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Abstract |
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Abstract
Introduction
Materials and methods
Results
Discussion
Conclusions
References
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Angiogenesis is a feature of chronic inflammation produced by Mycoplasma pulmonis infection of the respiratory tract. The mechanism of this angiogenesis is unknown, but cellular growth factors and matrix remodeling proteases produced by inflammatory cells are likely to be involved. The goal of this study was to determine the relationship between changes in the number, shape, and distribution of ED2-immunoreactive macrophages and the development of angiogenesis in the tracheal mucosa of Wistar rats after M. pulmonis infection. In pathogen-free rats, ED2-positive cells were scattered in the airway mucosa (261 ± 42 cells/mm2 of surface, mean ± SE). Most cells were irregularly shaped and had moderate ED2 immunoreactivity. No lymphoid tissue was present. The number of ED2-positive cells increased rapidly after infection, was 120% above baseline at 1 wk, and remained significantly increased throughout the 4-wk study (P < 0.05). Angiogenesis was first detected at 2 wk, and at 3 wk the vessel length density was nearly 8-fold the pathogen-free value. At 3 and 4 wk, focal sites of angiogenesis coincided with discrete clusters of round, strongly immunoreactive ED2-positive cells (1,340 ± 124 cells/mm2) in polyp-like collections of mucosal lymphoid tissue. The close association of distinctive ED2-positive cells with angiogenic blood vessels suggests a relationship between a subset of tissue macrophages and the angiogenesis associated with M. pulmonis infection. The time course of the changes indicates that the initial influx of ED2-positive macrophages precedes the angiogenesis, and the rounding of the cells parallels the growth of new vessels.
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Introduction |
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Abstract
Introduction
Materials and methods
Results
Discussion
Conclusions
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Angiogenesis, the formation of new blood vessels from existing ones, is a key step in the growth of tumors and the development of chronic inflammation and is recognized as a potential therapeutic target (1, 2). Many factors that stimulate or inhibit angiogenesis have been identified, but their cellular sources are only now being identified (3, 4).
Macrophages are likely to be involved in angiogenesis
under some conditions (5, 6). These cells are present in
most tissues, are recruited to sites of inflammation, and produce substances that have angiogenic activity (5). Among
these are tumor necrosis factor alpha (TNF-
), vascular
endothelial growth factor (VEGF), transforming growth
factor alpha (TGF-), and interleukin (IL)-8 (7). Macrophages also release proteases that remodel the extracellular matrix and can synthesize extracellular matrix molecules such as fibronectin or proteoglycans (12, 13).
Mycoplasma pulmonis infection of the respiratory tract of rats and mice has been used as a model of chronic inflammatory disease with mononuclear cell infiltration and angiogenesis in the airway mucosa (14). The amount of angiogenesis reflects the severity of the infection (16, 17). Most of the angiogenic vessels are tortuous, capillary-sized, and readily distinguished from normal vessels in the airway mucosa (16). The angiogenic blood vessels are also abnormally sensitive to substance P, because of increased expression of NK1 receptors on their endothelial cells (16, 18). This property has been used to identify the angiogenic vessels, which become very leaky after exposure to substance P and thereby can be labeled with tracers such as Monastral blue (16, 17). Alternatively, the angiogenic blood vessels can be identified after staining in situ with perfusion of biotinylated or fluorescent Lycopersicon esculentum lectin through the vasculature (19).
There is no information on whether tissue macrophages participate in the process of angiogenesis in the airways of M. pulmonis-infected rats. In pathogen-free rats, however, macrophages are located in the airway mucosa, lung parenchyma, and alveolar lumen (22). Although alveolar macrophages are readily sampled under normal or pathologic conditions (23, 24), those in the airway mucosa are more difficult to isolate and have received less attention (25, 26).
The goal of the present study was to determine whether tissue macrophages are involved in the development of angiogenesis in the airway mucosa after M. pulmonis infection. Our approach was to relate the number, shape, and distribution of tissue macrophages to the location and amount of angiogenesis. Tissue macrophages were identified by immunohistochemistry with the ED2 monoclonal antibody (27), and angiogenic blood vessels were made visible by lectin binding (19, 20) or by Monastral blue labeling after an injection of substance P (16). Morphometric measurements were made on blood vessels and ED2-immunoreactive (ED2-positive) cells in tracheal whole mounts from Wistar rats infected with M. pulmonis for 1 to 4 wk (16).
