Interferometry with Bose-Einstein condensates in microgravity.

Atom interferometers covering macroscopic domains of space-time are a spectacular manifestation of the wave nature of matter. Because of their unique coherence properties, Bose-Einstein condensates are ideal sources for an atom interferometer in extended free fall. In this Letter we report on the realization of an asymmetric Mach-Zehnder interferometer operated with a Bose-Einstein condensate in microgravity. The resulting interference pattern is similar to the one in the far field of a double slit and shows a linear scaling with the time the wave packets expand. We employ delta-kick cooling in order to enhance the signal and extend our atom interferometer. Our experiments demonstrate the high potential of interferometers operated with quantum gases for probing the fundamental concepts of quantum mechanics and general relativity.

Epithelial transformations in the establishment of the blood-gas barrier in the developing chick embryo lung.

The tall epithelium of the developing chick embryo lung is converted to a squamous one, which participates in formation of the thin blood-gas barrier. We show that this conversion occurred through processes resembling exocrine secretion. Initially, cells formed intraluminal protrusions (aposomes), and then transcellular double membranes were established. Gaps between the membranes opened, thus, severing the aposome from the cell. Alternatively, aposomes were squeezed out by adjacent cells or were spontaneously constricted and extruded. As a third mechanism, formation and fusion of severed vesicles or vacuoles below the aposome and their fusion with the apicolateral plasma membrane resulted in severing of the aposome. The atria started to form by progressive epithelial attenuation and subsequent invasion of the surrounding mesenchyme at regions delineated by subepithelial alpha-smooth muscle actin-positive cells. Further epithelial attenuation was achieved by vacuolation; rupture of such vacuoles with resultant numerous microfolds and microvilli, which were abscised to accomplish a smooth squamous epithelium just before hatching.

In situ localization of TNFalpha/beta, TACE and TNF receptors TNF-R1 and TNF-R2 in control and LPS-treated lung tissue.

Tumor necrosis factor (TNF) has been implicated in several infectious and inflammatory lung diseases. Two closely related variants, TNFalpha and TNFbeta, elicit various cellular responses via two distinct TNF receptors, the 55-kDa TNF-R1 and the 75-kDa TNF-R2. Recently, a TNFalpha-converting enzyme (TACE) was described, which cleaves and releases the membrane-bound TNFalpha. In the present study in normal rat and human lung tissue, the constitutive expression of TNFalpha/beta, TACE and TNF-R1/R2 was investigated by immunohistochemical techniques. In addition, TNFalpha and TNFbeta mRNA were localized by in situ hybridization. Both TNFalpha and TNFbeta were detected in various lung cell types. Expression of TNFalpha was particularly prominent in bronchial epithelial cells and vascular smooth muscle cells, next to alveolar macrophages. Both in situ hybridization for TNFalpha message and TACE immunostaining matched this expression profile. TNFbeta-so far only known to be produced by lymphocytes-was demonstrated in alveolar macrophages, bronchial epithelial cells, vascular smooth muscle cells and endothelial cells at the protein and the message level. Both TNF receptors were detected, with TNF-R1 being prominent on bronchial epithelial cells and endothelial cells, and TNF-R2 being expressed by nearly all cell types. Following LPS stimulation in isolated rat lungs TNFalpha/beta signal intensity was largely reduced due to liberation of stored TNFalpha/beta, while TACE immunoreactivity remained unchanged or was enhanced, demonstrating increased TNF generation. We conclude that both TNFalpha and TNFbeta are constitutively expressed by several non-leukocytic cell types in the human and rat lung. In concert with the expression of TACE and the TNF receptors R1 and R2, this finding suggests in addition to the known role of the TNF system in inflammation physiological functions of the TNF system in different compartments of the adult lung, with the vasculature and the bronchial tissue being of particular interest in addition to the leukocyte/macrophage populations.

Comparison of different detection methods in quantitative microdensitometry.

