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Departments of Human Anatomy and Histology, and Preclinical and Clinical Pharmacology (E.M.), University of Florence, and Prosperius Institute (M.B.), Florence, Italy
Address all correspondence and requests for reprints to: Prof. Tatiana Bani Sacchi, Dipartimento di Anatomia Umana e Istologia, Sezione di Istologia, V. le G. Pieraccini 6, I-50139 Firenze, Italy. E-mail: histology{at}cesit1.unifi.it
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Abstract |
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Guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received RLX (30 µg/kg BW, twice daily for 4 days) before antigen challenge. Other animals received inactivated RLX in place of authentic RLX. Respiratory abnormalities, such as cough and dyspnea, were analyzed as were light and electron microscopic features of lung specimens.
RLX was shown to reduce the severity of respiratory abnormalities, as well as histological alterations, mast cell degranulation, and leukocyte infiltration in sensitized guinea pigs exposed to ovalbumin aerosol. RLX was also found to promote dilation of alveolar blood capillaries and to reduce the thickness of the air-blood barrier.
This study provides evidence for an antiasthmatic property of RLX and raises the possibility of new therapeutic strategies for allergic asthma in humans.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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On these grounds, we hypothesize that RLX may counteract the pathogenic events underlying allergic asthma. In fact, it has been established that the release of IgE-dependent mediators from inflammatory cells, namely granule-associated mast cell mediators (histamine, eosinophil, and neutrophil chemotactic factors) and membrane-derived agents from activated mast cells, platelets, and macrophages (leukotrienes, PGs, and platelet-activating factors) play a major role in the pathogenesis of allergic asthma (10, 11, 12). This disease is characterized by bronchoconstriction, hypersecretion of mucus, and inflammation (13) mainly caused by IgE-mediated hypersensitivity reactions to inhaled antigens. Moreover, the ability of RLX to stimulate the production of nitric oxide by mast cells and platelets further strengthens the hypothesis of a beneficial action of RLX in asthma, as nitric oxide has been shown to cause relaxation of lung airways and blood vessels and, hence, to improve asthmatic symptoms and favor lung perfusion (14).
The results obtained from the current study first show that RLX counteracts the respiratory and histopathological abnormalities of an experimentally induced asthma-like reaction in guinea pigs. In fact, in the guinea pig, repeated exposure to antigen has been demonstrated to cause airway hyperresponsiveness and leukocyte infiltration of lung tissue mimicking histological and pharmacological correlates of asthma in humans (15, 16, 17).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Treatments
A group of 12 guinea pigs was injected with water (5 ml/kg BW,
ip, plus 5 ml/kg BW, sc). Two weeks later, these animals were treated
with an aerosol of ovalbumin (Fluka, Buchs, Switzerland) suspended in
water (5 mg/ml) and used as controls. They are referred to as group
1.
The remaining 58 guinea pigs were sensitized with 100 mg/kg BW, ip, plus 100 mg/kg BW, sc, ovalbumin, suspended in water (20 mg/ml). Two weeks later, they were challenged with an aerosol of ovalbumin (5 mg/ml water) to verify that sensitization had occurred. The animals were withdrawn from antigen exposure at the first sign of respiratory abnormality. Forty-eight of 58 guinea pigs developed airway hyperresponsiveness to the inhaled antigen. These animals are referred to as sensitized animals. They were selected for further treatments, as indicated below.
Group 2.
Three days after ovalbumin challenge, 12 guinea
pigs were treated with two daily sc injections (at 0900 and 1600
h) of 1 ml PBS for 4 days. Finally, the animals underwent a second
challenge with ovalbumin aerosol, as described below.
Group 3.
Twelve guinea pigs were treated as described for
group 2, but received two daily sc injections of RLX (30 µg/kg BW),
dissolved in 1 ml PBS, for 4 days instead of PBS injections. Pure
porcine RLX, prepared according to the method of Sherwood and O’Byrne
(18), was provided by Dr. O. D. Sherwood. This dose of RLX was chosen
because it was recognized to be effective in previous in
vivo studies in mice and rats (19, 20). At the end of the
treatment, the animals underwent a second challenge with ovalbumin
aerosol, as described below.