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Materials and Methods |
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Introduction
Materials and methods
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Discussion
Conclusions
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Animals
Male Wistar rats (175 to 200 g) (Charles River, Hollister, CA), approximately 7 wk of age, were housed under barrier conditions in filter-top microisolator cages. Daily on three consecutive days, the rats were anesthetized (ketamine 50 mg/kg and xylazine 2 mg/kg intramuscularly), and 100 µl of culture medium containing M. pulmonis (strain 5782C, 7.5 × 109 colony-forming units per milliliter; University of Alabama, Birmingham, AL) was inoculated into each nostril (17). All experimental procedures were approved by the Committee on Animal Research of the University of California, San Francisco (San Francisco, CA).
Experimental Plan
At 1, 2, 3, or 4 wk after the first inoculation, rats were anesthetized with sodium pentobarbital (50 mg/kg intraperitoneally; Nembutal; Abbott Laboratories, North Chicago, IL), and 1 ml of blood was withdrawn for measurement of serologic antibody titers. Angiogenic blood vessels were labeled by injecting Monastral blue (30 mg/kg intravenously [i.v.] over 10 s; Sigma Chemical Co., St. Louis, MO) followed immediately by substance P (5 µg/kg i.v. over 20 s; Peninsula Laboratories, Belmont, CA) (17). Five minutes later, fixative was perfused through the ascending aorta at a pressure of 120 mm Hg. Tissues were then processed for lectin staining of the microvasculature or for ED2 immunohistochemistry. Alternatively, the trachea was removed, mucosal lymphoid tissue was stained by esterase histochemistry, and the tissue was prepared as a flattened whole mount (28).
Lectin Staining of Microvasculature
The vasculature in the airway mucosa was made visible by staining the endothelium by perfusion of biotinylated L. esculentum lectin (no. B-1175; Vector, Burlingame, CA) (19). In these animals, the fixative (1% paraformaldehyde plus 0.5% glutaraldehyde in phosphate-buffered 0.9% NaCl [PBS], pH 7.4) was perfused for 5 min. The vasculature was then perfused with PBS for 1 min, PBS plus 1% bovine serum albumin (BSA; Sigma) for 1 min, biotinylated lectin (5 to 10 µg/ml in 50 ml PBS/BSA) for 1 min, PBS/BSA for 1 min, and PBS for 1 min (19). Tracheas were removed and opened along the ventral midline, were pinned with the mucosal surface up on Sylgard-coated (Dow Corning, Midland, MI) plastic dishes, and permeabilized by overnight incubation in PBS containing 0.3% Triton X-100 at room temperature. The pinned tracheas were incubated for about 24 h with avidin-peroxidase complex (Vector) diluted 1:200, washed for 2 h with 50 mM Tris-HCl buffer (pH 7.4) containing 1% Triton X-100, incubated for 5 min with 0.05% 3,3'-diaminobenzidine tetrahydrochloride (DAB; Sigma) in Tris-HCl buffer, then incubated for 10 min with 0.05% DAB and 0.01% hydrogen peroxide in Tris-HCl buffer at room temperature. Tracheas were dehydrated, flattened, cleared, and mounted with the mucosal surface upward (19).
ED2 Immunohistochemistry
In rats used for immunohistochemistry, the fixative (1% paraformaldehyde in PBS, pH 7.4) was perfused for 2 min. Tracheas were exposed, incised along the ventral midline, removed, and fixed in 4% paraformaldehyde overnight at 4°C. Tracheas were washed in PBS (pH 7.6) containing 0.3% Triton X-100 (Sigma) and 0.01% thimerosal, cut transversely into three parts, stretched, and pinned flat onto Sylgard slabs (29). Whole mounts were incubated in 5% normal goat serum blocking antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 2 h and then incubated with the ED2 primary antibody (1:10,000; Serotec, Washington, DC) for 36 h (27). After washes in PBS, specimens were incubated in goat antimouse immunoglobulin G coupled to horseradish peroxidase (1:200; Jackson ImmunoResearch) for 24 h. After washes in PBS followed by 0.05 M Tris-HCl buffer (pH 7.6), specimens were incubated for 10 min in Tris-HCl buffer containing 0.05% DAB and for another 10 min in the same solution plus 0.01% hydrogen peroxide. After washes in deionized water, specimens were dehydrated, flattened, cleared, and mounted (29).
