Leukotrienes cause eosinophil emigration into conjunction tissue

The ability of LTB₄, LTC₄, the 5S,6R and 5R,6S LTD₄ stereoisomers, and LTE₄ to evoke leukocyte infiltration into the conjunctiva was demonstrated in the guinea pig by histological andl ight microscopy techniques. LTD₄ and LTE₄ demonstrated a dose-dependent and predominantly eosinophilic infiltrate over the selected dose range (10ng to 1000ng), while there was only a minimal response to LTC₄. LTB₄ produced marked eosinophil inflitrates only at the highest dose; scattered neutrophil infiltrates were also noted at the high dose of LTB₄. The 5R,6S LTD₄ stereoisomer did not evoke any leukocyte infiltrantion. The SRS-A antagonist, FPL 55712, abolished pre-treatment had no inhibitory effect, indicating direct mediation of this response by LTs. Histamine caused a comparable eosinophilia over a dose range of 10μg to 1000μg. LT-induced eosinophil emigration was directed to the conjunctival epithelium; the cells appeared intact and no tissue damage was observed. These results may have relevance in the areas of allergic conjunctivitis and asthma research.     

Leukotrienes cause mesentric vasoconstriction and hemoconcentration in rats without activating thromboxane receptors

Thromboxane A₂/prostaglandin H₂ (TP)-receptor activation has been reported to participate in some of the response to peptide leukotrienes (LT). We examined the effect of TP-receptor antagonism on LT-induced mesenteric vasoconstriction and hemoconcentration in anesthetized rats. The antagonist used in these studies, SQ 30,741, was shown to have high selectivity and potency for vascular TP-receptors in the rat. Arterial (i.a) injection of LTC₄ and D₄ elicited dose-dependent and transient reductions in mesenteric blood flow without changes in arterial blood pressure. These responses were unaffected by a dose of SQ 30,741 which produced 99% inhibition of similar responses to U-46,619. In contrast, LT-induced mesenteric vasoconstriction was inhibited 90% by two LT antagonists, LY 171,883 and SKF 104,353. In other experiments i.v. infusion of LTD₄ caused increases in hematocrit and reductions in arterial blood pressure that were not influenced by SQ 30,741. These data suggest that increases in mesenteric vascular resistance and hemoconcentration in response to LTs are not the result of TP-receptor activation.     

Immunohistochemical artifact for nitrotyrosine in eosinophils or eosinophil containing tissue

Immunohistochemical artifacts for nitrotyrosine were investigated in eosinophils with regard to fixatives. Immunoreactivity for nitrotyrosine was revealed in separated eosinophils and in gastric mucosa fixed with periodate, lysine-paraformaldehyde (PLP). The increase in immunoreactivity by PLP was due to periodate itself, a component of PLP. Nitrotyrosine formed by peroxidase using NO 2 - and H 2 O 2 or by peroxynitrite was not completely inhibited by 100 mM dithionite but the immunoreactivity for nitrotyrosine antibodies by PLP was completely inhibited by 5.7 mM dithionite. Although untreated eosinophils or ovalbumin (OVA) did not show protein tyrosine nitration in a standard Western blot, the treatment of the blotted membrane with PLP increased the reactivities of proteins from eosinophils with anti-nitrotyrosine antibodies. The increase in immunoreactivity of OVA with anti-nitrotyrosine antibodies by PLP did not change with pre-treatment with dithionite but was abolished by treatment with dithionite after PLP fixation. In HPLC assays, periodate did not generate nitrotyrosine from l -tyrosine and aminotyrosine. These results suggest that the treatment of eosinophils or eosinophil-containing tissues with PLP fixative augments the immunoreactivity of nitrotyrosine antibodies with eosinophils due to the formation of epitopes similar to nitrotyrosine by an oxidation reaction of periodate, which evokes an artifact in nitrotyrosine immunohistochemistry.     

Ostertagia ostertagi: excretory secretory chemotactic substance from infective larvae as cause of eosinophil locomotion

The infective (third stage) larva of Ostertagia ostertagi produced an excretory secretory substance that is chemotactic for bovine eosinophils. The eosinophil chemotactic substance was present in supernatants of larval cultures within 6 hr of incubation in Eagle's Minimum Essential Medium containing 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid. The substance responsible for eosinophil chemotaxis has activity in small amounts, a molecular weight (MW) greater than 2000, and is heat labile at 100 C. Bovine eosinophils appeared to have a receptor for the chemotactic excretory secretory substance which is either identical or structurally similar to the previously described eosinophil chemotactic substance present in soluble third stage larval extracts. The larval substance may cause eosinophil accumulation in the abomasal tissue of cattle with ostertagiasis.     

