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Abstract |
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Abstract
Introduction
Methods
Results
Discussion
References
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Studies from our laboratory have shown that chronic cigarette smoke exposure causes a neutrophilia associated with a shortening of the mean transit time of polymorphonuclear leukocytes (PMN) though the postmitotic pool of the marrow. The present study was designed to test the hypothesis that PMN newly released from bone marrow by smoke exposure preferentially sequestered in pulmonary microvessels. The thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) was used to label dividing PMN in the marrow of rabbits; their appearance in the circulation was measured using immunocytochemistry, and their sequestration in lung tissue was determined using standard morphometric techniques. Animals exposed to 11 d of cigarette smoke (n = 6) compared with sham-exposed control animals (n = 4) showed no increase in circulating PMN counts but showed an increase in both the percentage of band cells (smoking, 9.8 ± 1.1% versus control, 5.5 ± 0.9%; P < 0.05) and BrdU-labeled PMN (PMNBrdU) in the circulation (smoking, 10.8 ± 0.6% versus control, 7.5 ± 0.3%; P < 0.05). There were more PMN sequestered in the lungs of smoke-exposed animals (51.7 ± 3.4 × 107/ml tissue) than in those of control animals (25.1 ± 1.8 × 107/ ml tissue) (P < 0.05) and a higher percentage of these cells were PMNBrdU (smoking, 16.9 ± 2.3% versus control, 9.6 ± 0.4%; P < 0.05). The percentage of PMNBrdU in the gravity-independent regions (11.7 ± 1.9%) of the lung was higher than gravity-dependent regions (7.8 ± 1.8%) in the smoke-exposure group (P < 0.05). Transmission electron microscopy showed pulmonary capillary endothelial damage with adherent PMN in the smoke-exposure group. We conclude that younger PMN released from the bone marrow by cigarette smoking preferentially sequestered in pulmonary microvessels and speculate that these PMN may contribute to the alveolar wall damage associated with smoke-induced lung emphysema.
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Introduction |
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Abstract
Introduction
Methods
Results
Discussion
References
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The destruction of alveolar walls that characterizes emphysema is closely linked to cigarette smoking (1). A proteolytic imbalance created by the inhalation of cigarette smoke is thought to be responsible for this parenchymal destruction. This imbalance has been attributed to both an increase in the release of proteolytic enzymes by inflammatory cells and a reduction in the function of the natural inhibitors of these enzymes due to oxidation of their active site (2). Although the exact site of this imbalance is not known, there is growing evidence that the polymorphonuclear leukocytes (PMN) delayed in the lung by the presence of cigarette smoke may damage the lung tissue from within the vascular space (6).
Cigarette smoking also increases the number of PMN in the circulation and the degree of deterioration in lung function correlated with the level of circulating PMN (10, 11). We have shown that chronic cigarette smoke exposure in rabbits causes a leukocytosis similar to that seen in human smokers (12). The study also demonstrates that this leukocytosis is associated with an increase in circulating PMN and band cells, confirming that a release of both mature and immature PMN from the bone marrow contributes to the rise in leukocyte count. This was associated with an increase in bone marrow turnover of PMN with a shortening of the mean transit time of PMN through the postmitotic pool of the marrow (12).
Maturation of PMN in the postmitotic pool of the bone marrow is associated with an increase in their mobility, deformability, and chemotactic responsiveness (13). Because the transit time of PMN through the postmitotic pool was shortened by the smoke exposure, immature PMN were released into the circulation. On the basis of previous studies showing that immature PMN are larger and less deformable (15), we postulate that there would be a preferential sequestration of these immature cells in the pulmonary microvessels during smoking. Previous studies from our laboratory have shown both sequestration (6, 7) and activation of PMN in the pulmonary microvessels during cigarette smoking (8). Using morphometric analysis, Finkelstein and colleagues have shown no correlation between the inflammatory cells in lung tissue and the presence of emphysema (16). This suggests that not just the burden of PMN but also their phenotypic and functional characteristics are important in the pathogenesis of emphysema. Prolonged retention and activation of these less deformable immature PMN in the lung could increase the burden of elastase release and contribute to alveolar wall destruction.
The present study was designed to test the hypothesis that PMN released from the bone marrow by smoke exposure was preferentially sequestered in the alveolar walls of lung tissue. To test this hypothesis, we used the thymidine analogue 5'-bromo-2'-deoxyuridine (BrdU) (17) to label the dividing myeloid cells in bone marrow and measure their release from the bone marrow and their sequestration in the lung.