To test the penetration of the antibodies into tracheal whole mounts, for comparison we used cross-sections of the two rostral-most tracheal rings from pathogen-free rats and rats infected with M. pulmonis for 4 wk (four rats per group). After fixation in 4% paraformaldehyde for 24 h, tracheal rings were embedded in 9% SeaPlaque agarose (FMC BioProducts, Rockford, ME) and cut into 100- to 150-µm-thick cross-sections with a Vibratome (Technical Products International, St. Louis, MO) or into 10-µm-thick cross-sections with a cryostat. Sections were processed for immunohistochemistry as described previously. As another control, whole mounts and Vibratome sections were processed as above, except the primary antibody was replaced with 1% normal goat serum.
Morphometric Methods
Length density of Monastral blue-labeled blood vessels. As an index of the amount of angiogenesis, the length density of Monastral blue-labeled blood vessels in the same tracheal whole mounts used for quantifying ED2-positive cells was measured morphometrically (29). This approach took advantage of the finding that angiogenic blood vessels in the airway mucosa of M. pulmonis- infected rats are abnormally sensitive to the leak-producing action of substance P (16). Therefore, the number of leaky vessels, as shown by Monastral blue labeling, reflects the amount of angiogenesis. Measurements were made with a computer-generated sine-wave test grid overlaid (spacing 40 mm) on digitized color video images of whole mounts viewed with a Zeiss Axiophot microscope (projected magnification ×370) (29, 30). Length density measurements are expressed as millimeters of vessel length per square millimeter of mucosa.
Quantification of ED2-positive cells. The number of ED2-positive cells was determined in tracheal whole mounts of pathogen-free rats (n = 11) and rats infected with M. pulmonis for 1, 2, 3, or 4 wk (n = 6 rats per group). Specifically, ED2-positive cells were counted in 12 consecutive regions, each measuring 0.152 mm2 as defined by an eyepiece grid, over cartilaginous rings in each whole mount. The number of ED2-positive cells was expressed per square millimeter of mucosal surface.
Shape index of ED2-positive cells.
The shape of ED2-positive cells was expressed quantitatively using a shape
index, which is a shape-sensitive parameter (4
A/P2,
where A is the projected cell area and P is the projected
cell perimeter) that expresses the ratio of area to perimeter relative to this ratio for a circle (30). Circular cells have
a shape index of 1, whereas irregularly shaped cells have a
smaller shape index that decreases toward zero as the perimeter increases with respect to the area. Projected areas
and perimeters of 20 ED2-positive cells in the rostral portion of each trachea of pathogen-free rats and 1-, 2-, 3-, and 4-wk infected rats (n = 6 rats per group) were measured on digitized color video images of whole mounts viewed with a Zeiss Axiophot microscope (projected magnification approximately ×1,800), using a digitizing tablet
and measuring software developed in the laboratory for
this purpose (30).
Measurements of mucosal thickness and polyps. Mucosal thickness was measured on color video images of Vibratome cross-sections of tracheas from pathogen-free and M. pulmonis-infected rats (n = 4 rats per group). In addition, the diameter and projected area of 10 polypoid regions of mucosal lymphoid tissue were measured in the rostral third of each tracheal whole mount of rats infected with M. pulmonis for 4 wk (n = 6). The number of ED2-positive cells in each polyp was counted, and the density of ED2-positive cells was expressed per square millimeter of polyp. Measurements were made at a projected magnification of ×220, using the same morphometry system used to measure shape index. The area density of mucosal polyps, expressed as a percentage of mucosal surface area, was measured by point counting on color video images of tracheal whole mounts from 4 wk-infected rats (1,134 points per trachea, magnification ×92, n = 6 rats).
Serologic Antibody Titers
Serologic antibody titers to M. pulmonis, Sendai virus (parainfluenza virus type 1), and rat coronavirus/sialodacryoadenitis virus were measured by enzyme-linked immunosorbent assays (Microbiological Associates, Rockville, MD).
Statistical Analysis
Values are expressed as means ± SE (n = 6 rats per group) unless specified otherwise. The significance of differences between groups was evaluated by analysis of variance and Fisher's test or Scheffé's F test for multiple comparisons or, for values that were not normally distributed, by the Mann-Whitney test. The relationship between area density of mucosal polyps and body weight or serologic antibody titer to M. pulmonis was assessed by linear regression. Differences were considered significant when P < 0.05.
