Effects of hydrogen peroxide on the guinea-pig tracheobronchial mucosa in vivo.

Lumenal entry of plasma (mucosal exudation) is a key feature of airway inflammation. In airways challenged with histamine-type mediators and allergen the mucosal exudation response occurs without causing epithelial derangement and without increased airway absorption. In contrast, reactive oxygen metabolites may cause mucosal damage. In this study, involving guinea-pig airways, we have examined effects of H2O2 on airway exudation and absorption in vivo. Vehicle or H2O2 (0.1 and 0.5 M) was superfused onto the tracheobronchial mucosal surface through an oro-tracheal catheter. 125I-albumin, given intravenously, was determined in tracheobronchial tissue and in lavage fluids 10 min after challenge as an index of mucosal exudation of plasma. The tracheobronchial mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA was superfused 20 min after vehicle or H2O2 (0.1 and 0.5 M) had been given. A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of airway absorption. The high dose of H2O2 (0.5 M) produced epithelial damage, increased the absorption of 99mTc-DTPA (P < 0.001), and increased the exudation of plasma (P < 0.001). Notably, it appeared that all extravasated plasma had entered the airway lumen within 10 min. These data demonstrate that H2O2 differs from exudative autacoids such as histamine by causing both epithelial damage and plasma exudation responses. These data also agree with the view that the epithelial lining determines the rate of absorption and is responsible for the valve-like function that allows lumenal entry of extravasated bulk plasma without any increased inward perviousness.

Plasma-derived proteins in airway defence, disease and repair of epithelial injury.

One significant characteristic of the airway mucosa in vivo, that cannot easily be mimicked in vitro, is its microcirculation, which generates a highly dynamic, biologically active milieu of plasma-derived molecules that may pass to the airway lumen in vivo. New data on the mechanisms of airway mucosal exudation indicate that the protein systems of circulating plasma may contribute significantly to the biology and immunology of the lamina propria, its surface epithelium and the luminal surface, not only in injured airways, but also in airways that are activated but display no sign of oedema, epithelial disruption, or increased absorption capacity. We suggest that present knowledge of the mechanisms of plasma exudation, together with rapidly emerging information (not detailed herein) on receptors, target cells and cellular responses to the plasma-derived molecules, must be considered in any realistic model that investigates "immuno-inflammatory" mechanisms of the airway mucosa.

Contribution of plasma-derived molecules to mucosal immune defence, disease and repair in the airways.

This review discusses recent observations, in health and disease, on the release and distribution of plasma-derived molecules in the airway mucosa. Briefly, the new data on airway mucosal exudation mechanisms suggest that the protein systems of plasma contribute significantly to the mucosal biology, not only in injured airways but also in such mildly inflamed airways that lack oedema and exhibit no sign of epithelial derangement. Plasma as a source of pluripotent growth factor, adhesive, leucocyte-activating, etc., molecules may deserve a prominent position in schemes that claim to illustrate immunological and inflammatory mechanisms of the airway mucosa in vivo.

Effects of topical platelet activating factor on the guinea-pig tracheobronchial mucosa in vivo.

Platelet activating factor (PAF) has been reported to produce a variety of airway effects including epithelial damage and increased airway-lung absorption of hydrophilic tracers. The present study examines effects of PAF on the guinea-pig tracheobronchial mucosa in vivo. Vehicle with and without PAF (4.0 and 8.0 nmol) was superfused onto the tracheobronchial mucosa. The levels of 125I-albumin, previously given intravenously, were determined in tracheobronchial lavage fluids as an index of mucosal exudation of plasma. The mucosa was also examined by scanning electron microscopy. In separate animals, 99mTc-DTPA (a low molecular weight, 492 Da, hydrophilic tracer) was superfused onto the mucosal surface through an oro-tracheal catheter, together with vehicle or PAF (8.0 nmol). A gamma camera determined the disappearance rate of 99mTc-DTPA from the airways as an index of mucosal absorption. PAF produced dose-dependent mucosal exudation of plasma up to 20-fold greater than control (P < 0.001). However, PAF did not damage the epithelium and the absorption ability of the airway mucosa was unaffected. The results, in contrast to previous reports, suggest that PAF may not readily damage the airway mucosa even at large exudative doses of the agent. The present finding support the view that the plasticity of the epithelial junctions allows the creation of valve-like paracellular pathways for unidirectional clearance of extravasated plasma into the airway lumen. We suggest that endogenous PAF may participate in first line respiratory defence reactions by causing lumenal entry of bulk plasma without harming the epithelium.

