Endothelial cells modulate eosinophil surface markers and mediator release.

Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells. The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C4 were evaluated. A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1beta slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-gamma; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1beta-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C4 than control cells (872.4 and 103.9 pg x mL(-1), respectively). Endothelial cell pretreatment with IL-4 or IL-1beta further increased leukotriene C4 release (1,789.1 and 2,895.1 pg x mL(-1), respectively), whereas pretreatment with IFN-gamma decreased it (293.7 pg x mL(-1)). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis.

Migration through basement membrane modulates eosinophil expression of CD44.

BACKGROUND: Tissue eosinophils express more membrane receptors and release more mediators than blood eosinophils, suggesting that migration from blood to tissue modulates eosinophil phenotype and functions. OBJECTIVE: We postulated that eosinophil passage through endothelial basement membrane, an important step of eosinophil migration into tissue, may be responsible for some of these changes. METHOD: We previously showed that 5-oxo-6, 8, 11, 14-eicosatetraenoic acid (5-oxo-ETE) in combination with IL-5 promotes eosinophil migration through Matrigel, a mouse tumour cell-derived basement membrane. Using this model, we evaluated the effect of trans-Matrigel migration on purified human blood eosinophil expressions of CD44, CD69 and HLA-DR that either increase or appear on activated eosinophils, and releases of peroxidase (EPO), leukotriene (LT) C(4) and granulocyte-monocyte colony stimulating factor (GM-CSF). RESULTS: IL-5, but not 5-oxo-ETE, increased eosinophil expression of CD44 and CD69. Migration of eosinophils through Matrigel significantly increased CD44 expression level over the one induced by IL-5 (P = 0.0001). Migration through Matrigel did not modify CD69 expression compared with the one obtained in the presence of IL-5 alone; however, incubation of eosinophils on Matrigel decreased IL-5-induced CD69 (P = 0.0001). Trans-Matrigel migration did not modify HLA-DR expression, nor EPO, LTC(4) and GM-CSF releases. CONCLUSION: These data show that in vitro trans-Matrigel migration and Matrigel contact modulate eosinophil membrane receptor expression. Consequently, they suggest that migration through basement membrane mediates changes in cell-surface phenotype observed on activated eosinophils and probably prepares them for interactions with tissue components and cells.

Airway and tissue behavior during early response in sensitized rats: role of 5-HT and LTD4.

We have recently demonstrated that tissue resistance increases during the early response (ER) to antigen challenge in sensitized Brown-Norway rats. The purpose of the present study was to investigate the role of the potential ER mediators 5-hydroxytryptamine (5-HT) and leukotriene D4 (LTD4) in the airway and tissue response. We sensitized the rats with ovalbumin (OA) and performed experiments on anesthetized, open-chested, mechanically ventilated [breathing frequency = 1 Hz, tidal volume = 12 ml/kg, positive end-expiratory pressure (PEEP) = 3 cmH2O] animals. We affixed alveolar capsules to the lungs to measure alveolar pressure and calculated the resistance of lung (RL), tissue (Rti), and airway (Raw). To assess the effects of LTD4 and 5-HT, we administered the antagonists methysergide (5-HT antagonist) and MK-571 (LTD4 antagonist) before challenge. To assess lung morphometry during the ER, the lungs of four animals from each group were frozen with liquid nitrogen (PEEP = 3 cmH2O). Airway constriction was assessed by measuring the ratio of the airway lumen to the ideally relaxed airway (Abm/A*bm). Tissue distortion was assessed by measuring the mean linear intercept between alveolar walls (Lm), an atelectasis index (ATI) derived by calculating the ratio of tissue to air space, and SD of the two (SD-Lm and SD-ATI). In all animals receiving OA but no antagonists, an ER was seen (RL, Rti, and Raw = 180.7 +/- 6.1, 155.4 +/- 8.2, and 223.1 +/- 14.0% of baseline, respectively). Methysergide significantly inhibited the ER (RL, Rti, and Raw = 117.0 +/- 5.9, 101.2 +/- 1.6, 133.7 +/- 10.2%, respectively), whereas MK-571 partially reduced the ER (RL, Rti, and Raw = 144.2 +/- 5.6, 132.9 +/- 5.7, and 155.5 +/- 9.2%, respectively). Abm/A*bm was significantly decreased, and SD-Lm and SD-ATI were significantly increased in animals receiving OA alone and in those receiving MK-571 before OA challenge. These data suggest that alterations in both airways and tissues contribute to the ER and that 5-HT and, to a lesser degree, LTD4 are important mediators of the ER in this rat model of extrinsic asthma.

Dynamic elastance and tissue resistance of isolated liquid-filled rat lungs.