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Methods |
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Abstract
Introduction
Methods
Results
Discussion
References
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Experimental Groups
The experiments were performed on adult female New Zealand white rabbits (weight 1.8 to 2.4 kg). Animals exposed to cigarette smoke for 11 d (n = 6) were compared with sham-exposed controls (n = 4). All experiments were approved by the Animal Experimentation Committee of the University of British Columbia.
Experimental Protocol
The animals were exposed to cigarette smoke using a technique that has been described previously in detail (12). Briefly, the smoke was generated from standard unfiltered cigarettes (Tobacco Manufacture Council, Montreal, PQ, Canada) using a machine that delivered smoke at a rate of 2 puffs/min. The nose of the mildly anesthetized rabbit was fixed into a small chamber (0.1 to 0.2 ml/kg Inovar-Ver; Janssen Pharmaceutical, Mississauga, ON, Canada) and rabbits in prone position were exposed to the smoke of six cigarettes for 20 min daily for 11 d (Figure 1). The rabbits in the control group were anesthetized in the same way and were exposed to room air. Baseline blood samples were obtained daily from the central ear artery before exposure to measure the total circulating white blood cell (WBC) and PMN counts. On Day 1, carboxyhemoglobin HbCO levels were measured before and after the initial cigarette exposure (IL-482 CO-Oximeter system; Instrumentation Lab Co., Lexington, MA). Sequential blood samples (1 ml) were obtained and collected in standard Vacutainer tubes containing potassium ethylenediaminetetraacetic acid (Becton Dickinson, Rutherford, NJ) for blood cell counts determined on a model SS80 Coulter Counter (Coulter Electronics, Hialeah, FL) and for differential WBC counts on Wright's stained blood smears.
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On Day 9, a single dose of BrdU (100 mg/kg) was infused through the marginal ear vein at a concentration of 20 mg/ml in normal sterile saline over a period of 15 min (Figure 1). On Day 11, blood samples were obtained before and after smoke exposure and animals were killed in prone position 40 h following the BrdU infusion with an overdose of sodium pentobarbitone. The time of 40 h was selected to kill animals on the basis of previous studies showing a rapid release of BrdU-labeled PMN (PMNBrdU) from the bone marrow into the circulation between 36 and 48 h after labeling (12). After the animals were killed, their chests were opened rapidly and the bases of the hearts were ligated to maintain the pulmonary blood volume. The tracheas and both lungs were separated from other organs. The right lungs were used for transmission electron microscopic (TEM) studies, and the left lungs were used for quantitative histology. The right lungs were inflated at 25 cm H2O by intratracheal instillation of 10% phosphate-buffered formalin and fixative for 2 h. After fixation, the lungs were cut into five slices perpendicular (at 90°) to the gravitational field, and randomly selected blocks of tissue 1 cm × 0.5 cm × 0.3 cm were processed in paraffin for histologic evaluation.
The blood samples were used to detect BrdU-labeled PMN. Samples were collected in acid-citrate-dextrose for preparation of leukocyte-rich plasma (LRP). Erythrocytes in the samples were allowed to sediment for 25 to 30 min after the addition of an equal volume of 4% dextran (average molecular weight, 162,000) (Sigma Chemical Co., St. Louis, MO) in PMN buffer (138 mM NaCl, 27 mM KCl, 8.1 mM Na2HPO4, 7 H2O, 1.5 mM KH2PO4, and 5.5 mM glucose [pH 7.4]). The resulting LRP was cytospun at 1,600 rpm for 4 min to obtain a monolayer of cells on slides coated with 3-aminopropyl-tri-ethoxysilane.
Immunocytochemical Detection of BrdU-labeled Cells
A mouse monoclonal antibody to BrdU and the alkaline phosphatase antialkaline phosphatase (APAAP) method were used to stain for presence of BrdU in PMN on cytospins made from LRP and lung sections. Cytospins were air-dried, fixed in acetone for 10 min, and then incubated at 37°C for 15 min in a 0.04% pepsin solution acidified to pH 2.5. The lung sections were deparaffinized, rehydrated, and digested for 10 min in 0.4% pepsin. DNA in both samples was denatured in 2 N HCl at 37°C for 1 h. This was followed by neutralization in three washes of 0.1 M borate buffer, pH 8.5, each for 10 min. After nonspecific binding sites were blocked by incubation with 5% normal rabbit serum for 15 min, the specimens were incubated with 2 µg/ml mouse anti-BrdU antibody (DAKO Laboratories, Copenhagen, Denmark) prepared with 1% bovine serum albumin in 50 mM Tris-Cl, 150 mM NaCl, pH 7.6 (Tris-buffered saline [TBS]) at room temperature in a humidified chamber for 1 h. Nonspecific mouse immunoglobulin G1 (IgG1) at 2 µg/ml was used as a negative control. Incubation in a 1:20 dilution of rabbit antimouse IgG (DAKO) for 30 min was followed by 30 min in a 1:50 dilution of a mouse monoclonal APAAP complex (DAKO). Slides were washed in 0.1% Tween 20 in TBS for 10 min after each antibody application. The alkaline phosphatase was developed for 20 min in 100 ml TBS at pH 8.7, after the addition of a mixture of 0.5 ml of 4% sodium nitrate, 0.2 ml of 5% fuschin (Merck, Rahway, NJ) in 2 M HCl, and 50 mg naphtol-AS-B1-phosphate (Sigma) dissolved in 0.3 ml N,N-dimethylformamide. Endogenous alkaline phosphatase was blocked by addition of 17.5 mg levamisole (Sigma) to the color reaction. The slides were counterstained with Mayer's hematoxylin for 25 s, dehydrated through graded alcohols from 70% to 100% and xylene, and mounted with a permanent mounting medium.