Quantitative evaluation of immunohistochemical staining has become a focus of attention in research applications and in pathological diagnosis, such as Her-2/neu assessment in mammary carcinoma. Reproducibility of immunostaining techniques and microscopical evaluation are prerequisites for a standardized and reliable quantitation of immunostaining intensity. In the present study, different staining and microscopical techniques, including fluorescence microscopy, epipolarization microscopy of immunogold-silver, and absorbance microdensitometry were compared concerning suitability for quantitative evaluation. We describe a staining procedure using alkaline phosphatase-based immunohistochemistry with the substrate Vector Red and subsequent microdensitometry with a custom-designed absorbance filter. We have characterized linearity of the staining intensity in dependence of development time, antibody concentration, and section thickness by means of artificial standards consisting of agarose blocks into which immunogold- or alkaline phosphatase-conjugated antibodies were incorporated. Applicability of the different techniques was tested by anti-CD45 immunostaining of leukocytes within rat lung tissue detected by immunofluorescence, immunogold-silver epipolarization microscopy, as well as alkaline phosphatase-based Vector Red absorbance or fluorescence measurement. Excellent qualities of Vector Red for quantitative microdensitometric evaluation of staining intensity were particularly obvious for absorbance microscopy. Applicability in paraffin-embedded tissue as well as in cryosections, excellent segmentation, linearity over a wide range, light stability, and feasibility for permanent mounting and for long-term storage are the outstanding features of this technique for use in routine quantitative evaluation.

Rat pulmonary cyclooxygenase-2 expression in response to endotoxin challenge: differential regulation in the various types of cells in the lung.

Cyclooxygenase (Cox), the key enzyme of prostanoid synthesis, consists of the two isoforms Cox-1 and Cox-2, both recently noted to be constitutively expressed in rat lungs with a distinct profile of cellular distribution. The responsiveness of pulmonary Cox-1 and Cox-2 expression to intravascular endotoxin lipopolysaccharide (LPS) administration was investigated in isolated, ventilated rat lungs, buffer-perfused with or without admixture of rat plasma. Immunohistochemical staining intensity was measured by a previously described method of silver enhancement and epipolarization image analysis. Both the Cox-1 mRNA, quantified in the whole lung homogenate, and the cellular localization of Cox-1 were unchanged in response to LPS. In contrast, time- and dose-dependent up-regulation of Cox-2 mRNA (lung homogenate) occurred, and differential LPS reactivity at the cellular level was observed. Up-regulation of Cox-2 in cell types expressing this enzyme already under baseline conditions was noted in bronchial epithelial cells, bronchial and vascular smooth muscle cells, cells within the BALT and myocytes of the large hilar veins. De novo induction of Cox-2 occurred in endothelial cells and the majority of alveolar macrophages. Down-regulation of Cox-2 was observed in perivascular and peribronchial macrophage-like cells. Moreover, differential impact of plasma components was noted: for the large majority of cells, CD14 surface expression correlated with Cox-2 responsiveness to LPS independent of plasma, whereas the presence of plasma components was a prerequisite for the LPS response in CD14-negative cells. LPS did not provoke physiological changes in the perfused lungs, but markedly enhanced baseline prostanoid generation. We conclude that LPS-induced Cox-2 regulation occurs in a complex, cell-specific manner, which may be relevant for pathogenetic sequelae in septic lung injury and acute respiratory failure.

In situ localization and regulation of thromboxane A(2) synthase in normal and LPS-primed lungs.

Thromboxane (Tx) A(2) synthase catalyzes the conversion of prostaglandin H(2) to the unstable metabolite TxA(2), which is a potent mediator of vasoconstriction and bronchoconstriction. The cellular localization of TxA(2) synthase was examined by immunohistochemistry and in situ hybridization in human and rat lung tissues. Bronchial epithelial cells, bronchial smooth muscle cells, peribronchial nerve fibers, single cells of bronchus-associated lymphoid tissue, single cells located in the alveolar septum, and alveolar macrophages exhibited positive immunostaining for TxA(2) synthase protein in lung tissue of both species. In addition, vascular smooth muscle cells of muscular and partially muscular vessels displayed strong (rat) and moderate (human) immunostaining for TxA(2) synthase. In situ hybridization performed in the rat lungs demonstrated TxA(2) synthase mRNA localization in accordance with the immunostaining pattern. Perfusing isolated rat lungs with endotoxin for 1 and 2 h resulted in a marked increase in TxA(2) synthase protein staining intensity in most cell types as measured by quantitative image analysis, whereas the in situ hybridization signal was unchanged. We conclude that the pulmonary distribution of TxA(2) synthase displays close similarity between rat and human lung tissues and matches well with the previously described immunolocalization of cyclooxygenase-1 and cyclooxygenase-2 in this tissue. Endotoxin challenge is suggested to cause a rapid upregulation of TxA(2) synthase at the posttranscriptional level. These data provide a morphological basis for the understanding of the role of TxA(2) in the regulation of lung bronchial and vascular tone and in immunologic events.