In vivo restitution of airway epithelium.

Epithelial shedding occurs in health and, extensively, in inflammatory airway diseases. This study describes deepithelialisation, reepithelialisation and associated events in guinea-pig trachea after shedding-like epithelial denudation in vivo. Mechanical deepithelialisation of an 800-microns wide tracheal zone was carried out using an orotracheal steel probe without bleeding or damage to the basement membrane. Reepithelialisation was studied by scanning- and transmission electron microscopy and light microscopy. Nerve fibres were examined by immunostaining. Cell proliferation was analysed by [3H]-thymidine autoradiography. Immediately after epithelial removal secretory and ciliated (and presumably basal) epithelial cells at the wound margin dedifferentiated, flattened and migrated rapidly (2-3 microns/min) over the denuded basement membrane. Within 8-15 h a new, flattened epithelium covered the entire deepithelialised zone. At 30 h a tight epithelial barrier was established and after 5 days the epithelium was fully redifferentiated. After completed migration an increased mitotic activity occurred in the epithelium and in fibroblasts/smooth muscle beneath the restitution zone. Reinnervating intraepithelial calcitonin gene-related peptide-containing nerve fibres appeared within 30 h. We conclude that (1) reproducible shedding-like denudation, without bleeding or damage to the basement membrane, can be produced in vivo; (2) secretory and ciliated cells participate in reepithelialisation by dedifferentiation and migration; (3) the initial migration is very fast in vivo; (4) shedding-like denudation may cause strong secretory and exudative responses as well as proliferation of epithelium, and fibroblasts/smooth muscle. Rapid restitution of airway epithelium may depend on contributions from the microcirculation and innervation.

Effects of topical budesonide on epithelial restitution in vivo in guinea pig trachea.

BACKGROUND: Continuous epithelial shedding and restitution processes may characterise the airways in diseases such as asthma. Epithelial restitution involves several humoral and cellular mechanisms that may potentially be affected by inhaled anti-asthma drugs. The present study examines the effect of a topical steroid on epithelial restitution in vivo in the guinea pig. METHODS: The airway epithelium was mechanically removed from well defined areas of guinea pig trachea without surgery and without damage to the basement membrane or bleeding. An anti-inflammatory dose of budesonide (1 mg) was administered repeatedly to the tracheal surface by local superfusion 24 hours before, at (0 hours), and 24 hours after the denudation. Migration of epithelial cells, formation of a plasma exudation-derived gel, and appearance of luminal leucocytes were recorded by scanning electron microscopy. Cell proliferation was visualised by bromodeoxyuridine immunohistochemistry and tissue neutrophils and eosinophils by enzyme histochemistry. RESULTS: Immediately after creation of the denuded zone ciliated and secretory cells on its border dedifferentiated, flattened out, and migrated speedily (mean (SE) 2.3 (0.3) micron/min) over the basement membrane. After 48 hours the entire denuded zone (800 microns wide) was covered by a tightly sealed epithelium; at this time increased proliferation was observed in new and old epithelium and subepithelial cells. Budesonide had no detectable effect on epithelial dedifferentiation, migration, sealing, or proliferation. Immediately after denudation and continuously during the migration phase plasma was extravasated creating a fibrinous gel rich in leucocytes, particularly neutrophils, over the denuded area. Budesonide had no effect on either the gel or the leucocyte density. CONCLUSIONS: These observations suggest that topical glucocorticoids may not interfere with a fast and efficient restitution of the epithelium in the airways.

Topical nitroprusside may reduce histamine-induced plasma exudation in human nasal airways.