Evaluation of BrdU-labeled PMN in the Blood
Any PMN on the cytospins that contained nuclear stain were counted as positive and designated PMNBrdU. All slides were evaluated on a Zeiss Universal Research light microscope (Model IIR; OberKochen, Germany) at ×400 magnification. The slides were coded and examined without knowledge of the group or the sampling time. Fields were selected in a systematic randomized fashion, and 100 cells were evaluated per specimen. All the PMN in a selected field were evaluated, except if the cell was broken or overlapping with neighboring cells where there was a tendency to trap stain between them.
Morphometric Evaluation of PMNBrdU in the Lung
Paraffin-embedded sections stained for BrdU were point counted at ×800 magnification using a Nikon Microphot-fx light microscope (Nikon, Tokyo, Japan) with Bioview, an image processing system (Infrascan Inc., Richmond, BC, Canada). In each animal, 10 random fields were evaluated on each slide from five different tissue blocks. The number of points falling on airspace, tissue, large blood vessels, and PMN (PMNtotal) and PMNBrdU in the airspace, in the tissue, or in the vessels were counted. Tissue in this context consists largely of alveolar walls and includes capillaries and interstitial spaces. The number of PMNtotal and PMNBrdU/ml tissue were calculated:
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where 143 fl is the assumed volume of a rabbit PMN (18). Similar calculations were made to calculate the PMNBrdU/ml tissue. The number of PMNtotal and PMNBrdU in gravity- independent, gravity-dependent, and whole-lung regions were calculated in each animal.
TEM
The right lungs of rabbits were inflated (through the trachea, pressure approximately 25 cm H2O) and immersion-fixed for 1 h with 2.5% (vol/vol) glutaraldehyde in 0.1 M Na cacodylate buffer (pH 7.3). Tissue samples (approximately 1 mm3), randomly collected from the upper and lower region of the lung, were further fixed for 1 h with 1% (vol/vol) OsO4, washed, ethanol-dehydrated, and embedded in LR White. Ultrathin sections were stained with uranyl acetate, citrated, and examined on a Philips 400 transmission electron microscope (Philips, Eindhoven, Netherlands).
Statistical Analysis
All values are expressed as means ± SEM. The leukocyte counts before and after smoke exposure were compared by using a paired t test. The number of PMN in gravity- independent and gravity-dependent regions in each animal, as well as the percentage of PMNBrdU in different lung regions, was compared through paired t tests. Differences between the smoke-exposure and control groups were determined by an analysis of variance with Bonferroni corrections for multiple comparisons, and P < 0.05 was accepted as statistically significant.
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Results |
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Abstract
Introduction
Methods
Results
Discussion
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Carboxy-Hemoglobin Levels
The HbCO levels increased from the baseline value of 7.9 ± 0.4% to 13.8 ± 1.7% in the smoke-exposure group (P < 0.05), and remained unchanged in the control group (7.5 ± 0.6% to 7.8 ± 0.6%).
Peripheral Blood Cell Counts
In both the smoke-exposed and control groups, the PMN counts did not change significantly during chronic smoke exposure or after the smoke exposure on Day 11 (Figure 2a). The percentage of circulating band cells increased with smoke exposure, but not in the control animals (Figure 2b, P < 0.05). On Day 11, smoke exposure caused a decrease in the percentage of band cells in the peripheral blood from 9.8 ± 1.1% to 3.7 ± 0.5% (Figure 2b, P < 0.05), with no change in the control rabbits.