Group 4.
Twelve guinea pigs were treated as described for
group 2, but received two daily sc injections of inactivated porcine
RLX (iRLX) at a dose of 30 µg/kg BW, dissolved in 1 ml PBS, for 4
days. RLX was inactivated according to the method of Büllesbach
and Schwabe (21). Briefly, 1 mg porcine RLX was dissolved in 0.1
M borate buffer, pH 8.9, with a 10-fold molar excess of
1,2-cyclohexanedione (Sigma Chemical Co., St. Louis, MO), which reacts
specifically with arginine residues (22), thus modifying the receptor
interaction site of the RLX molecule. Excess reagent was removed by
dialysis against distilled water. Successful inactivation was assessed
by the inability of iRLX to increase the coronary flow in isolated and
perfused guinea pig heart, at variance with authentic RLX (7). At the
end of the treatment with iRLX, the guinea pigs underwent a second
challenge with ovalbumin aerosol, as described below.
Group 5.
The remaining 12 sensitized guinea pigs were
excluded from any further treatment and were used as controls for
groups 2, 3, and 4 in the morphological studies of lung tissue
samples.
Evaluation of respiratory activity
The guinea pigs in groups 1–4 were placed, one by one, in a
whole body respiratory chamber, as described previously (23, 24). The
changes in inner pressure in the respiratory chamber induced by
breathing were monitored with a high sensitivity pressure transducer
(Battaglia-Rangoni, Bologna, Italy; pressure and linearity ranges from
-10 to +50 mm Hg) connected with a PC2400A channel of a polygraph
(Battaglia-Rangoni). Upon stabilization of the breath pattern (usually
occurring within 30–60 sec), the guinea pigs from groups 1–4 were
challenged with an aerosol of ovalbumin (5 mg/ml in water) for 10 sec.
With this device, very small aerosol particles can be obtained that can
easily reach the lower respiratory airways, as assessed in previous
tests carried out with aerosolization of trypan blue dye dissolved in
water (Ballati, L., personal observation). The nonsensitized guinea
pigs of group 1 were included in the aerosolic challenge to reveal
possible alterations of the breath pattern due to aspecific stimulation
of the airways by the aerosol droplets. The changes in the respiratory
activity of the animals subjected to the different treatments were
recorded for 5 min after the aerosol administrations. Evaluation of the
following parameters was achieved (24): latency time (seconds),
assessed as the time between the onset of aerosolization and the first
cough stroke, a cough stroke being assumed as a respiratory movement
whose amplitude exceeded at least 10% that of normal breath preceding
the cough stroke; and cough severity, assessed as the product of cough
frequency and mean cough amplitude, assuming as cough frequency the
number of cough strokes per min and as cough amplitude the excess
pressure (millimeters of Hg) over the normal breath preceding the cough
stroke. In addition, the occurrence of dyspnea, recognized in breath
recordings as a series of irregular breaths of abnormally elevated or
reduced amplitude compared with the basal breath, was reported.
Morphological analysis
Once extracted from the respiratory chamber, the guinea pigs of
groups 1–4 were killed by lethal ip injections of sodium thiopenthal
(Pentothal, Abbott, Latina, Italy). The sensitized guinea pigs of group
5, which were excluded from aerosol administration, underwent the same
fate. At death, the thorax was opened, allowing for the gross
appearance of lungs to be examined, and tissue specimens from the
middle lobe of the right lung were excised from each animal and
processed for light and electron microscopy.