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Angiogenesis after M. pulmonis Infection
The tracheal vasculature, stained by perfusion of biotinylated L. esculentum lectin, was conspicuously different in pathogen-free and infected rats. The vasculature in pathogen-free rats had a simple, organized pattern, and most of the capillaries were short, relatively straight, and located in regions of mucosa overlying cartilaginous rings (Figure 1A). By comparison, blood vessels in infected rats were disorganized and much more abundant as a result of angiogenesis (Figure 1B). Many of the newly formed vessels were the size of capillaries but did not have the normal location or arrangement of capillaries. Tortuous, capillary-sized angiogenic blood vessels were particularly numerous in focal, polyp-like regions of mucosa (Figure 1B).
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Infection-related differences in the tracheal vasculature were even more conspicuous when mediator sensitivity was tested by injecting substance P and then using Monastral blue to label the leaky vessels. In the tracheal mucosa of pathogen-free rats, most of the labeled vessels were postcapillary venules and collecting venules located between cartilaginous rings (Figure 1C). In infected rats, labeled blood vessels were much more numerous. Most resembled tortuous capillaries with a conspicuously abnormal architecture (compare Figures 1C and 1D). The labeled vessels were widely scattered but were particularly abundant in polyp-like regions of mucosal lymphoid tissue (Figure 1D).
The association of Monastral blue labeling with angiogenesis (17, 18) was used in measuring the progression in vessel growth after M. pulmonis infection. The amount of angiogenesis, inferred from the length density of Monastral blue-labeled blood vessels (combined length of blue vessels per square millimeter of mucosa), was significant at 2 wk. The amount doubled between 2 and 3 wk, and at 3 and 4 wk the mean values were nearly 8 times the pathogen-free value (Figure 2). The value at 1 wk tended to be higher than that for pathogen-free rats, but the difference was not statistically significant.
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values
significantly different from 1- and 2-wk groups as well as pathogen-free group; analysis of variance and Fisher's test, P < 0.05.
Changes in Tracheal Mucosa after M. pulmonis Infection
The thin tracheal mucosa of pathogen-free rats (Figure 3A), which averaged 56 ± 3 µm (n = 4 rats) in thickness and contained no lymphoid tissue, was conspicuously different from the thick mucosa with abundant lymphoid tissue in M. pulmonis-infected rats (Figure 3B). At 4 wk the mucosa averaged 196 ± 17 µm (n = 4 rats) in thickness and was 3.5 times the thickness in pathogen-free rats.
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Beginning about 2 wk after infection, lymphoid tissue
accumulated in the tracheal mucosa. Initially the accumulations were small and limited to the rostral trachea, but at
3 and 4 wk they were prominent and some had the appearance of mucosal polyps. At 4 wk mucosal polyps had a
mean diameter of 200 ± 9 µm, occupied 22 ± 5% of the
mucosal surface, and gave the mucosa a cobblestone-like appearance. The number of mucosal polyps was related
to the severity of disease, as reflected by antibody titer
and body weight. In evidence of this relationship, the proportion of tracheal surface occupied by polyps at 4 wk
was directly proportional to the serologic antibody titer to
M. pulmonis (correlation coefficient = 0.94; P < 0.01) and
inversely proportional to total body weight (correlation coefficient = 
0.84; P < 0.05).
Distribution of ED2-Positive Cells before and after M. pulmonis Infection
In pathogen-free rats, ED2-positive cells were scattered in the mucosa, both over cartilaginous rings (Figure 3C) and between the rings. The number located between the rings was greater than elsewhere because these regions were thicker, consisting of mucosa, submucosa, intercartilaginous connective tissue, and adventitia, all of which contained ED2-positive cells.
At 1 wk after infection, ED2-positive cells were twice as numerous as in pathogen-free rats. Most were uniformly distributed, but some accumulated near the border of cartilaginous rings. At 2 wk, ED2-positive cells were as numerous as at 1 wk. However, at 3 and 4 wk, when mucosal polyps were numerous, ED2-positive cells had a distinctly nonuniform distribution due to the presence of discrete clusters of cells in polyps (Figure 3D). These cells were closely associated with tortuous, Monastral blue- labeled angiogenic blood vessels in the polyps. ED2-positive cells were also scattered in the tissue between the polyps and in deeper regions of the mucosa.
