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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A pulmonary Cryptococcus neoformans (Cne: strain 52D, ATCC24067) infection model in mice was used to examine the possible role for T cell-mediated immunity in regulating vascular adhesion molecules on lung endothelium during development of granulomatous inflammation. Resolution of pulmonary Cne infection in C.B-17 mice begins by Day 14 following intratracheal inoculation and depends on T cell-mediated recruitment of monocytes followed by their activation. C.B-17 scid/scid (SCID) mice mount a less exuberant pulmonary inflammatory response, recruit fewer monocytes into their lungs, and fail to clear the infection. Recruitment of leukocytes into infected tissue is mediated by both the interaction of adhesion molecules expressed on the surface of activated vascular endothelial cells with ligands on circulating cells, and the directed response of these leukocytes to chemotactic factors. The kinetics of expression of the endothelial cell adhesion molecules E-selectin, vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), all previously shown to regulate monocyte recruitment, were examined in the lungs of infected C.B-17 and SCID mice during pulmonary infection to determine if T cells were necessary for their upregulation. Immunohistochemical analysis showed that upregulation of E-selectin, VCAM-1, and ICAM-1 did not differ significantly between C.B-17 and SCID mice at any time during infection. Maximal expression in C.B-17 and SCID mice was noted between Days 5 and 7 for all three molecules and preceded maximal influx of leukocytes into the lung. Thus, the inability of SCID mice to recruit optimal numbers of monocytes into infected lungs was not the result of a failure to express the critical adhesion molecules early in infection, but likely reflected absence of immune dependent chemotactic factors.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Pulmonary infection with Cryptococcus neoformans (Cne) in immunocompetent humans is often subclinical and limited to the lung. However, in HIV-infected patients, as the level of CD4+ T cells decreases, the yeast is more likely to disseminate from pulmonary foci to establish extrapulmonary infections, particularly within the central nervous system (1). A murine model of pulmonary Cne infection has provided insight into important host responses that restrict yeast cell growth within the lung. Effective anticryptococcal defense in this model was shown to depend on a specific T-cell immune response mediated by both CD4+ and CD8+ T cells (4).
During a pulmonary Cne infection in immunocompetent BALB/c and C.B-17 mice, cellular recruitment into the lungs peaked in both strains during the second week of infection, which corresponded with initiation of resolution (5). T cells were present in large numbers at this time, but macrophages, neutrophils, and eosinophils were also recruited into infected lungs. BALB/c mice depleted of either CD4+ or CD8+ T cells or both, recruited fewer monocytes (7). Lovchik and colleagues (8) demonstrated that C.B-17 scid/scid (SCID) mice were incapable of resolving infection and recruited fewer cells into their lungs. It is possible that the role of T cells is to mediate recruitment by both secreting chemotactic factors and regulating the expression of vascular adhesion molecules.
Adhesion to activated endothelial cells is the initial
phase for leukocytes migrating into infected tissue and has
been described as a three-step process that includes rolling
(mediated by selectins and their carbohydrate ligands [9],
although a contribution by 
1 integrins has been demonstrated in vitro [10]) followed by attachment and migration; the latter two events are mediated by integrins and
adhesion molecules of the immunoglobulin gene family (11). In a Cne-infected lung, the major effector cells that must efficiently utilize this process are monocytes. The adhesion molecules expressed on endothelial cells, vascular
cell adhesion molecule-1 (VCAM-1; CD106) and intercellular adhesion molecule-1 (ICAM-1; CD54), have been
shown to be important for the emigration of monocytes
(12, 13). The role of E-selectin (CD62E) in monocyte migration is controversial and has been based on in vitro
models that have produced conflicting results depending
on the assay conditions (14, 15). Monocytes extravasate
and migrate through tissue to infectious foci attracted by
chemoattractants such as RANTES, macrophage inflammatory protein (MIP-1
and ), and monocyte chemotactic protein-1 (MCP-1), which are produced at sites of infection (16, 17). In particular, recent evidence has shown MCP-1 to be important in the resolution of a pulmonary
Cne infection, because mice depleted of MCP-1 were unable to recruit monocytes into infected lungs (18).
The current study characterizes the development of some of the major relevant adhesion molecules after intratracheal (IT) inoculation with Cne. C.B-17 and SCID mice were compared to determine whether the infection was sufficient to upregulate E-selectin, VCAM-1, and ICAM-1, or whether an intact immune response was necessary for the expression of these molecules. Semiquantitative assessment of the expression of these adhesion molecules during the course of infection showed that both SCID and C.B-17 mice were able to upregulate lung vascular endothelial cell adhesion molecules during infection to an equivalent degree. Thus the role for T cells in effective monocyte recruitment is likely related primarily to regulating chemoattractant production.