For light microscopic study of lung mast cells, tissue samples were fixed by immersion in Mota fluid, dehydrated, and embedded in paraffin. Sections 5-µm thick were cut and stained with toluidine blue to reveal metachromasia of mast cell granules. For routine histology and cytochemical study of leukocytes, other tissue samples were fixed in Bouin’s fluid. Sections 5 µm thick were stained with hematoxylin and eosin or subjected to the cytochemical reaction for peroxidase to identify infiltrating leukocytes. Briefly, dewaxed sections were incubated for 5 min in a solution of 3,3'-diaminobenzidine tetrahydrochloride (Sigma; 0.15%) in Tris buffer (0.2 M; pH 7.4), added with drops of 3% H2O2 just before use. After thorough rinsing in Tris buffer and then in water, the sections were dehydrated and mounted in Permount (Fisher Scientific, Fairlawn, NJ). Peroxidase-containing cells could be identified by dark brown oxidized 3,3'-diaminobenzidine tetrahydrochloride precipitate.
For electron microscopy, the tissue samples were fixed in cold 4% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 3 h at room temperature and postfixed in 1% osmium tetroxide in 0.1 M phosphate buffer, pH 7.4, for 1 h at 4 C. They were then dehydrated in graded acetone, passed through propylene-oxide, and embedded in Epon 812 (Fluka). Ultrathin sections were stained with uranyl acetate and alkaline bismuth subnitrate (25) and examined under a Siemens Elmiskop 102 electron microscope (Siemens, Berlin, Germany) at 80 kV.
Computer-assisted morphometry
A series of determinations was carried out on histological
sections stained with toluidine blue to evaluate the optical density of
lung mast cells in terms of light transmittance across their
cytoplasms, which is inversely related to the content of secretory
granules. Determinations were performed according to the method
described previously for similar purposes (8). The mast cells were
viewed by a CCTV television camera (Sony, Tokyo, Japan) applied to a
Reichert-Jung Microstar IV light microscope (Cambridge Instruments,
Buffalo, NY) with a x100 oil immersion objective and interfaced with
an Apple Macintosh LC III personal computer through a Videospigot card
(Supermac, Sunnyvale, CA). The card allows for the light transmitted
across the microscope slide to be determined within a range of 256 gray
levels between 0 (black level) and 255 (white level). The card also
allows for a digitized image to be reproduced on the basis of the
values estimated. Measurements of transmittance were carried out using
a NIH 1.49 image analysis program. It allows for measurement of the
staining intensity of mast cells to be obtained and, therefore, the
amount of mast cell granules to be evaluated. In each experimental
group, the transmittance of 120 randomly chosen mast cells, 10 from
each animal in the group, was analyzed, and the mean transmittance
value (±SE) was calculated.
A second series of determinations was carried out on histological sections stained with the histochemical reaction for peroxidase to evaluate the number of infiltrating leukocytes. In each experimental group, 60 randomly chosen microscopic fields, 5 from each animal of the group, were analyzed with a light microscope with a x40 objective. In each field, the number of peroxidase-positive cells was counted. The surface area of the lung tissue, with the exclusion of the spaces occupied by air, was measured by computer-assisted morphometry through an optical density cut-off, using the same device described above for mast cell morphometry. The number of leukocytes infiltrating the lung was expressed as number of cells per 104 µm2 of actual lung tissue area, independent of the amount of air trapping.
A third series of determinations was carried out on semithin sections from the tissue samples processed for electron microscopy to evaluate the surface area of microvessel lumina. Semithin sections (2 µm thick) were cut and stained with toluidine blue-sodium tetraborate. This procedure enables the lung blood capillaries to be easily recognized on the basis of the histological structure of their wall, as described by Simionescu and Simionescu (26). In particular, capillaries have a flat endothelium and a few or no pericytes included in the basal lamina. In each animal, five randomly chosen microscopic fields were photographed using a Leitz light microscope (Leitz, Rockleigh, NJ) with a x63 objective in oil immersion. Photoprints at x1385 final magnification were obtained, and the outlines of capillary lumina were traced onto superimposed acetate sheets. The profiles obtained were measured by computer-assisted morphometry using the same device and program described above. The program enables the areas encircled by each profile to be measured and the mean surface area (±SE) to be calculated.