Number of ED2-Positive Cells before and after M. pulmonis Infection
The mean number of ED2-positive cells in the mucosa overlying cartilaginous rings of tracheas of pathogen-free rats was 261 ± 42 cells/mm2 of luminal surface (Figure 4). At 1 wk after infection, the number of ED2-positive cells in this region increased 120%. The number remained at about this level throughout the 4-wk study (Figure 4). At 4 wk there were 1,340 ± 124 ED2-positive cells/mm2 in mucosal polyps, compared with an overall density of 543 ± 30 cells/mm2 in these tracheas. The density of ED2-positive cells in polyps was five times the density in the tracheal mucosa of pathogen-free rats. No immunoreactive cells were present in tracheal specimens processed without the primary antibody.
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Shape of ED2-Positive Cells before and after M. pulmonis Infection
Most ED2-positive cells in pathogen-free rats had an irregular shape and as many as five thin, branching cytoplasmic processes (Figure 5A). Consistent with their irregular shape, the cells had a mean shape index of only 0.21 (Figure 6). The cell bodies were 10 to 25 µm in greatest dimension, and the processes ranged from 3 to 30 µm in length. ED2-immunoreactivity on the cell surface tended to be granular.
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At 1 wk after infection, ED2-positive cells still had an irregular shape, but they had shorter processes (Figure 5B) and a commensurate increase in shape index (0.34; P < 0.05 compared with the pathogen-free value). At 2 wk many of the cells lacked processes, which further increased the shape index (0.52; Figure 6). The most conspicuous changes in cell shape were evident at 3 and 4 wk after infection, when most ED2-positive cells were rounded, lacked processes (Figure 5C), and had the highest shape indices (0.70 and 0.79, respectively; Figure 5). These cells also had stronger ED2-immunoreactivity than corresponding cells in pathogen-free rats (compare Figures 5A and 5C).
Antibody Titers
Pathogen-free rats had no detectable serologic antibody titers to M. pulmonis. The infected rats had no significant titers to M. pulmonis at 1 wk after infection, but thereafter the titers increased progressively (0.16 ± 0.04 at 2 wk, 0.61 ± 0.08 at 3 wk, and 0.71 ± 0.12 at 4 wk; n = 6 rats per group). None of the rats had significant antibody titers to Sendai virus or rat coronavirus/sialodacryoadenitis virus.
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The major new finding in this study was the concentration of ED2-positive cells at sites of angiogenesis in focal, polyp-like collections of lymphoid tissue in the airways of rats with M. pulmonis infection. The mucosal polyps contained ED2-positive cells that were rounder, had stronger ED2-immunoreactivity, and were five times as densely packed as those in pathogen-free rats. The polyps also contained tortuous, capillary-sized angiogenic blood vessels that were closely associated with ED2-positive cells. The number of ED2-positive cells doubled during the first week after infection and remained at the increased level during the remainder of the 4-wk study. Angiogenesis occurred gradually during the first 2 wk, increased rapidly between the second and third weeks, and was a prominent feature after the third week. These observations raise the possibility that the influx of tissue macrophages contributes to the development of angiogenesis at sites of chronic inflammation in the airway mucosa after M. pulmonis infection.
Experimental Model
Chronic nature of M. pulmonis infection. Immediately after infection, M. pulmonis activates airway macrophages, leading to phagocytosis of organisms, cytokine secretion, and other changes associated with the initial inflammatory response (31). However, the initial immune response directed against M. pulmonis does not clear the infection, and the persistence of the organisms triggers further alterations of the immune system and the development of bronchus-associated lymphoid tissue (14, 34, 35). Chronic disease ensues, characterized by mucosal remodeling, mucous gland hyperplasia, and angiogenesis (14, 16, 17, 36). Because of its persistent natural history, M. pulmonis infection provides an opportunity to examine the cellular and molecular mechanisms involved in angiogenesis associated with chronic inflammation.
Identification of macrophages with ED2 antibody. ED2 is a well-characterized monoclonal antibody that recognizes a membrane antigen on rat tissue macrophages (27, 37). ED2-positive cells with a macrophage phenotype are abundant in the airways and other organs of pathogen-free animals (27). However, ED2 does not recognize all types of mononuclear phagocytes, as ED2-negative subsets have been identified with other monoclonal antibodies (27). For example, the monoclonal antibody ED1, which recognizes all cells of the mononuclear phagocyte system of rats, including dendritic cells, stains many more cells than ED2 (27). ED3 recognizes a subset of macrophages that is restricted to lymphoid organs and inflamed tissues (38), and OX6 recognizes dendritic cells as a population distinct from most ED2-positive macrophages (39, 40). ED2 was the focus of the present study because it has the greatest selectivity for tissue macrophages (27). A logical next step would be to examine changes in other subsets of macrophages, dendritic cells, and lymphocytes after M. pulmonis infection.