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mice
Severe combined immunodeficient C.B-17 scid/scid mice and their coisogenic C.B-17 (H-2d) counterparts were either purchased from Taconic (Germantown, NY) or were progeny of a University of New Mexico (UNM) breeding colony. The animals were maintained in a filtered-air environment and given sterile food and water ad libitum. Mice were infected at 8 to 12 wk of age. All procedures involving mice were carried out under aseptic conditions.
Organism
The yeast strain used in the experiments was described previously (5). Briefly, Cne strain 52D, obtained from the American Type Culture Collection (24067; ATCC, Rockville, MD) was grown for 36 to 40 h at room temperature in Sabouraud dextrose broth (1% neopeptone, 2% dextrose; Difco, Detroit, MI). Prior to inoculating mice, the organism was washed in sterile saline, counted on a hemocytometer, and diluted to approximately 105 colony forming units/ml in sterile nonpyrogenic saline (Baxter Healthcare Corporation, Deerfield, IL).
Intratracheal Inoculation
Mice were anesthetized with an intraperitoneal injection of tribromoethanol (Aldrich Chemical Company, Milwaukee, WI), and the trachea was surgically exposed. A 50-µl volume was inoculated through a bent 30-gauge needle (Becton Dickinson, Rutherford, NJ) attached via polyethylene tubing (Intramedic; Clay Adams, Parsippany, NJ) to a tuberculin syringe (Monoject, St. Louis, MO). The incision was closed, and the mice were allowed to recover. To control for the possibility that cell adhesion molecules may be upregulated after surgery, a group of mice were also inoculated with 50 µl of sterile nonpyrogenic saline (Baxter Healthcare) and followed for the duration of the experiment.
Antibodies
The following monoclonal antibodies (mAbs) were used in this study: anti-VCAM-1 (Clone M/K-2, rat IgG2a; ATCC, CRL1909), anti-ICAM-1 (Clone YN1/1.7, rat IgG2a; ATCC, CRL1878) and anti-E-selectin (Clone 9A9, rat IgG2b, a kind gift from Dr. B. Wolitzky, Hoffmann-La Roche Inc., Nutley, NJ; and Clone 10E9.6, rat IgG2a, Pharmingen, San Diego, CA). For immunohistochemistry, mAbs were purified by treating tissue-culture supernatants with ammonium sulfate and then passing the solubilized precipitate through a Protein G column (MAbTrap G II; Pharmacia, Uppsala, Sweden). Isotype-specific control rat mAbs were purchased from Zymed Laboratories (San Francisco, CA). The conjugated antibody used for the detection of mAbs for immunohistochemistry was horseradish peroxidase (HRP)-conjugated goat-antirat IgG (H&L) (Jackson Immunoresearch Laboratories, West Grove, PA).
Preparation of Tissue for Immunohistochemistry and Histology
Lungs to be frozen were excised from mice after their pulmonary cavity was opened and the trachea exposed. Prior
to excision, lungs were inflated with a solution of Optimal
Cutting Temperature (OCT) diluted 1:3 in phosphate-buffered saline (PBS), pH 7.4, and immediately placed
into 2-methylbutane (Aldrich Chemicals) that had been
cooled in a methanol/dry-ice bath. Lungs were subsequently stored at 
70°C. For histologic preparation, lungs
were excised as described previously, except they were inflated with formalin (Sigma Chemical Co., St. Louis, MO)
and stored in formalin until embedded in paraffin. The
sections were stained with hematoxylin and eosin (H&E)
in the histology laboratory at UNM Hospital. Photomicrographs were taken with an Olympus AH-2 microscope
(Tokyo, Japan).
Immunohistochemistry
To investigate the expression of cell adhesion molecules
throughout the infection, lungs that had been frozen were
embedded in OCT and 6-µm sections were cut with a cryostat (Bright Hacker, Fairfield, NJ) and mounted onto Superfrost/Plus microscope slides (Fisher Scientific, Pittsburgh, PA). Sections were allowed to dry by incubation in
a slide drier. If staining was not performed immediately,
slides were stored at 70°C in a sealed slide box. For staining, slides were brought to room temperature, fixed in acetone for 5 min, and then rehydrated for 15 min with PBS,
pH 7.4. To prevent nonspecific interactions of the antibodies, sections were treated with 20% normal goat serum in
PBS (NGS/PBS) for 30 min at room temperature in a humid chamber. Sections were then incubated with the primary antibody in NGS/PBS for 60 min at room temperature in a humid chamber. All antibodies were used at
previously determined optimal concentrations. Slides were
washed three times in PBS, the HRP-conjugated secondary antibody (goat antirat IgG [H&L]-HRP, Jackson Immunoresearch Laboratories) was added, and slides were
incubated as described previously. After washing in PBS
and then acetate buffer, slides were developed in 3-amino-9-ethylcarbozole (Aldrich Chemicals) that had been resuspended in acetate buffer. Sections were counterstained
with Gill's Hematoxylin (No. 1; Fisher Scientific).