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PMNBrdU in the Circulation
Smoke exposure stimulated the PMN release from the bone marrow and increased the percentage of PMNBrdU in the circulation compared with the control animals (Figure 3, P < 0.05). On Day 11, smoke exposure caused a decrease in the percentage of PMNBrdU in the peripheral blood from 10.8 ± 0.6% to 7.3 ± 0.5% (Figure 3, P < 0.05), with no change in the control animals. There was no difference between the number of circulating PMNBrdU of control and smoking animals at the time of killing (P = not significant).
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PMN in the Lung
Figure 4 shows that there were more PMNtotal in the lung tissues (alveolar wall) of the smoke-exposed animals than in those of controls. This increase was evident in both gravity-dependent and gravity-independent regions of the lung (P < 0.05). Figure 5 shows more PMNBrdU in the lung tissues of the smoke-exposed than the control animals. This was evident in all regions of the lung (P < 0.05). Correcting these numbers for the small difference in circulating PMNBrdU between groups (Figure 3) did not change these results. In smoke-exposed animals, there were also more PMNBrdU in the gravity-independent than the gravity-dependent regions (P < 0.05) (Figures 5 and 6).
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Figure 6 shows the comparison between the fraction of PMNBrdU in large lung vessels (representing circulating blood) and lung tissues (alveolar wall) in different lung regions. The percentage of PMNBrdU in large lung vessels was not different between the smoke-exposed and control animals. There was a significantly higher percentage of PMNBrdU in the gravity-independent than the gravity- dependent lung regions in the smoke-exposed animals (P < 0.05). This preferential retention of PMNBrdU was not seen in the control group.
TEM
Figure 7a shows a TEM photomicrograph of a lung section of control rabbit, demonstrating platelets and erythrocytes in pulmonary capillary. Figures 7b to 7d show representative TEM sections of the lungs of rabbits exposed to cigarette smoke for 11 d. Figure 7b shows capillary endothelial cells with high surface electron density and vacuoles signifying endothelial damage. Figure 7c shows a platelet and a neutrophil adjacent to damaged endothelium, and Figure 7d shows PMN that have migrated into the alveolar wall.
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Discussion |
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Abstract
Introduction
Methods
Results
Discussion
References
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Epidemiologic studies have shown a relationship between chronic cigarette smoking and elevated circulating leukocyte counts (10, 11). The magnitude of this leukocytosis is directly related to the decline in lung function (19, 20), suggesting a pathogenic role for the increased number of circulating leukocytes in cigarette smoke-related lung disease.
Previous studies from our laboratory have shown that
chronic cigarette smoke exposure stimulates the bone
marrow to release PMN into the circulation (12). These
PMN have a shortened mean transit time through the marrow, resulting in the release of mature and immature PMN
into the circulation (12). This stimulation of the bone marrow by cigarette smoke could be mediated by soluble factors released from the lung. The alveolar macrophage is
the most likely cell source of these mediators because it
has been shown to produce tumor necrosis factor-
, interleukin (IL)-1, IL-6, and IL-8 when stimulated, all of which
are known to stimulate the bone marrow (21, 22). These
factors could either stimulate the bone marrow directly,
or cause an intramedullary production of hematopoietic
growth factors such as granulocyte macrophage colony-stimulating factor and granulocyte colony-stimulating factor (23).
Chronic smoke exposure increased both the percentage of band cells and PMNBrdU in the circulation that are compatible with an increased bone marrow turnover of granulocytes. The acute smoke exposure before the rabbits were killed (Day 11) decreased the percentage of band cells and PMNBrdU in the systemic circulation, suggesting sequestration of these PMN. Studies on lung tissue provided evidence that this decrease could be due to the preferential sequestration of the PMNBrdU in the pulmonary microvessels (Figures 5 and 6).
Studies have shown that active cigarette smoking activates PMN in pulmonary capillaries (8) and causes a delay in PMN in the lung microvessels that reverses when smoking is stopped (6, 7). PMN are normally sequestered in pulmonary capillaries because of the discrepancy in size between the cells and the pulmonary capillary segments. This requires PMN to deform in order to cross the pulmonary vascular bed that slows their passage through the lung (18). Changes in the deformability of PMN following smoke exposure in vitro (24) suggests that smoking changes the deformability of PMN, which leads to their retention in pulmonary capillaries. This study confirms these observations and extends them by showing that the population of PMN that sequester are predominantly younger PMN recently released from the bone marrow. This preferential sequestration of younger PMN was not seen in the control animals, implying that PMN released from the bone marrow under control conditions are similar to their normal circulating counterparts.