Tissue whole mounts for studying macrophage-vessel associations. Although some cellular events involved in angiogenesis are readily appreciated in histologic sections, possible relationships between macrophages and angiogenic blood vessels are difficult to investigate with tissue sections because of the complex geometry of the vasculature. In the present study, we circumvented this problem by using tracheal whole mounts in combination with ED2-immunohistochemical staining and Monastral blue labeling of angiogenic blood vessels. This approach revealed a clear colocalization of a subset of macrophages and angiogenic blood vessels. It also allowed straightforward determination of the three-dimensional shape of the macrophages, quantifying of the number and distribution of the cells, and quantifying of the amount of angiogenesis. Whole mounts do, however, have the limitation of favoring immunohistochemical staining of cells near the mucosal surface due to limited penetration of the reagents. This was not a serious problem in the present study because the cells of interest were located superficially.
Macrophages in Acute and Chronic Inflammation
M. pulmonis infection induced a rapid increase in the number of tissue macrophages. The number peaked 1 wk after infection and remained elevated for at least 4 wk. This increase in number of macrophages is similar to changes observed after Sendai virus infection, which causes a rapid influx of macrophages and other inflammatory cells (41). Macrophage recruitment after Sendai virus infection begins about Day 2, peaks on Day 5, and then returns to baseline.
Unlike Sendai virus infection, M. pulmonis infection results in chronic disease (14, 16, 17, 36). The onset of the chronic phase coincided with the accumulation of lymphoid tissue and the presence of macrophages that were more rounded, strongly immunoreactive for ED2, and concentrated at focal sites of angiogenesis. These focal collections of macrophages appeared to be ideally positioned for participating in angiogenesis, possibly through the release of angiogenic growth factors, matrix remodeling proteases, or chemotactic factors. Differences between the rounded macrophages associated with angiogenic vessels and the irregularly shaped ones in the normal mucosa are consistent with the influx or differentiation of a functionally specialized subset of macrophages during the chronic phase of M. pulmonis infection.
The heterogeneity of macrophages in M. pulmonis- infected rats is consistent with the functional diversity of macrophages in rheumatoid arthritis (42, 43). Only one of three subsets of macrophages isolated from arthritic synovial tissue in Percoll gradients induces angiogenesis in the rat cornea (42, 43). Macrophage subsets have also been identified in other models of chronic inflammation (26, 44, 45), but to our knowledge none has previously been shown to be associated with sites of angiogenesis in situ.
Angiogenesis after M. pulmonis Infection
Consistent with previous reports (17), the present study showed that most of the angiogenesis occurred after the second week of M. pulmonis infection. This period coincides with the accumulation of mucosal lymphoid tissue (14, 46). The length density of Monastral blue-labeled blood vessels at 3 and 4 wk was significantly greater than at 1 or 2 wk and was nearly eight times the value for pathogen-free rats. These length density measurements serve as a useful index of the number of newly formed blood vessels (16, 17). When exposed to substance P, the new vessels leak Monastral blue particles, which become trapped in the endothelial basement membrane and label the vessel wall (16, 47). The heavy labeling of the angiogenic blood vessels is the consequence of their unusual sensitivity to substance P due to the overexpression of NK1 receptors on endothelial cells (18).
The amount of angiogenesis in the airway mucosa is related to the severity of the M. pulmonis infection. For example, there is a close correspondence between the number of Monastral blue-labeled vessels, as indicated by area density measurements, and disease severity as reflected by titer of M. pulmonis antibodies in the blood (16). Area density measurements of the labeled vessels can increase 46-fold under conditions of severe infection (16).
The proliferation of blood vessels evident at 3 and 4 wk after infection totally changed the appearance of the tracheal microvasculature. Many new vessels were present, and most of these were tortuous, capillary-sized, and labeled with Monastral blue. Where the mucosal remodeling was most severe, the new vessels were concentrated in polypoid collections of lymphoid tissue. These vessels differed from normal tracheal capillaries, which do not have NK1 receptors and are unresponsive to substance P (18, 30, 48). In the normal tracheal vasculature, NK1 receptors, substance P responsiveness, and Monastral blue labeling are limited to postcapillary venules and collecting venules (48).