The effect of the surface forces of the alveolar air-liquid interface on the dynamic behavior of lung tissue was investigated in five isolated liquid-filled rat lungs. The lungs were subjected to 0.04-Hz sinusoidal oscillation (1.5-ml tidal volume) at lung volume (VL) levels ranging from volume at zero pressure (V0) + 4 ml to V0 + 10 ml. Oscillations were performed at each VL after inflation of the lungs from V0. Alveolar pressure (PA) was measured with an alveolar capsule attached to the visceral pleura. Dynamic elastance (Edyn), tissue resistance (Rti), and hysteresivity [eta = Rti omega/Edyn, where omega is angular frequency (2 pi x frequency)] were computed from PA and VL changes. Edyn was 59.6 +/- 4.3 Pa/ml at V0 + 4 ml and varied little up to V0 + 7 ml. Thereafter, Edyn increased markedly with VL, reaching 102 +/- 16 Pa/ml at V0 + 10 ml. No significant difference was found between elastance computed from PA and that computed from pressure recorded at the airway opening. Rti was 35.2 +/- 3.6 Pa.s.ml-1 and exhibited a VL dependence similar to that of Edyn. As a result, eta was 0.16 and did not vary significantly in the explored VL range. We conclude that PA can be reliably measured in the liquid-filled lung by means of alveolar capsules. In the liquid-filled lung, Edyn was smaller than and eta was similar to values reported for air-filled lungs. Hence, surface tension accounts for a considerable part of elastance and Rti of the air-filled lung within the volume range of normal breathing.

In vitro airway and tissue response to antigen in sensitized rats. Role of serotonin and leukotriene D4.

We have recently demonstrated that tissue resistance increases during the early response (ER) to antigen challenge in sensitized Brown-Norway rats. The purpose of the present study was to investigate the in vitro airway and tissue responses to antigen and the involvement of the potential mediators serotonin (5-HT) and leukotriene D4 (LTD4). We sensitized Brown-Norway rats with ovalbumin (OA) and subsequently challenged bronchial rings and subpleural parenchymal strips with OA in the organ bath. In selected experiments tissues were incubated with methysergide (a 5-HT receptor antagonist), ketanserin (a 5-HT2 receptor antagonist), MK-571 (a LTD4 receptor antagonist), or MK-886 (5-lipoxygenase inhibitor) prior to challenge. Both bronchial rings and parenchymal strips constricted in response to OA. Methysergide and ketanserin completely inhibited OA-induced constriction of bronchial rings. The effect of MK-571 was not significant, whereas MK-886 partially blocked OA-induced bronchial constriction, suggesting a potential role for LTC4 in antigen-induced airway constriction. In parenchymal strips, methysergide, ketanserin, MK-571, and MK-886 all partially inhibited the OA response, whereas the combinations of methysergide and MK-571 or ketanserin and MK-886 completely ablated the response. These data suggest that both bronchial rings and parenchymal strips constrict after OA challenge but that the relative contributions of 5-HT and LTD4 to the allergic response in central airways and parenchymal tissues differ.

Structural composition of lung parenchymal strip and mechanical behavior during sinusoidal oscillation.

The lung parenchymal strip is comprised of many different anatomic elements, including small vessels, small airways, and alveolar walls. We questioned whether the relative amounts of these different structures are important in determining the mechanical behavior of this preparation during dynamic oscillations. We studied 16 parenchymal strips (10 x 2 x 2 mm) from 12 Sprague-Dawley rats. The strips were suspended in an organ bath filled with Krebs solution, bubbled with 95% O2-5% CO2, and maintained at 37 degrees C. One end of the strip was attached to a force transducer, and the other end was attached to a lever system that effected length (L) changes. We oscillated the strips at various resting tensions (T) (0.9 and 1.5 g), frequencies (0.1, 0.3, 0.6, and 1.0 Hz), and amplitudes (1.1, 2.4, and 5.3% of optimal L). We obtained T vs. L curves and calculated the resistance, elastance, and hysteresivity (ratio of energy dissipated to energy stored) of the tissue. At the end of the experiment, the strips were fixed in Formalin at T = 1 g. Histological sections were examined, and the amounts of airway, blood vessel, and alveolar wall were quantified using point counting techniques. We found that whereas resistance varied significantly with frequency and T, elastance and hysteresivity varied with only T. The fractional areas of alveolar, blood vessel, and bronchial wall were 86.3 +/- 0.5 (SE), 8.4 +/- 0.3, and 5.3 +/- 0.4%. Only hysteresivity and the fractional area of alveolar wall were significantly correlated at the lower resting tension (r = -0.76, P = 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)

Airway and tissue responses to antigen challenge in sensitized brown Norway rats.