Mucosal exudation of nonsieved bulk plasma is a key feature of airway defense and inflammation. We have previously observed in guinea pig tracheobronchial airways that endogenous nitric oxide (NO) of the mucosa may tonically suppress the permeability of the subepithelial microcirculation, and that topical administration of the NO donor nitroprusside may reduce plasma exudation responses. The present study examines whether nitroprusside affects histamine-induced mucosal exudation of plasma in the human nasal airway. In a dose-finding tolerability experiment, using changes in nasal patency as response, placebo and nitroprusside (1.2 and 3.6 mg per nasal cavity) were applied on the mucosal surface with a nasal-spray device. Nasal peak expiratory flow (PEF) rates were measured before the application and thereafter every third minute for 15 min. Nitroprusside produced a dose-dependent decrease in nasal PEF rates compared to placebo. Placebo or nitroprusside (7.2 mg) was then given to the right nasal cavity, followed 3 min later by challenge with saline or histamine (600 micrograms). The drug and the challenge were both applied with a nasal-spray device. With a nasal pool-device, the same large part of the nasal mucosal surface was lavaged before and after the treatment/challenge. The lavage fluid levels of alpha 2-macroglobulin were measured as an index of mucosal exudation of bulk plasma. The histamine-induced lavage fluid level of alpha 2-macroglobulin was significantly higher after treatment with placebo than with nitroprusside. The present data indicate that nitroprusside may have antiexudative effects in human airways. Hence, unlike other microvascular permeability active agents, this pharmacologic principle may be active in both guinea pig and human airways.

Epithelial pathways for luminal entry of bulk plasma.

Inflammatory challenges of the airway mucosa cause luminal entry of bulk plasma. Extravasation of plasma is well described but the routes for epithelial passage of plasma are largely unknown. Using colloidal gold (5 nm) as tracer we have now examined the fate of extravasated plasma in the airways. The tracer was given intravenously to anaesthetized, ovalbumin-sensitized guinea-pigs 2 min prior to airway mucosal challenge with 12 pmol ovalbumin (the dose was selected from a separate dose-response study). Tissue specimens were collected 30 s, 3 and 6 min after end of challenge (separate time course experiments suggested that the peak rate of entry of plasma occurred at about 5 min). The colloidal gold particles were visualized by autometallographic silver intensification. The gold produced no circulatory disturbance and had a uniform vascular distribution with negligible adherence to vascular endothelium. After challenge gold was first widely distributed in the lamina propria. At 3 and 6 min the tracer was also in the epithelium and airway lumen. It appeared that plasma was moved distinctly between and all around each epithelial cell. Bright field-, scanning-, and transmission electron-microscopy indicated that the luminal entry of plasma did not affect the integrity of the epithelial lining. This study demonstrates that the plasma exudate moves across an intact epithelial layer through ubiquitous paracellular pathways.(ABSTRACT TRUNCATED AT 250 WORDS)

Microcirculation-derived factors in airway epithelial repair in vivo.

Airway epithelial repair, by cell migration over a denuded, intact basement membrane, occurs rapidly in vivo. The present study examines microcirculation-derived factors in the reepithelialization process in the guinea pig. A well-defined tracheal zone was gently deepithelialized; no bleeding occurred and the basement membrane was left intact. Plasma exudation was visualized by use of iv colloidal gold (diameter: 5 nm) or fluoresceinisothiocyanate-labeled dextran. Scanning and transmission electron microscopy confirmed the migration of epithelial cells and, additionally, allowed us to examine the presence of an extracellular matrix gel and leukocytes on the denuded basement membrane. Fibronectin was analyzed by immunocytochemistry. Following epithelial removal plasma promptly extravasates and produces a fibrin-fibronectin gel to cover the denuded basement membrane. Epithelial cells dedifferentiate, flatten, and migrate rapidly (several micron/min) beneath the plasma-derived gel. Within 30 min the gel contains numerous leukocytes, some of which are eosinophils. Plasma exudes into the gel until about 8 hr by which time the entire denuded zone (800 microns) is covered by squamous epithelium. The fibrin-fibronectin gel is suggested to be exclusively plasma-derived. In conclusion, reepithelialization in vivo occurs beneath a gel containing adhesive plasma proteins and leukocytes. We suggest that a plasma exudate provides immediate cover of denuded airway basement membrane and that plasma- and leukocyte-derived factors contribute essentially to reepithelialization in vivo.

Mucosal nitric oxide may tonically suppress airways plasma exudation.