Previous studies have shown that M. pulmonis infection not only increases the total number of vessels (angiogenesis) but also increases the amount of Monastral blue labeling of individual vessels (16). The conspicuously abnormal vascular architecture indicates that most of the change at 3 and 4 wk is due to an increased number of vessels. However, the present studies confirmed that the architecture of the tracheal microvasculature is relatively normal at 1 and 2 wk after infection, even though the amount of Monastral blue labeling tends to be increased (17). Much of the increase in vessel labeling during this early period may result from increased vessel sensitivity to substance P due to increased NK1 receptor expression (18).
Macrophages and Angiogenesis
The chronic inflammation established during M. pulmonis
infection favors the continued synthesis and release of factors that stimulate angiogenesis, and macrophages are a
potential source of such factors (5, 49). Macrophages produce TNF-, which can stimulate blood vessel formation
in the rat cornea at very low doses (8, 9). Macrophages in
rheumatoid arthritic tissue express VEGF messenger RNA
and have VEGF immunoreactivity (11). IL-8, which is a
chemotactic factor for neutrophils and lymphocytes, has
angiogenic activity, and antibodies to IL-8 block the angiogenic activity of macrophages isolated from arthritic synovial tissue (10). TGF- is another potent angiogenic factor
from macrophages (7).
The unmistakable geographic relationship between macrophages and focal sites of angiogenesis in polypoid collections of mucosal lymphoid tissue is consistent with the role of macrophages in angiogenesis after M. pulmonis infection. What about the temporal relationship between the influx and shape change of macrophages and the onset of angiogenesis? After the initial influx of ED2-positive cells during the first week, the population underwent a progressive change in shape. The replacement of irregularly shaped ED-positive cells by rounded cells coincided with the burst of angiogenesis. The rounded, strongly ED2-positive cells may represent a distinct subset of macrophages responsible for sustaining vessel growth during the chronic phase of the immune response. It is unknown which of the chemical mediators that can change the shape of macrophages (50) are responsible for inducing the rounded phenotype at sites of angiogenesis.
M. pulmonis infection results in the infiltration of the airway mucosa by many types of inflammatory cells, including T and B lymphocytes, dendritic cells, and granulocytes (53). Any of these cells, as well as the organisms themselves, could contribute angiogenic stimuli. It is therefore likely that products of multiple cell types participate in the formation of new blood vessels under these conditions. The M. pulmonis model, in combination with immunohistochemistry, in situ hybridization, genetically altered mice, and other methods, should make it possible to identify these angiogenic factors and to determine the contribution of various cell types to their production.
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Conclusions |
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Abstract
Introduction
Materials and methods
Results
Discussion
Conclusions
References
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M. pulmonis infection induces the influx of ED2-positive macrophages into the airway mucosa. The influx during the first week coincides with the acute inflammatory response to the infection. This is followed by the development of chronic inflammation with mucosal remodeling and angiogenesis. A distinctive feature of the chronic phase is the clustering of round, strongly ED2-immunoreactive macrophages at foci of angiogenesis in polyp-like collections of lymphoid tissue. The distinctive shape, immunoreactivity, and distribution of these cells suggest that a particular subset of macrophages is involved in the angiogenesis triggered by M. pulmonis infection.
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Footnotes |
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Address correspondence to: Donald M. McDonald, M.D., Ph.D., Cardiovascular Research Institute, University of California, San Francisco, CA 94143-0130. E-mail: dmcd{at}itsa.ucsf.edu
(Received in original form July 7, 1997 and in revised form May 20, 1998).
Abbreviations: diaminobenzidine tetrahydrochloride, DAB; interleukin, IL; phosphate-buffered 0.9% NaCl, PBS; vascular endothelial growth factor, VEGF.Acknowledgments: This work was supported in part by NIH Grant HL-24136 and Novartis, Basel, Switzerland. One author (Å.D.) was supported in part by the Swedish Society of Medicine, Astra-Draco, Sweden; Swedish Medical Research Council; Swedish Society of Otolaryngology-Head and Neck Surgery; and University of Umeå, Sweden. The authors thank Dr. J. Russell Lindsey and Ms. Julie Erwin of the University of Alabama, Birmingham, for supplying the M. pulmonis organisms; and Dr. Peter Baluk for help with the immunohistochemistry.
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