CT-assisted versus silicone rubber cast morphometry of the lower respiratory tract in healthy amazons (genus Amazona) and grey parrots (genus Psittacus).

The objective of this study was to examine the normal respiratory tract of grey parrots and amazons by using two different methods. The lower respiratory tract of five amazons and four grey parrots, all healthy, were investigated applying computerised tomography (CT). Volumes and densities of the body, the body cavities, the normal lungs, and the airsacs in the living animals were defined as reference values of healthy birds to give a basis for future CT-diagnosis of respiratory diseases and their precise locations in parrots. In a parallel study, the lung and air sac volumes of six amazons and two grey parrots were measured using silicone rubber casts produced after the method described by H.-R. Duncker. Values for identical respiratory structures gained by these different methods were compared.

Cyclooxygenase isoenzyme localization and mRNA expression in rat lungs.

Prostanoid generation may proceed via both isoforms of cyclooxygenase, Cox-1 and Cox-2. Cox-1 is thought to be ubiquitously expressed, whereas Cox-2 is mostly assumed to be dynamically regulated, responding to inflammatory stimuli. The cellular localization of Cox-1 and Cox-2 in the lung, an organ with high cyclooxygenase activity, is not known. In normal rat lungs the expression and localization of Cox-1 and Cox-2 were examined with immunogold-silver staining and the RT-PCR technique. Quantitative image analysis of the staining intensity was performed by measuring mean gray values of digitized epipolarization images. Expression of both Cox-1 and Cox-2 was readily detectable in rat lungs. Cox-1 immunoreactivity localized predominantly to bronchial epithelial cells, smooth muscle cells of large hilum veins, and (with lower expression) to alveolar macrophages and pulmonary artery endothelial cells. The most intense Cox-2 staining was noted in macrophage- and mast cell-like cells, detected in close vicinity to the bronchial epithelium and in the connective tissue surrounding the vessels. In addition, strong Cox-2 expression was found in smooth muscle cells of partially muscular vessels and large veins of the hilum. Bronchial epithelial cells displayed Cox-2 immunoreactivity with limited intensity. Alveolar macrophages and alveolar septal cells were only occasionally stained with anti-Cox-2 antibodies. Both Cox-1 and Cox-2 are constitutively expressed in several cell types of normal rat lung, but display clearly different patterns of cellular localization. Cox-2 may not be related only to lung inflammation, but is suggested to be implicated in regulatory processes under physiological conditions as well.

Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin.

The role of the endothelial cytoskeleton for the structural integrity of the pulmonary gas exchange area was probed with the use of Clostridium botulinum C2 toxin. This agent causes selective loss of nonmuscle F-actin. In buffer-perfused rabbit lungs, vascular pressures were kept within physiological ranges. In different groups, low-dose [0.3 (C2,I)/0.6 (C2,II) ng/ml] and high-dose [10 (C2,I)/20 (C2,II) ng/ml] toxin were applicated into the buffer fluid; experiments were terminated after a total weight gain of either 1 or 7.5 g. Electron microscopy revealed extensive attenuations, undulations, and protrusions of the endothelial layer, suggestive of "remodeling" and "flowing" of the cell membrane in low C2 toxin-treated lungs accompanied by few disruptions of the endothelial layer and edema formation. In addition, endothelial cells displayed vesiculation and bleb formation. Lungs that were exposed to high-toxin doses displayed marked attenuations of the endothelial layer in addition to large endothelial cell disruptions, which did not include interendothelial junctions. Interestingly, type II epithelial cells displayed fusion of lamellar bodies. Collectively, these data suggest that the actin microfilament system is instrumental in supporting endothelial cell membrane configuration and integrity and maintains the intimal barrier function of the lung microvasculature.

Role of endothelial cytoskeleton in high-permeability edema due to botulinum C2 toxin in perfused rabbit lungs.