Statistical analysis
The reported data are expressed as the mean ±
SE. In both functional and morphometrical assays, the
distribution of the measured values in the various experimental groups
was assessed to be Gaussian. Statistical analysis was performed by
one-way ANOVA, followed by the Student-Newman-Keuls multiple comparison
test. Calculations were carried out using a GraphPad Prism 2.0
statistical program (GraphPad Software, San Diego, CA).
P < 0.05 was considered significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. There were no substantial abnormalities in
the nonsensitized guinea pigs after inhalation of ovalbumin aerosol
(group 1), apart from sporadic cough strokes arising about 2 min after
the onset of the aerosol. Challenge of sensitized guinea pigs with the
ovalbumin aerosol (group 2) resulted in striking abnormalities of the
respiratory pattern, consisting of a significant reduction of the cough
latency time and a significant increase in the severity of cough.
Episodes of dyspnea were also found in the breath recordings from 6 of
12 animals in group 2. Conversely, treatment with RLX of sensitized
guinea pigs before antigen inhalation (group 3) resulted in a marked
reduction of the respiratory abnormalities compared with those in the
sensitized animals not receiving RLX in group 2. In particular, the
cough latency time was significantly increased, and the severity of
cough was significantly reduced. Clear-cut signs of dyspnea in the
breath recordings were not detected in any of the animals of this
group. Administration of iRLX in place of authentic RLX to the
sensitized guinea pigs of group 4 resulted in clear-cut respiratory
abnormalities after antigen challenge. In fact, compared with the
animals given authentic RLX, the severity of cough was significantly
increased. Cough latency time also decreased, although it did not reach
statistical significance due to large individual variations. Episodes
of dyspnea were not observed.
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Light microscopy of lung tissue showed that the intrapulmonary bronchi
and respiratory air spaces of control guinea pigs in groups 1 and 5 had
a normal appearance. In particular, intrapulmonary bronchi showed open
lumina with bronchial mucosa forming short folds, and the respiratory
air spaces were evenly small sized (Fig. 1A).
Conversely, lung tissue from the sensitized guinea pigs challenged with
the antigen (group 2) mostly showed a reduction of the lumen of
intrapulmonary bronchi, with long mucosal folds expanding into the
lumen. Moreover, in large areas of the lung parenchyma, the respiratory
air spaces were markedly dilated by accumulation of entrapped air (Fig. 1B). In the sensitized guinea pigs treated with RLX (group 3), the
histological lung abnormalities were nearly abrogated. In fact, the
intrapulmonary bronchi usually showed no appreciable signs of
constriction, and the respiratory air spaces were not dilated (Fig. 1C). Only in small areas of the lung parenchyma were attenuated signs
of bronchoconstriction and dilation of respiratory air spaces observed.
In the sensitized guinea pigs treated with iRLX in place of authentic
RLX (group 4), the histological features of the lung tissue were
similar to those of the sensitized animals of group 2, but
bronchoconstriction and air spaces dilation were usually less severe
(Fig. 1D).
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).
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).
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).
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). In contrast, in the sensitized guinea pigs treated
with RLX of group 3, the alveolar blood capillaries were strongly
dilated, and the air-blood barrier was markedly reduced in large areas
of the alveolar wall due to extreme reduction of the thickness of the
cytoplasmic processes of endothelial cells and type I pneumocytes and
of the basement lamina (Fig. 5B). When iRLX was given in place of
authentic RLX (group 4), the above changes in the air-blood barrier
were not found.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Concerning the mechanisms by which RLX exerts its beneficial action, it is possible that they chiefly rely on the ability of this hormone to inhibit degranulation of lung mast cells and, hence, the release of bronchoconstrictor and proinflammatory mediators by these cells. This view is also in keeping with the results of our previous studies showing that RLX depresses histamine release and granule exocytosis by serosal mast cells (8).