Semiquantitative Analysis of Lung Sections for Inflammation
To assess the total inflammatory response for each lung, a low-power objective was used to scan the entire H&E-stained lung and the lung was scored depending on the percentage of the lung that contained inflammatory cells. The scoring system used was based on the percentage of lung that contained inflammatory cells and was as follows: 1, 6-20%; 2, 21-40%; 3, 41-60%; 4, 61-80%; 5, 81-100%. Lungs were also scanned to determine the relative density of infiltrating cells in the inflamed areas using a graded coding system similar to that used to assess the percentage of inflamed lung. The two values for each lung were then multiplied to obtain a possible total score of 25. For example, if 50% of the lung were involved with inflammation, but in each of these areas the cells occupied only 15% of the area, the total score would be 3 × 1 = 3. Sections from infected C.B-17 and SCID mice from four different experiments (one or two mice per time point per group, for each experiment) were blinded and read by two individuals. A score for each mouse lung was the average of both readers' scores, and the data were expressed as the mean ± SD for each mouse lung per group per time point over the four experiments.
Semiquantitative Analysis of Lung Sections for Expression of Endothelial Adhesion Molecules
A system was developed for assessing the expression of endothelial adhesion molecules on pulmonary arteries, arterioles, venules, and veins by immunohistochemistry from frozen lung sections from two mice per group per time point per experiment and is defined in Table 1. Sections were scanned to locate the area of greatest inflammation, and vascular endothelial staining was scored within the large field where inflammation was maximal. We also scored epithelial staining and staining of alveolar septi separately. Alveolar capillary staining was difficult to distinguish from alveolar epithelial staining and thus was not considered in the "endothelial" staining category. For naive and saline-inoculated lungs, four to five areas of the section were scanned and a value based on vascular endothelial staining within these areas was determined.
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Isolation of Recruited Lung Cells
Isolation of lung cells from Cne-infected lungs was performed by the method described previously by Lovchik and colleagues (8). From three experiments, three mice per group at Day 0 of infection (harvested 2 h after inoculation), nine mice per group at Day 7, and 14 C.B-17 mice and 12 SCID mice at Day 14 of infection were killed, and the lungs were enzyme-digested with collagenase A (Boehringer Mannheim, Indianapolis, IN) and deoxyribonuclease I (Sigma). After enzyme digestion, cells were obtained as a single-cell suspension. Total viable cell numbers were determined using trypan blue, and cell types were assessed by staining cytospin preparations with a Leukostat Stain Kit (Fisher Diagnostic, Pittsburgh, PA).
Statistical Analysis
Analysis of data in Figure 2 was performed using the Mann-Whitney U test. Student's t test statistical analysis was performed on the data in Figures 3 and 4 for individual time points. StatView (Abacus Concepts, Inc., Berkeley, CA) was used for all of the analyses.
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Abstract
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Materials and Methods
Results
Discussion
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Histologic Examination of Pulmonary Inflammatory Response during Cne Infection in C.B-17 and SCID Mice
Two hours after IT inoculation of Cne (Day 0), no inflammatory cells were present in the lungs of C.B-17 mice (Figure 1A). Similarly, Cne were not detected until Day 3 when clumps and discrete organisms were noted in alveolar spaces. Cellular infiltrates were still not present at that time, but first became evident at Day 5 of infection (not shown). By Day 7 in C.B-17 mice, alveolar spaces contained predominantly neutrophils, some mononuclear cells, and Cne (Figure 1C). Neutrophils surrounded peribronchial vessels and were present in adjacent alveolar spaces. Between Days 5 and 14 of infection, lymphocytes were predominantly within peribronchial areas and around vessels but were also scattered throughout the lung in alveolar spaces (Figures 1C and 1E). By Day 14 in C.B-17 mice, lymphocytes and macrophages were increased relative to neutrophils. The majority of organisms were associated with inflammatory cells, which consisted largely of aggregates of macrophages. At Day 35, lymphocytes and macrophages were the predominant infiltrating cells, with lymphocytes located prominently in areas surrounding bronchi and in granulomas consisting of both macrophages and lymphocytes within alveoli (Figure 1G). Very few organisms were present at this time, but those present were within macrophages. Large areas of normal lung surrounded the residual inflammation.