The cytoskeleton of the endothelial cell has been suggested to regulate endothelial barrier function. We investigated the role of actin in the maintenance of pulmonary capillary integrity in perfused rabbit lungs. As a tool for selective perturbation of actin, we employed Clostridium botulinum C2 toxin, which is composed of a membrane translocation component (C2II) and a component (C2I) effecting ADP-ribosylation of nonmuscle G-actin. ADP-ribosylated actin no longer capable of polymerization but acts as a barbed end-capping protein, thereby effecting selective loss of the nonmuscle F-actin content. In buffer-perfused rabbit lungs, combined application of both toxin components (range 50 pg/ml-5 ng/ml C2I) resulted in a time- and dose-dependent increase in the capillary filtration coefficient (Kfc) with concomitant edema formation. Only 300:600 pg/ml C2I:II sufficed to induce a > 10-fold rise of Kfc values within 110 min. This severe lung permeability increase occurred in the absence of vasomotor responses and potassium release or lactate dehydrogenase release. Application of each single toxin component displayed markedly reduced efficacy. Similar to the C2 toxin effect, severe permeability increase without concomitant hemodynamic changes was evoked by cytochalasin D, known to possess F-actin-disrupting properties. Preloading of lung cells with phallacidin, which in opposition to C2 toxin decreases F-actin depolymerization, significantly reduced the C2 toxin-induced increase in vascular permeability. Electron microscopic examination of C2 toxin-poisoned lungs showed early, extensive endothelial cell attenuations, followed by disruptions of the endothelial layer and marked interstitial edema formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Morphometric analysis of pulmonary intracapillary leukocyte pools in ex vivo-perfused rabbit lungs.

Characterization and quantification of lung intracapillary leukocytes is of interest for a better understanding of immunological and inflammatory features in this organ. We developed a technique of computer-assisted measurement of digitalized electron-microscopic images and electronic data processing for morphometry of intracapillary leukocyte pools in rabbit lungs (L. Ermert, W. Seeger, and H.-R. Duncker, Cell Tissue Res. 271: 469-476, 1993). Measurements were undertaken in buffer-perfused isolated lungs (avoiding any reentry of washed-out cells); perfusion fixation was performed 7.5, 35, and 185 min after onset of artificial circulation (n = 5 each). Data were compared with that of nonperfused lungs fixed by tracheal instillation (baseline). Total lung capillary neutrophil counts were 1.41 x 10(9), 1.35 x 10(9), 1.37 x 10(9), and 0.69 x 10(9) (baseline, 7.5, 35, and 185 min perfusion, respectively). Corresponding data for intracapillary lymphocytes were 1.07 x 10(9), 0.84 x 10(9), 0.81 x 10(9), and 0.57 x 10(9); and for microvascular monocytes, data were 0.21 x 10(9), 0.19 x 10(9), 0.18 x 10(9), and 0.08 x 10(9). Ratios of cell volume and surface variables of the different intracapillary leukocyte types did not change during ex vivo lung perfusion. We conclude that the rabbit pulmonary capillary bed harbors large pools of different leukocytes, which surpass pool sizes of corresponding circulating cells and display very slow washout kinetics under conditions of lung-buffer perfusion. A major impact of these intracapillary leukocyte pools on immunological and inflammatory events in isolated-perfused and transplanted lungs must be assumed.

Effects of stroma-free hemoglobin solutions on pulmonary vascular resistance and mediator release in the isolated perfused rabbit lung.

The aim of this study was to evaluate the effect of an ultrapure bovine stroma-free hemoglobin (SFH) on pulmonary vascular resistance and mediator release and to analyze potential mechanisms of action in the isolated perfused rabbit lung model. Repetitive bolus applications of small amounts of bovine SFH were examined which resulted in a reproducible acute increase of pulmonary vascular resistance of approx. 9 mmHg (controls, n = 6). It was tested whether the platelet-activating factor (PAF) antagonist WEB 2086 (50 microM; n = 6), the cyclooxygenase blocker diclofenac (10 micrograms/ml; n = 6), the iron-chelating agent deferoxamine (500 micrograms/ml, n = 6) and the radical scavenger catalase (5000 U/ml; n = 6) exert a protective effect on vasoconstrictor response to SFH. The pressure increase was completely suppressed in the lungs pretreated with WEB 2086, whereas diclofenac, deferoxamine and catalase failed to inhibit the vasoconstriction due to SFH. No significant differences in either TXB2 generation or in histamine release were found in the WEB 2086 group compared with untreated lungs. The results point towards the crucial role of PAF in mediation of vasoconstrictor side effects due to SFH.