In a search for airway epithelial mechanisms that may affect the subepithelial microcirculation, we examined plasma exudation responses to NG-nitro-L-arginine-methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor. L-NAME was applied topically on the tracheal mucosa of guinea pigs that had previously received 125I-albumin and/or colloidal gold particles (5 nm) intravenously. Luminal entry of plasma was determined by the levels of 125I-albumin in tracheal lavage fluid. Topical L-NAME (2.2, 9, and 22 mumol), but not intravenous L-NAME (375 mumol/kg), produced plasma exudation into the airway lumen (p < 0.01 to p < 0.001). The L-NAME enantiomer NG-nitro-D-arginine-methyl ester (D-NAME, 9 mumol) produced no exudative response. Coadministration of L-arginine (27 mumol) abolished the L-NAME-induced exudation. The extravasated plasma was distributed in the lamina propria and between epithelial cells (colloidal gold). The epithelial surface structure (scanning electron microscopy) appeared intact. Staining with nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase suggested that epithelial basal may contain nitric oxide synthases. We suggest that endogenously released nitric oxide from epithelial or other superficial cells tonically suppresses the macromolecular permeability of the subepithelial microcirculation.

Allergen-induced biphasic plasma exudation responses in guinea pig large airways.

In this study involving sensitized guinea pigs (anesthetized intramuscularly with a 3:2 mixture of ketamine+xylazine, 1 ml/kg), we applied allergen (ovalbumin) selectively to the tracheobronchial mucosa (sparing the nasal passages and the terminal airways) and examined the occurrence of immediate and late-phase inflammatory exudation of plasma and plasma-derived mediators (bradykinins) into the airway lumen. The experiments were terminated 10 to 480 min after challenge. A selective lavage that sampled the surface liquids of the extrapulmonary bronchi and the lower trachea was performed. The amount of plasma (microliter) was determined by analysis of a plasma tracer, [125I]albumin, in lavage fluid and blood (plasma) samples. Ovalbumin, 3 to 12 pmol, and histamine, 5 and 10 nmol, produced a dose-dependent immediate exudation response (p < 0.001). The effects were nonneurogenic because they were not affected by topical lidocaine given in a dose (3 nmol) that prevented the exudative effect of capsaicin. The 6- and 12-pmol doses of ovalbumin (but not 3 pmol) produced a significant late-phase exudative response at 5 h (p < 0.001), and both the immediate and late phases were associated with increased (p < 0.01 to p < 0.001) levels of bradykinin in the lavage fluids. Histamine, even in doses that produced a greater early response than the allergen, did not produce a late-phase response. A single topical dose of an antiasthma steroid (budesonide, 12 mumol/kg) administered just before ovalbumin (6 pmol) had little effect on the immediate response but inhibited the late-phase response (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of nicotine on the human nasal mucosa.

BACKGROUND: Topical application of nicotine and stimulation of tachykinin containing sensory nerves have been shown to produce mucosal exudation of plasma and derangement of the epithelial lining in guinea pig and rat airways. If this occurred in man these effects might contribute to the pathogenesis of airway disease. This study, performed in healthy volunteers without atopy, examined whether nicotine affects the plasma exudation response and the mucosal absorption permeability of the human nasal airway. METHODS: The acute effects of increasing topical doses of nicotine (0.08-2.0 mg) were examined (n = 8) on nasal symptoms (pain), mucosal exudation of plasma (albumin), mucosal secretion of mucin (fucose), and mucosal exudative responsiveness (histamine induced mucosal exudation of albumin). A separate placebo controlled study was carried out to determine whether frequent applications of the high dose of nicotine (2.0 mg given eight times daily for nine days) had any deleterious effects on the airway mucosa detectable as altered responses to histamine challenge. Both mucosal exudation of plasma (n = 12) and mucosal absorption of chromium-51 labelled EDTA (n = 8) were thus examined in nasal airways exposed to both nicotine and histamine. RESULTS: Nicotine caused nasal pain and produced dose dependent mucosal secretion of fucose but failed to produce any mucosal exudation of albumin. The exudative responsiveness to histamine was, indeed, decreased when the challenge was performed immediately after administration of acute doses of nicotine, whereas the responsiveness was unaffected when histamine challenges were carried out during prolonged treatment with nicotine. The nasal mucosal absorption of 51Cr-EDTA in the presence of histamine did not differ between subjects receiving either placebo or nicotine treatment for nine days. CONCLUSIONS: The results indicate that nicotine applied to the human airway mucosa produces pain and secretion of mucin, but inflammatory changes such as mucosal exudation of plasma and epithelial disruption may not be produced. Neurogenic inflammatory responses, which are so readily produced in guinea pig and rat airways, may not occur in human airways.

Plasma exudation and solute absorption across the airway mucosa.