IL-16 activates plasminogen-plasmin system and promotes human eosinophil migration into extracellular matrix via CCR3-chemokine-mediated signaling and by modulating CD4 eosinophil expression

Increased eosinophil counts are a major feature of asthmatic airways. Eosinophil recruitment requires migration through epithelium and tissue extracellular matrix by activation of proteases. We assessed the capacity of IL-16, a CD4(+) cell chemotactic factor, to induce migration of eosinophils through a reconstituted basement membrane and evaluated the proteases, mediators, and receptors involved in this migration. IL-16 added to lower chambers of Invasion Chambers elicited eosinophil migration through Matrigel. This effect was decreased by inhibition of the plasminogen-plasmin system (Abs against urokinase plasminogen activator receptor or plasminogen depletion), but not by anti-matrix metalloproteinase-9 Abs. Abs against CD4 also inhibited IL-16-induced eosinophil migration. At the baseline level, few eosinophils (4.6% positive cells with a mean fluorescence of 0.9) expressed surface membrane CD4, while most permeabilized eosinophils (68% positive cells with a mean fluorescence of 18) express the CD4 Ag. TNF-pretreatment increased surface membrane CD4(+) expression by 6-fold as previously described, and increased IL-16-induced cell migration by 2.2-fold. Incubation of eosinophils with IL-16 also increased surface membrane CD4 expression by 5.4-fold, supporting the role of CD4 as receptor for IL-16. Abs against CCR3, eotaxin, or RANTES blocked IL-16-induced migration. In conclusion, IL-16 promotes eosinophil migration in vitro, by activating the plasminogen-plasmin system and increasing the membrane expression of its receptor. This effect is initiated via CD4 and mediated via the release of CCR3 ligand chemokines. Interestingly, most eosinophils express intracellular CD4. Hence, IL-16 may play an important role in the recruitment of blood eosinophils to the bronchial mucosa of asthmatics.     

Metaplasia of aortic tissue into tracheal tissue. Surgical perspectives

Tracheal reconstruction after extensive resection remains an unsolved surgical problem. Numerous attempts have been made using tracheal grafts or prosthetic conduits with disappointing results. In this study, we propose a new alternative using an aortic autograft as tracheal substitute. In a first series of experiments, a half circumference of two rings was replaced with an autologous carotid artery patch. In a second series, a complete segment of trachea was replaced with an autologous aortic graft supported by an endoluminal tracheal stent. No dehiscence or stenosis was observed. Microscopic examinations at 3 and 6 months showed the replacement of the aortic tissue by tracheal tissue comprising neoformation of cartilage and mucociliary or non-keratinizing metaplastic polystratified squamous epithelium. Although these results need to be confirmed by a larger series of experiments, they showed that a vascular tissue placed in a different environment with a different function can be submitted to a metaplastic transformation which tends to restore a normal structure adapted to its new function. These remarkable findings offer new perspectives in tracheal reconstruction in human.     

Complement activated granulocytes can cause autologous tissue destruction in man

Activation of polymorphonuclear granulocytes (PMNs) by C5a is thought to be important in the pathogenesis of multiple organ failure during sepsis and after trauma. In our experiment exposure of human PMNs to autologous zymosan activated plasma (ZAP) leads to a rapid increase in chemiluminescence. Heating the ZAP at 56( degrees )C for 30 min did not alter the changes, while untreated plasma induced only baseline activity. The respiratory burst could be completely abolished by decomplementation and preincubation with rabbit antihuman C5a antibodies. Observation of human omentum using electron microscopy showed intravascular aggregation of PMNs, with capillary thrombosis and diapedesis of the cells through endothelial junctions 90 s after exposure to ZAP. PMNs caused disruption of connections between the mesothelial cells. After 4 min the mesothelium was completely destroyed, and connective tissue and fat cells exposed. Native plasma and minimum essential medium did not induce any morphological changes. These data support the concept that C5a activated PMNs can cause endothelial and mesothelial damage in man. Even though a causal relationship between anaphylatoxins and organ failure cannot be proved by these experiments C5a seems to be an important mediator in the pathogenesis of changes induced by severe sepsis and trauma in man.     

Linear bulk diffusion into heterogeneous tissue

In this paper an expression is derived which describes the transient overall uptake of an inert solute by a section of tissue excised with parallel faces and placed upon an impermeable base. The approach diverges from the conventional analyses for perfused tissue (Morales and Smith,Bull. Math. Biophysics,6, 125–141, 1944;7, 47–99, 1945) because the extravascular zone is regarded as a heterogeneous diffusion medium. Account for this is taken by regarding tissue as effectively composed of two phases—a continuous (extracellular) phase similar to water, and a dispersed phase comprising cells of irregular profile. In both phases the relevant mode of uptake is taken as bulk diffusion rather than surface permeation, thus emphasizing the influence of the internal geometry of the tissue upon its overall exchange response.     