Effect of hypertonic NaCl-starch-solution on oedema of different pathogenesis.

Small volumes of hypertonic NaCl-solutions have been proven to restore haemodynamics in hypovolemic shock patients. Topic of this study was to investigate whether bolus application of 7.5% NaCl-6.5% starch-solution (HSS) apart from its relevance in shock might be an effective therapy in oedema. Considering differential therapeutic aspects, the volume effects of 7.2 ml HSS were tested in three types of oedema: hydrostatic oedema induced by venous congestion (n = 6), oedema caused by bradykinin injection (n = 6), and proteinase-induced oedema (n = 6). The arterial, venous pressure and weight changes indicating volume shifts between intra- and extravascular space were continuously monitored in 36 isolated perfused rabbit hindlimbs. Oedema formation was induced corresponding to a weight gain of 18-20 g. Subsequently 7.2 ml HSS were injected into the extracorporeal circulation system containing 200 ml cell free, isoosmotic perfusate. Six experiments of each oedema group without HSS-application served as controls. 75-100% of oedema formation could be remobilised via bolus application of HSS within 5 min in all types of oedema. A persisting weight reduction was detectable in the hydrostatic and bradykinin oedema, whereas in the elastase oedema the initial weight loss was followed by a regain of weight up to 180% of initial oedema formation at 120 min after HSS-application. The results show that, due to the osmotic gradient induced by bolus application of HSS, the hydrostatic and bradykinin oedema can be permanently remobilised, whereas the therapeutic effect during proteinase oedema is only short-lasting due to an irreversible damage of barrier function.

Modulation of pulmonary vascular resistance and edema formation by short-term infusion of a 10% fish oil emulsion.

BACKGROUND: The aim of this study was to investigate whether the pulmonary response to inflammatory stimulation, resulting in increased vascular resistance and permeability, could be attenuated by short-term infusion of triglycerides containing omega-3 fatty acids. With the concept of altering the composition of membrane phospholipids in such a manner that stimulation resulted in the release of less vasoconstrictive and permeability-enhancing metabolites of eicosapentaenoic acid instead of those of arachidonic acid (AA), the parenteral application of a lipid emulsion prepared from fish oil (Omegavenös) was tested in comparison with a soy oil preparation (Lipovenös). METHODS: Isolated lungs from anesthetized rabbits were ventilated and recirculatingly perfused (200 ml/min) with 200 ml cell-free buffer solution to which either 2 ml saline (controls, n = 6), 2 ml Lipovenös 10% (n = 6) or 2 ml Omegavenös 10% (n = 6) were added. To study the possible metabolic alterations in states of an enhanced AA turnover, lungs of each group were stimulated with smaller doses of A23187 (10(-8) M) during the 180-min lipid perfusion period, followed by a 10 times higher calcium ionophore A23187 (10(-7) M) challenge after washing out the lipids by exchange of perfusion fluid. Pulmonary artery pressure (PAP) and the lung weight gain indicating edema formation were monitored, and eicosanoids were analyzed in samples of the perfusate. RESULTS: Upon A23187 injection lung weight gain and PAP increase were significantly reduced (50%) in Omegavenös-perfused lungs in comparison with controls and Lipovenös treatment. The vascular reactions were accompanied by a shifting from LTC4 to LTC5 during and after Omegavenös perfusion. CONCLUSION: The data demonstrate that omega-3 fatty acids seem to be incorporated into the phospholipid pool of the pulmonary tissue, even after short-term infusion (3 h) resulting in an attenuated pressure reaction and edema formation due to an altered spectrum of metabolites in the case of inflammatory stimulation.

Effects of hemorrhage, hypoxia, and intravascular coagulation on bacterial clearance and translocation.