Previous studies have shown longer transit times for PMN in gravity-independent regions of the lung during acute smoke exposure in rabbits (7). This longer transit time is thought to be related to biophysical factors resulting in smaller pulmonary capillary segments in these gravity-independent regions of the lung (25, 26) as well as changes in the deformability of PMN as a result of smoking (24). In our model of chronic smoking, there was diffuse sequestration of PMN in both gravity-dependent and -independent regions of the lung (Figure 5). However, the PMN released from the bone marrow preferentially sequester in gravity-independent regions of the lung (Figure 6), suggesting that these younger PMN could be the population of PMN instrumental in the pathogenesis of smoke-related emphysema.
Several functional and biophysical factors could be responsible for this preferential sequestration of younger PMN in the lung. Lichtman and Weed have shown that PMN harvested from the postmitotic pool in the bone marrow are larger and less deformable than PMN in the peripheral circulation (15). This study suggests that younger PMN released from the marrow by smoke exposure have similar phenotypic characteristics to those in the postmitotic pool in the marrow, causing them to sequester in lung microvessels. Alternatively, these younger PMN could be more sensitive to activation by smoke exposure that would retain them in lung capillaries. Previous studies from our laboratory have shown that these younger PMN are slow to migrate (27), which suggests that, similar to bone marrow granulocytes, they are less responsive to chemoattractants (14).
Altered adhesive properties of younger PMN could also contribute to sequestration of PMN in pulmonary capillaries. PMN released into the circulation with bone marrow stimulation by complement fragments (28) and pneumococcal pneumonia (29) express high levels of the adhesion molecule L-selectin (27). Although L-selectin contributes to the recruitment of PMN in different models of acute lung inflammation (30), there is no evidence that L-selectin on PMN is involved in the sequestration of PMN in lung microvessels. Furthermore, in vitro filtration studies of PMN (31) and in vivo studies in rabbits (32) have shown that blocking the CD11/CD18 complex did not influence the retention of PMN in either micropore filters or lung capillaries, respectively. Taken together, neither the adhesive properties of PMN nor their sensitivity to activating stimuli seem to be associated with enhanced sequestration in lung microvessels. We suspect that the deformability characteristic of younger PMN is the major factor responsible for their sequestration in the lung.
PMN have been implicated in lung destruction because
of their ability to produce oxygen free radicals and to release proteolytic enzymes (33). Activation of PMN in
the microvessels of the lung during cigarette smoking is
associated with upregulation of the adhesion molecule
CD18/CD11 (8) in the gravity-independent regions of the
lung. In these studies, Klut and colleagues observed "pocket
areas" between the PMN and endothelium containing the CD18/CD11 molecule (8), and subsequent studies showed
degranulation of primary granules into these pockets (9).
It has been postulated that the formation of these pockets
is crucial for creating a proteolytic imbalance, as they provide an environment in which proteolytic enzymes are
protected from natural plasma inhibitors such as 1-proteinase inhibitor (1-PI). Because these younger PMN have
a longer transit time through lung capillaries, they are more
vulnerable to subsequent intravascular cell activation by
cigarette smoke that could promote pocket formation. Qualitative analysis of alveolar wall damage using electron microscopy shows endothelial damage associated with sequestered PMN in pulmonary capillaries. These qualitative
observations suggest that free oxidant radicals produced either by the cigarette smoke or by PMN trapped in pulmonary capillaries could produce a proteolytic imbalance in
these pockets by functionally inactivating 1-PI (5). We
speculate that these newly released PMN may contribute to the alveolar wall destruction that is the hallmark of cigarette smoke-induced lung emphysema.
In summary, our results show that chronic cigarette smoke exposure stimulates the bone marrow to release PMN that preferentially sequestered in alveolar walls, especially in gravity-independent lung regions. Activation of these younger PMN that are delayed in the lung during cigarette smoking may be accompanied by the release of cytotoxic chemicals such as oxygen radicals and hydrolytic enzymes. Qualitative observations showed damage of pulmonary capillary endothelium, and we postulate that sequestration and activation of these younger PMN could be important in creating the proteolytic imbalance responsible for alveolar destruction resulting in smoke-induced lung emphysema.
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Footnotes |
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Abbreviations: 5'-bromo-2'-deoxyuridine, BrdU; interleukin, IL; leukocyte-rich plasma, LRP; polymorphonuclear leukocytes, PMN; BrdU- labeled PMN, PMNBrdU; Tris-buffered saline, TBS; transmission electron microscopy, TEM.
(Received in original form December 17, 1997 and in revised form April 20, 1998).
Acknowledgments: This work was supported by grant MRC 4219 from the Medical Research Council of Canada and the British Columbia Lung Association. The authors especially thank Stuart Greene for photography and Lorraine Verbrugt for statistical analysis.
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Abstract
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Methods
Results
Discussion
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