Radial bulk diffusion into heterogeneous tissue

An expression is derived which describes the transient distribution of solute diffusing into heterogeneous tissue from a fully-stirred cylindrical region in which there has been a step change in solute concentration. This is envisaged as a model for the uptake of drugs by vessel walls, although the same approach has been extended for estimating the mode of total uptake of substances by annular specimens of tissue. Tissue is regarded as effectively composed of two phases—an extracellular (continuous) phase, similar to water, and a dispersed phase comprising cells of irregular profile. In each phase the relevant mode of uptake is taken as bulk diffusion rather than the permeation of a surface membrane.     

Translating cell polarity into tissue elongation

Planar cell polarity, the orientation of single-cell asymmetries within the plane of a multicellular tissue, is essential to generating the shape and dimensions of organs and organisms. Planar polarity systems align cell behavior with the body axes and orient the cellular processes that lead to tissue elongation. Using Drosophila as a model system, significant progress has been made toward understanding how planar polarity is generated by biochemical and mechanical signals. Recent studies using time-lapse imaging reveal that cells engage in a number of active behaviors whose orientation and dynamics translate planar cell polarity into tissue elongation. Here we review recent progress in understanding the cellular mechanisms that link planar polarity to large-scale changes in tissue structure.     

An alternate STAT6-independent pathway promotes eosinophil influx into blood during allergic airway inflammation

Background

Enhanced eosinophil responses have critical roles in the development of allergic diseases. IL-5 regulates the maturation, migration and survival of eosinophils, and IL-5 and eotaxins mediate the trafficking and activation of eosinophils in inflamed tissues. CD4⁺ Th2 cells are the main producers of IL-5 and other cells such as NK also release this cytokine. Although multiple signalling pathways may be involved, STAT6 critically regulates the differentiation and cytokine production of Th2 cells and the expression of eotaxins. Nevertheless, the mechanisms that mediate different parts of the eosinophilic inflammatory process in different tissues in allergic airway diseases remain unclear. Furthermore, the mechanisms at play may vary depending on the context of inflammation and microenvironment of the involved tissues.

Methodology/Principal Findings

We employed a model of allergic airway disease in wild type and STAT6-deficient mice to explore the roles of STAT6 and IL-5 in the development of eosinophilic inflammation in this context. Quantitative PCR and ELISA were used to examine IL-5, eotaxins levels in serum and lungs. Eosinophils in lung, peripheral blood and bone marrow were characterized by morphological properties. CD4⁺ T cell and NK cells were identified by flow cytometry. Antibodies were used to deplete CD4⁺ and NK cells. We showed that STAT6 is indispensible for eosinophilic lung inflammation and the induction of eotaxin-1 and -2 during allergic airway inflammation. In the absence of these chemokines eosinophils are not attracted into lung and accumulate in peripheral blood. We also demonstrate the existence of an alternate STAT6-independent pathway of IL-5 production by CD4⁺ and NK cells that mediates the development of eosinophils in bone marrow and their subsequent movement into the circulation.

Conclusions

These results suggest that different points of eosinophilic inflammatory processes in allergic airway disease may be differentially regulated by the activation of STAT6-dependent and -independent pathways.     

Experimental evidence suggesting that nitric oxide diffuses from tissue into blood but not from blood into tissue

The aim of this study was to evaluate in vivo whether nitric oxide (NO) is able to diffuse from blood into tissues and vice versa from tissues into blood. We used an in vivo model of intestinal ischemia (superior mesenteric artery occlusion) selectively increasing NO levels in intestinal tissue and an infusion of L-arginine selectively increasing NO levels in blood. In this model we followed formation of nitrosyl complexes of hemoglobin (Hb-NO) in blood and nitrosyl-diethyldithiocarbamate-iron complexes (DETC--Fe--NO) in ischemic intestine and normoxic tissues by means of electron paramagnetic resonance spectroscopy. NO trapping by DETC--Fe in the tissues resulted in a reduction of Hb--NO levels in blood accompanied by the formation of water-insoluble DETC--Fe-NO complexes in ischemic intestine and normoxic tissues both during ischemia and during reperfusion. Administration of L-arginine increased NO levels in blood but neither in ischemic intestine nor in normoxic tissue. Our data suggest that NO released in blood from endothelial cells does not diffuse into tissue. In contrast, NO formed in tissue diffuses into blood. The latter indicates that NO formed in tissues may exert its biological activities systematically.     