OBJECTIVE: This study was undertaken to discover if impaired blood clearance functions and killing capacity of the reticuloendothelial system contribute to the high occurrence rate of septic complications after shock, trauma, and thermal injury. DESIGN: Prospective, randomized, controlled trial. SETTING: Experimental laboratory in a university teaching hospital. SUBJECTS: Thirty-three standard-breed rabbits of either sex. INTERVENTIONS: Defined numbers of Escherichia coli (1.3 x 10(8)) colony-forming units were injected intravenously 60 min after induction of hypoxia, standardized by defined reduction of oxygen uptake (60% to 75% of baseline) induced by hypoventilation (n = 6) or hemorrhage (n = 6), after complete defibrination caused by the snake toxin, ancrod (n = 6), and after 60 mins without intervention (controls, n = 6). At 180 mins after bacterial injection, the animals were killed and tissue samples of liver, kidney, spleen, and lung were collected for microbiological examinations. MEASUREMENTS AND MAIN RESULTS: Bacterial elimination from the blood and distribution pattern of viable bacteria in liver, spleen, kidney, and lung were investigated in hemorrhagic, hypoxic, and defibrinated rabbits. Compared with controls, there was a distinct alteration of the elimination kinetics of bacteria from the circulating blood in the experimental groups. First, the initial counts of viable E. coli were up to 300% (p < .05) higher in the defibrination, hemorrhage, and hypoxia groups than in controls. Second, greater numbers of E. coli were found in the blood for a significantly (p < .001) longer period, coupled with up to four times higher counts in organ homogenates in the hemorrhagic and defibrinated groups (p < .01) and more than 100 times higher counts than control values in the hypoxic animals (p < .001). CONCLUSION: Hemorrhage, hypoxia, and intravascular coagulation induce impaired bacterial clearance from the blood that is associated with altered organ distribution patterns, thus reflecting dysfunction of the reticuloendothelial system.

Alterations of bacterial clearance induced by endotoxin and tumor necrosis factor.

The purpose of the study was to investigate the potential influence of endotoxin and tumor necrosis factor (TNF) on immune function in terms of systemic clearance and organ distribution of injected Escherichia coli in a rabbit model. To enable quantification of the clearance process, defined numbers of exogenous E. coli (1.3 x 10(8) CFU) were injected intravenously 60 min after bolus application of TNF (4 x 10(5) U, n = 6), after infusion of endotoxin (40 micrograms/kg of body weight) for 1 h (n = 6) or 4 h (n = 6), or after saline infusion (controls, n = 6). Parameters monitored were arterial pressure, oxygen uptake, and rates of bacterial elimination from the blood. At 180 min after E. coli injection, the animals were sacrificed, and tissue samples of liver, kidney, spleen, and lung were collected for bacterial counts. Endotoxin infusion produced a significant delay in blood clearance compared with saline and TNF pretreatment. The diminished systemic bacterial elimination was associated with significantly higher numbers of E. coli in the organs, thus reflecting reticuloendothelial system dysfunction. TNF had no major influence on the elimination kinetics of bacteria but affected the tissue distribution pattern with increased accumulation of E. coli in the lung (up to 100-fold of control values; P < 0.001).

Influence of a pentoxifylline-analogue on the granulocyte-mediated pulmonary mediator release and vascular reaction.

The influence of a novel pentoxifylline-analogue, HWA 138, on the polymorphonuclear neutrophil leukocyte (PMN)-mediated changes in pulmonary resistance and mediator release was investigated in the isolated perfused and ventilated rabbit lung model. Isolated, washed human granulocytes were injected into the pulmonary artery and stimulated by either 10(-6) mol/L N-formyl-L-leucin-methionyl-L-phenylalanine (FMP) or 3 x 10(-8) mol/L phorbol 12-myristate 13-acetate (PMA) in the presence or absence (controls) of HWA 138 (10(-4) mol/L). Shortly after granulocyte activation, there was a massive generation and release of thromboxane (> 110 pg/ml) and histamine (150-400 nmol/L), with an acute increase of pulmonary artery pressure (> 8 mmHg) in the control groups. Application of HWA 138 almost completely suppressed mediator formation and release as well as pulmonary vascular reaction in the FMP stimulated group. In contrast to this, HWA 138 was unable to influence either mediator release, the pulmonary pressure reaction or interstitial edema formation following PMA stimulation. In the present model, HWA 138 is supposed to be effective via granulocytes by decreasing mediator release, obviously due to burst reaction.

Computer-assisted morphometry of the intracapillary leukocyte pool in the rabbit lung.