Besides mast cells, activated platelets are also involved in the pathogenesis of asthma through the release of proinflammatory mediators with bronchoconstrictor activity (12). Therefore, it is likely that the beneficial effect of RLX on airway hyperresponsiveness to inhaled antigen in the sensitized guinea pigs may also depend on the ability of this hormone to inhibit platelet activation, as evidenced by us in previous studies of human and rabbit platelets (9). In this context, it has been reported that platelet depletion depresses the asthmatic reaction in antigen-challenged sensitized guinea pigs (28) and even abolishes the lethal consequences of allergen challenge in the rabbit (29, 30). It is possible that the antiasthmatic effect of RLX may also rely at least in part on its thrombocytopenic property, which has been recognized by us in previous studies in the rat (20).
The current findings also show that RLX treatment causes a marked reduction of the recruitment of leukocytes in the lungs compared with that in the sensitized guinea pigs not given RLX. Infiltration of eosinophils is a prominent feature of asthmatic lungs, and inflammatory mediators released by these cells largely contribute to the pathogenesis of allergic asthma (31, 32, 33, 34). It is likely that in our experiments, the reduction of leukocyte infiltration is a consequence of the RLX-induced inhibition of mast cell and platelet activation, leading to a reduction of the release of chemotactic factors by these cells (10, 33).
A marked dilation of alveolar blood capillaries leading to a concurrent decrease in the thickness of the air-blood barrier was observed in the sensitized guinea pigs treated with RLX. These findings suggest that RLX can increase lung perfusion and gas exchanges. In turn, pulmonary vasodilation has been said to contribute in ameliorating asthma by removing inflammatory mediators (35). The effect of RLX on lung blood vessels is in keeping with the previously reported vasodilatory action of the peptide in other organs (3, 4, 5, 6, 7). The RLX-induced increase in lung blood supply may suggest a possible physiological role for RLX produced and secreted by right atrial cardiocytes (36). In fact, once released into the blood, cardiac RLX might reach the pulmonary circulation and act as a regulatory agent of lung perfusion.
As expected, administration of iRLX in place of RLX has little or no effect on the amelioration of the asthma-like reaction induced by antigen inhalation in the sensitized guinea pigs compared with the effect of the authentic peptide. This allows us to exclude the possibility of an aspecific action of RLX in counteracting the asthma-like reaction.
It has been shown that iRLX, which is stable in acidic or strongly alkaline solutions, progressively decomposes to regenerate the original molecule in neutral and weakly alkaline solutions (22), and hence, it can maintain a residual biological activity when administered in vivo (21). It is conceivable that in our experiments, part of iRLX had been converted to authentic RLX, thus providing a possible explanation for our findings that the respiratory and morphological parameters in the guinea pigs given iRLX are not entirely superposable on those in the animals that only received the PBS vehicle.
In previous studies by our group, RLX has been demonstrated to evoke the response of its targets through the stimulation of endogenous production of nitric oxide (6, 7, 8, 9, 37). Nitric oxide, in turn, has been shown to exert beneficial effects on asthma by acting on the lung components at multiple levels (14, 38). It also inhibits leukocyte adhesion to microvascular endothelium and emigration (39) and is a powerful vasodilatory agent (40, 41). Therefore, the antiasthmatic properties of RLX may also rely on the ability of this hormone to stimulate nitric oxide production by cells in the lungs.
The effects of RLX evidenced in the current study may explain previous clinical reports of a subjective and objective improvement in asthma during pregnancy (42, 43, 44), when the circulating levels of RLX are elevated (45, 46, 47), and deterioration of asthma in the puerperium (44), when a drop in circulating RLX occurs.
As a future perspective, recognition of the antiasthmatic properties of RLX may provide new therapeutic strategies for the treatment of allergic asthma.
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Acknowledgments |
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Footnotes |
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Received September 25, 1996.
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