Identification and tissue localization of an eosinophil 17 kDa protein accumulating in rat uterus upon estradiol treatment

In a previous paper (J. Steroid Biochem. 29 (1988) 475-480), the isolation of a 17 kDa protein that was dramatically induced in the uterus of estrogen-treated spayed rats was presented. We now describe a new purification procedure that is compatible with microsequencing of the 17 kDa protein. The protein partial N-terminal amino acid sequence analysis gave 28 residues that revealed a strong homology to the human major basic protein (MBP) of eosinophils described by Wasmoen et al. (J. Biol. Chem. 263 (1988) 12559-12563). Polyclonal rabbit antibodies were raised against this protein and used for tissue or blood cell analysis after electrophoresis and Western blotting. The 17 kDa protein was found to be constitutively present in the stomach and small intestine of the rat and guinea-pig. Estrogen treatment had a clearcut effect in guinea-pig uterus, but not as drastic as that observed in rat uterus. The protein was abundant in purified rat eosinophils. The antibodies cross-reacted with human MBP and an equivalent molecular weight human polymorphonuclear leukocyte protein. Immunohistochemical staining of rat uterus sections showed that the protein was first only associated with eosinophils that emigrate upon estrogen treatment; it then spread throughout the stroma and the deep glandular epithelium. It was not found in the myometrium. In conclusion, the appearance of a 17 kDa protein that is presumably the rat MBP is clearly regulated in the rat uterus.     

IL-13 is required for eosinophil entry into the lung during respiratory syncytial virus vaccine-enhanced disease

Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract disease in children. Children previously vaccinated with a formalin-inactivated RSV vaccine experienced enhanced morbidity and mortality upon natural RSV infection. Histological analysis revealed the presence of eosinophils in the pulmonary infiltrate of the vaccinated children. Eosinophils are characteristic of Th2 responses, and Th2 cells are known to be necessary to induce pulmonary eosinophilia in RSV-infected BALB/c mice previously immunized with a recombinant vaccinia virus (vv) expressing the RSV G protein (vvG). Using IL-13-deficient mice, we find that IL-13 is necessary for eosinophils to reach the lung parenchyma and airways of vvG-immunized mice undergoing RSV challenge infection. IL-13 acts specifically on eosinophils as the magnitude of pulmonary inflammation, RSV G protein-specific CD4 T cell responses, and virus clearance were not altered in IL-13-deficient mice. After RSV challenge, eosinophils were readily detectable in the blood and bone marrow of vvG-immunized IL-13-deficient mice, suggesting that IL-13 is required for eosinophils to transit from the blood into the lung. Pulmonary levels of CCL11 and CCL22 protein were significantly reduced in IL-13-deficient mice indicating that IL-13 mediates the recruitment of eosinophils into the lungs by inducing the production of chemokines important in Th2 cell and eosinophil chemotaxis.     

Transferring isolated mitochondria into tissue culture cells

We have developed a new method for introducing large numbers of isolated mitochondria into tissue culture cells. Direct microinjection of mitochondria into typical mammalian cells has been found to be impractical due to the large size of mitochondria relative to microinjection needles. To circumvent this problem, we inject isolated mitochondria through appropriately sized microinjection needles into rodent oocytes or single-cell embryos, which are much larger than tissue culture cells, and then withdraw a ‘mitocytoplast’ cell fragment containing the injected mitochondria using a modified holding needle. These mitocytoplasts are then fused to recipient cells through viral-mediated membrane fusion and the injected mitochondria are transferred into the cytoplasm of the tissue culture cell. Since mouse oocytes contain large numbers of mouse mitochondria that repopulate recipient mouse cells along with the injected mitochondria, we used either gerbil single-cell embryos or rat oocytes to package injected mouse mitochondria. We found that the gerbil mitochondrial DNA (mtDNA) is not maintained in recipient rho0 mouse cells and that rat mtDNA initially replicated but was soon completely replaced by the injected mouse mtDNA, and so with both procedures mouse cells homoplasmic for the mouse mtDNA in the injected mitochondria were obtained.     

Blockade of JAK2 by tyrphostin AG-490 inhibits antigen-induced eosinophil recruitment into the mouse airways

We studied the effect of tyrphostin AG-490, a specific Janus kinase 2 (JAK2) inhibitor, on antigen-induced eosinophil recruitment into the airways of sensitized mice and on IL-5-induced chemokinesis and adhesiveness of eosinophils. The in vivo administration of AG-490 prevented antigen-induced eosinophil infiltration in the airways of sensitized mice in a dose-dependent manner. However, the administration of AG-490 did not affect antigen-induced IL-5 production in the airways nor in vitro antigen-induced IL-5 production and T cell proliferation of spleen cells. Furthermore, AG-490 inhibited IL-5-induced chemokinesis and beta1-integrin adhesiveness of eosinophils in vitro. Because antigen-induced eosinophil recruitment into the airways is mediated by IL-5, these results indicate that JAK2 activation is critical for antigen-induced, IL-5-dependent mobilization of eosinophils into the tissue.