Computer-assisted morphometry was performed to evaluate the number and cell characteristics of capillary and alveolar leukocytes in rabbit lungs. An image-processing system and a programmable spread-sheet program were used, which allowed morphometric analysis of a large reference area. Neutrophils represented the largest intracapillary leukocyte population (2.2 x 10(7)/ml parenchyma, which corresponds to an approximately 104-fold microvascular enrichment of this cell type related to cell counts calculated for the capillary blood volume). In addition, large numbers of intracapillary lymphocytes (1.7 x 10(7)/ml parenchyma; 47-fold enrichment) and monocytes (0.3 x 10(7)/ml parenchyma; 86-fold enrichment) were detected. The total count of pulmonary leukocytes thus approximated the total number of pulmonary endothelial cells; and the total circulating pools of the different leukocytes were surpassed by the corresponding lung capillary pools, 3.2-fold for neutrophils, 1.2-fold for lymphocytes and 4.8-fold for monocytes. In contrast, alveolar cell numbers ranged from 1-2% of the capillary counts for all types of leukocytes. We conclude that the rabbit lung microvasculature harbours large pools of immunocompetent cells, which may contribute to host-defense mechanisms at the gas-exchange area.

Alterations of filtration coefficients in pulmonary edema of different pathogenesis.

Different pathomechanisms in the development of pulmonary edema are being discussed. We investigated the effect of pathogenetically varying forms of edema on lung vascular barrier function in isolated cell-free perfused rabbit lungs. As an index of permeability, capillary filtration coefficients (Kfc) were determined from the slope of lung weight change over periods of stepwise venous pressure elevation (5, 7.5, and 10 mmHg) before (controls) and 60 min after edema induction. Edema was induced by venous congestion (n = 6), by application of arachidonic acid in the presence of diclofenac sodium (n = 6), and by elastase application (n = 6). Control values ranged from 0.28 to 0.51 ml.min-1 x mmHg-1 x 100 g-1. Kfc was significantly enhanced after edema induction up to 243% of control value in the hydrostatic edema, 357% in the arachidonic acid edema, and 594% in the elastase edema. When the alterations in capillary filtration due to the different types of edema were compared, Kfc was significantly higher in the proteinase edema, indicating an irreversibly damaged barrier function. These data exemplify different pathophysiological characteristics due to the pathogenesis of interstitial edema formation.

Induction of severe vascular leakage by low doses of Escherichia coli hemolysin in perfused rabbit lungs.

S. aureus alpha-toxin and E. coli hemolysin (Hly) represent two prototypes of pore-forming cytolysins. Both are established virulence factors and have been implicated in the development of septic lung failure. Low doses of these agents cause thromboxane-mediated vasoconstriction and edema formation in isolated perfused rabbit lungs. In a preceding investigation, we observed that alpha-toxin causes overt endothelial cell damage in these lungs, as demonstrable by electron microscopy (Seeger W, Birkemeyer RG, Ermert L, Suttorp N, Bhakdi S, Duncker HR: Lab Invest 63:341, 1990). Here, we present results of a parallel study conducted with E. coli hemolysin. Thromboxane-dependent pulmonary hypertension was suppressed by the addition of acetylsalicylic acid to the perfusion fluid in all cases. Administration of 0.2 hemolytic units (HU; i.e., 20 ng/ml protein) resulted in progressive weight gain after a lag period of 10 to 15 minutes, and 30 minutes after toxin application the gravimetrically determined capillary filtration coefficients (Kfc) were increased greater than 10-fold. Perfusion was terminated when the total lung weight gain surpassed 20 gm. 0.12 HU/ml E. coli hemolysin caused 2- to 3-fold increased capillary filtration coefficients values within 110 minutes, concomitant with intermediate quantities of edema formation (9.7 +/- 2.7 gm). Potassium liberation in the absence of lactate dehydrogenase release occurred in all toxin treated lungs. Electron microscopic examination after perfusion fixation revealed interstitial edema formation in areas remote from the blood-gas exchange barrier. Increased numbers of endothelial plasmalemmal vesicles were visualized at the very onset of edema formation in lungs exposed to 0.2 HU/ml, and after a 110-minute exposure to 0.12 HU/ml of the toxin, but not in lungs exhibiting severe edema (greater than 20 gm weight gain). In contrast to our previous results with alpha-toxin, endothelial cells displayed normal electron density here and were not detached from the fused basal lamina. Hence, although both pore formers provoke severe vascular leakage in our experimental model, the underlying pathways probably divert fundamentally from each other.