It has recently been shown in several species that lung tissue resistance increases after administration of exogenous bronchoconstrictors. This finding suggests the possibility that lung parenchymal tissues could be involved in the pathophysiology of pulmonary allergic responses. To test this hypothesis, we sensitized Brown Norway rats with ovalbumin (OA) and performed experiments in anesthetized, open-chested, mechanically ventilated (respiratory frequency [f] = 1 Hz, tidal volume [VT] = 9 ml/kg, positive end-expiratory pressure [PEEP] = 3 cm H2O) animals. We affixed alveolar capsules to the lungs to measure alveolar pressure and calculated the resistance of lung (RL), tissue (Rti), and airway (Raw) under control conditions and after aerosol administration of saline (S) (n = 10) or OA (n = 14). To assess lung morphometry during the late response, the lungs of six S and six OA animals were frozen with liquid nitrogen (PEEP = 3 cm H2O) and processed via freeze substitution. Airway constriction was assessed by measuring the ratio of the airway lumen (A) to the ideally relaxed airway (Ar). Tissue distortion was assessed by measuring the mean linear intercept between alveolar walls (Lm), an atelectasis index (ATI) derived by calculating the ratio of tissue/airspace, and the standard deviation (SD) of Lm and ATI. In the OA group, all animals demonstrated an early response (ER; RL, Rti, Raw = 183.5 +/- 7.7, 159.7 +/- 9.9, 232.5 +/- 17.2% baseline, respectively) and 11 animals showed a late response (LR; RL, Rti, Raw = 178.9 +/- 5.1, 191.3 +/- 11.5, 176.6 +/- 17.3% baseline, respectively). Neither ER nor LR were observed in the saline group.(ABSTRACT TRUNCATED AT 250 WORDS)

Airway and tissue responses during hyperpnea-induced constriction in guinea pigs.

It has been reported that hyperpnea-induced bronchoconstriction in guinea pigs is a potential model for exercise-induced asthma in humans. On the basis of recent studies that show increases in tissue resistance after allergen exposure in sensitized rats, we hypothesized that lung tissues might also be involved in the pathophysiology in this asthma model. We measured tracheal pressure (Ptr) and alveolar pressure (PA) using alveolar capsules in open-chested, mechanically ventilated (respiratory frequency [f] = 1 Hz, tidal volume [VT] = 9 ml/kg, positive end-expiratory pressure [PEEP] = 4 cm H2O) guinea pigs under control conditions (regular breathing of warm, humidified air) and after dry gas hyperpnea challenge (HC, mixture of 95% O2 and 5% CO2, 150 breaths/min, 7 min). We calculated lung elastance (EL) and resistance of lung (RL), tissue (Rti), and airway (Raw) by fitting the equation of motion to changes in Ptr and PA. To assess the effects of volume history, we applied a single deep inflation (three times VT) in five HC animals. We performed morphometric analysis in five control and five HC animals, freezing the lungs with liquid nitrogen and processing the tissues via freeze substitution. HC significantly increased RL, Rti, Raw, and EL (424 +/- 62, 771 +/- 230, 287 +/- 33, 259 +/- 31% baseline, respectively). A deep inflation reduced RL, Rti, Raw, and EL by 30 +/- 4, 31 +/- 4, 29 +/- 6, 23 +/- 5%, respectively. In HC animals, the degree of airway constriction was most prominent in the larger airways; extensive tissue distortion was also observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis of the allylic regulatory steroid, 3 alpha-hydroxy-4-pregnen-20-one, by rat granulosa cells and its regulation by gonadotropins.

The production of the allylic regulatory steroid 3 alpha-hydroxy-4-pregnen-20-one (3 alpha HP) in the rat ovary was examined and compared to progesterone levels through use of specific RIAs that had been validated by capillary gas chromatography-mass spectrometry (GC/MS). Results showed that serum levels of 3 alpha HP are comparable to levels of progesterone at all ages examined. In the 4-day cycling rat, serum levels of 3 alpha HP were highest during diestrus and lowest during proestrus and estrus, while serum FSH levels were highest during proestrus/estrus and lowest during diestrus. Hypophysectomy resulted in decreases in ovarian and serum 3 alpha HP. Treatment of hypophysectomized rats with eCG, but not hCG, increased ovarian and serum 3 alpha HP, while serum progesterone was elevated by treatment with hCG. Ovariectomy resulted in a 55-60% reduction in serum 3 alpha HP, indicating that ovaries are a substantial, but not exclusive, source of 3 alpha HP in serum. As further evidence, cultures of preparations consisting primarily of either granulosa cells or granulosa/theca "shells" produced 3 alpha HP in time-dependent amounts comparable to those of progesterone. Granulosa cells in culture showed a significant increase in accumulation of 3 alpha HP (and progesterone) due to treatment with FSH, but not LH. In contrast to the granulosa-only cell cultures, follicle shells consisting of theca and granulosa cells responded to either LH or FSH treatment with marked increases in 3 alpha HP; increases resulting from combined treatment (FSH + LH) were significantly greater than those due to each hormone alone, but the increases were not additives.(ABSTRACT TRUNCATED AT 250 WORDS)