillary channels in these networks may allow alveolar blood flow to bypass marginated leukocytes. Hence, this interrelationship may be relevant for maintenance of adequate alveolar perfusion and low capillary network resistance despite excessive leukocyte margination in the pulmonary microvasculature. Local microhemodynamic factors may play a regulatory role in the spatial distribution of leukocyte margination

TGF-b2 induction regulates invasiveness of theileria-transformed leukocytes and disease susceptibility

Theileria parasites invade and transform bovine leukocytes causing either East Coast fever (T. parva), or tropical theileriosis (T. annulata). Susceptible animals usually die within weeks of infection, but indigenous infected cattle show markedly reduced pathology, suggesting that host genetic factors may cause disease susceptibility. Attenuated live vaccines are widely used to control tropical theileriosis and attenuation is associated with reduced invasiveness of infected macrophages in vitro. Disease pathogenesis is therefore linked to aggressive invasiveness, rather than uncontrolled proliferation of Theileria-infected leukocytes. We show that the invasive potential of Theileria-transformed leukocytes involves TGF-b signalling. Attenuated live vaccine lines express reduced TGF-b2 and their invasiveness can be rescued with exogenous TGF-b. Importantly, infected macrophages from disease susceptible Holstein-Friesian (HF) cows express more TGF-b2 and traverse Matrigel with great efficiency compared to those from disease-resistant Sahiwal cattle. Thus, TGF-b2 levels correlate with disease susceptibility. Using fluorescence and time-lapse video microscopy we show that Theileria-infected, disease-susceptible HF macrophages exhibit increased actin dynamics in their lamellipodia and podosomal adhesion structures and develop more membrane blebs. TGF-b2-associated invasiveness in HF macrophages has a transcription-independent element that relies on cytoskeleton remodelling via activation of Rho kinase (ROCK). We propose that a TGF-b autocrine loop confers an amoeboid-like motility on Theileria-infected leukocytes, which combines with MMP-dependent motility to drive invasiveness and virulence

Maternal and infant infections stimulate a rapid leukocyte response in breastmilk

Breastmilk protects infants against infections; however, specific responses of breastmilk immune factors to different infections of either the mother or the infant are not well understood. Here, we examined the baseline range of breastmilk leukocytes and immunomodulatory biomolecules in healthy mother/infant dyads and how they are influenced by infections of the dyad. Consistent with a greater immunological need in the early postpartum period, colostrum contained considerable numbers of leukocytes (13–70% out of total cells) and high levels of immunoglobulins and lactoferrin. Within the first 1–2 weeks postpartum, leukocyte numbers decreased significantly to a low baseline level in mature breastmilk (0–2%) (P\u3c0.001). This baseline level was maintained throughout lactation unless the mother and/or her infant became infected, when leukocyte numbers significantly increased up to 94% leukocytes out of total cells (P\u3c0.001). Upon recovery from the infection, baseline values were restored. The strong leukocyte response to infection was accompanied by a more variable humoral immune response. Exclusive breastfeeding was associated with a greater baseline level of leukocytes in mature breastmilk. Collectively, our results suggest a strong association between the health status of the mother/infant dyad and breastmilk leukocyte levels. This could be used as a diagnostic tool for assessment of the health status of the lactating breast as well as the breastfeeding mother and infant

Severity of experimental escherichia-coli mastitis in ketonemic and nonketonemic dairy-cows.

The severity of experimental Escherichia coli mastitis in relation to in vitro chemotaxis of polymorphonuclear leukocytes was investigated in cows during negative energy balance. The negative energy balance was induced by feed restriction. Cows were classified into two groups, ketonemic and nonketonemic, based on the beta-hydroxybutyrate concentration in the peripheral blood at the moment of inoculation. Bacterial growth in the inoculated quarter was used as a parameter to indicate the severity of experimental mastitis. In the nonketonemic cows, experimental mastitis ranged from moderate to severe. Severity of experimental mastitis was negatively related to preinfection chemotactic response of polymorphonuclear leukocytes. In contrast, the course of experimental mastitis in the ketonemic group was relatively severe in all cows, regardless of preinfection chemotactic response

The effects of stress on the enzymes of peripheral leukocytes

Previous work showed an early response of rabbit and human leukocyte enzymes to the stress of bacterial infection. Since these represented a mixed population of leukocytes and since polymorphonuclear leukocytes (PMN) increased in these preparations, it was necessary to establish whether the observed increase in lactate dehydrenase (LDH) and protein was the result of an increase in any one particular cell type or in all cells. The need for the development of a simple reproducible method for the differential separation of peripheral leukocytes for the furtherance of our own studies was apparent. It was also becoming increasingly apparent that morphologically similar cells, such as small lymphocytes (L) and macrophages, were capable of different biological functions. A dextran gradient centrifugation method was developed which has provided an easily reproducible technique for separating L from PMN. During the course of this work, in which over 250 rabbits were examined, the pattern of daily leukocyte protein and enzyme variation became increasingly more apparent. This information could have some impact on future work with leukocyte enzymes, by our group and by other workers. The differences in normal protein and enzyme levels maintained by some individuals, and some inbred strains, were evaluated and reported separately. It has been shown that one type of leukocyte may react more to a given stress than other leukocytes