The airway mucosa responds to inflammatory provocations with bulk exudation of plasma into the airway tissue (vascular exudation) and lumen (mucosal exudation). The intensity and time course of the exudative response can be relevantly examined by sampling and analysing airway surface liquids, because the luminal entry of plasma proteins/tracers promptly and quantitatively reflects the exudative response of the airways. The process of mucosal exudation of plasma is a prominent feature of airway inflammation and has been demonstrated in rhinitis, asthma, and bronchitis. Inflammatory mediators and allergen produce mucosal exudation of plasma into the airway lumen (outward permeability) whereas the solute absorption across the mucosa (inward permeability) is unaffected. Hence, in contrast to current views, we have demonstrated that in airway inflammation the solute absorption across the airway mucosa is not increased. The findings suggest the plasma exudation response also as a first line respiratory mucosal defence, allowing potent plasma protein systems to appear on an airway mucosa functionally intact as a barrier toward undue luminal material. Our data on plasma exudation and solute absorption across the mucosa of upper and lower airways further suggest the human nasal airways as a model relevant also for the tracheobronchial airways.

Appearance of airway absorption and exudation tracers in guinea pig tracheobronchial lymph nodes.

This study examines the fate of extravasated plasma in inflammatory stimulus-challenged large tracheobronchial airways of ketamine-xylazine-anesthetized guinea pigs. Entry of plasma tracers into the airway lumen was determined by a validated noninjurious airway lavage technique. Removal by airway lymphatics was assessed by tracheobronchial lymph node levels of plasma tracers. Mucosal challenges with histamine (5 nmol), bradykinin (5 nmol), capsaicin (0.4 nmol), or allergen (ovalbumin, 3 pmol) increased the appearance of a plasma tracer (131I-labeled albumin previously injected intravenously) in the airway lumen within 10 min (10-20 times control; P < 0.001), whereas the contractile agent carbachol (8 nmol) was without exudative effect. The mediators were without effect, and capsaicin and allergen only slightly increased the lymph node level of plasma exudation tracer (1.5 times control; P < 0.05). Hence, removal via the lymphatic route of plasma macromolecules may be negligible in the acute and postacute phases of an airway exudation response. Experiments were also carried out with luminally applied macromolecular tracers. These were absorbed from the mucosal surface into the circulation, but a small portion was also transported to the lymph nodes, demonstrating the interconnections between the mucosa and the sampled nodes. Only capsaicin produced an increased node level of absorption tracer. Immunohistochemistry showed that the tracheobronchial tissue and lymph nodes are endowed with nerve fibers containing substance P, the release of which may have mediated lymph transport, vascular, and exudative effects of capsaicin in the present studies.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sensitivity to toluene diisocyanate (TDI) in airways previously exposed to low doses of TDI.

Repeated airway exposures to toluene diisocyanate (TDI) may cause sensitization and asthma. This study has examined the acute inflammatory response to TDI in guinea-pig tracheobronchial airways, the development of increased sensitivity to TDI and the effects of xanthines and a glucocorticoid on these responses to TDI. A restricted surface area of the tracheobronchial mucosa of Ketalar-Xylazin anaesthetized guinea-pigs was exposed to TDI, dissolved in olive oil, by means of 1 min infusions through an oral catheter. The TDI-induced inflammatory process was quantified by determination of airway luminal entry of plasma. Already 3 nl (approximately 20 pmol) of TDI produced a significant and sustained exudation response (P less than 0.001 to P less than 0.01, 5 and 17 hr after exposure). Pretreatment with intravenous enprofylline (25 mumol/kg) intraperitoneally or 26 mumol/kg by tracheal superfusion) was without effect. Two repeated exposures to TDI 3 nl (on days 1 and 8) made the animals hyperresponsive to TDI so that on day 15 a previously subthreshold dose of TDI (0.3 nl) produced significant exudation both at 5 and 17 hr after exposure (P less than 0.001 to P less than 0.01). Similarly, two repeated dermal exposures to a large dose of TDI (20 microliters) lowered the threshold for tracheal provocation with TDI. Budesonide (2.6 mumol/kg orally) given daily during the topical airway 'sensitization' regimen (days 1-14) significantly reduced the response to the subsequent 0.3 nl challenge dose of TDI (P less than 0.05). The effects of daily treatments with either theophylline (100 mumol/kg) or enprofylline (50 mumol/kg) were not significant.(ABSTRACT TRUNCATED AT 250 WORDS)