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SCID mouse lungs were not different from control lungs 2 h after inoculation (Figure 1B). Lungs from SCID mice at Days 3 and 5 of infection also showed a lack of inflammation compared with those of C.B-17 mice (data not shown). Between Days 7 and 14 of infection, Cne were seen throughout the lung, including some in foci where neutrophils and macrophages had accumulated (Figures 1D and 1F). Unlike C.B-17 mice, in which organisms were typically surrounded by cells, SCID mice often demonstrated the presence of organisms in areas of lung with few or no inflammatory cells. By Day 35 of infection, Cne were readily visible throughout the lung, often unaccompanied by any inflammatory cells (Figure 1H).
Lung histologic sections from C.B-17 and SCID mice were compared semiquantitatively for their ability to recruit leukocytes into Cne-infected lungs. H&E-stained histologic sections were assessed in a blinded fashion for the degree of inflammation and the relative density of infiltrating cells in the areas of involved lung (i.e., cellularity). Time points from Day 0 to Day 35 of infection were examined by this method to determine the extent of inflammation after the expression of immunity in C.B-17 mice and whether there was a decrease in the inflammatory response in SCID mice as the infection progressed. Analysis of the total lung involvement is shown in Figure 2, comparing the total inflammatory response and cellularity between C.B-17 and SCID mice lungs during infection. C.B-17 mice had a significantly greater response at Day 14 (P < 0.01) when compared with the response seen in lungs of SCID mice at the same time. After Day 14 of infection the inflammatory response decreased in the lungs of both strains, despite the fact that the organism was not cleared from SCID mice.
In summary, the lung inflammatory response to infection began at Day 5, reached a maximum at Day 14, and gradually decreased by Days 28 to 35 in both C.B-17 and SCID mice. There was a more pronounced inflammatory response in the C.B-17 mice at Day 14, but SCID mice also showed increased pulmonary inflammation in response to Cne infection, consisting predominantly of neutrophils and macrophages.
Enumeration of Leukocytes Recruited into the Lung during Pulmonary Cne Infection in C.B-17 and SCID Mice
Semiquantitative analysis of Cne-infected lung sections revealed that macrophages were recruited into the lungs of C.B-17 mice more effectively than those of SCID mice (Figures 1E and 1F). To obtain a more accurate assessment of the number of cells recruited into the lungs of both C.B-17 and SCID mice, cells were isolated from enzyme-digested lungs at Days 0, 7, and 14 of infection and examined for the total cell number (Figure 3A) and cell type (Figure 3B). In accordance with previous data (8), total leukocyte recruitment into the lungs of C.B-17 mice increased during the initial 14 days of infection and was significantly greater than in SCID mice at this time point (P < 0.05). Both strains recruited similar numbers of neutrophils at each time point (Figure 3B), but C.B-17 mice had significantly greater numbers of macrophages in their lungs at Day 14 of infection than SCID mice (P < 0.05). As expected, C.B-17 mice also recruited significantly greater numbers of lymphocytes than SCID mice at Days 7 and 14 of infection (P < 0.005).
Semiquantitative Analysis of Immunohistochemical Detection of E-selectin, VCAM-1, and ICAM-1 in Cne-Infected Lungs of C.B-17 and SCID Mice
Frozen lung sections from various stages of infection in both strains of mice were stained for vascular endothelial adhesion molecules, and the kinetics and density of expression of E-selectin, VCAM-1, and ICAM-1 were compared (Figures 4A, 4B and 4C). Experiments in which C.B-17 mice were inoculated with either Cne or nonpyrogenic saline showed no significant difference in the expression of adhesion molecules at 2 h after inoculation (Day 0 on figures), either between the two groups of mice or as compared with naive C.B-17 mice (Figures 4A, 4B and 4C). The kinetics of expression and density of these adhesion molecules on lung vascular endothelium of C.B-17 and SCID mice in areas of inflammation only were not significantly different from each other at any time point. Peak expression occurred 5 to 7 d after inoculation, prior to maximal influx of inflammatory cells at Day 14 of infection.
E-selectin expression increased by Day 7 of infection in both groups and then decreased (Figure 4A). When the values for infected mice were compared with those of naive mice for E-selectin, a significant difference was observed at Days 5, 7, and 14 of infection for both groups (SCID: Days 5, 7, and 14 P < 0.05; C.B-17: Day 5 P < 0.05, Days 7 and 14 P < 0.006). Compared with C.B-17 mice inoculated with saline, both SCID mice and C.B-17 mice had significantly increased levels of expression of E-selectin at Days 7 (P < 0.05) and 14 (P < 0.005). Overall, however, E-selectin staining was much less prominent than VCAM-1 and ICAM-1 in both strains (Figures 5B and 5C).
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VCAM-1 expression on vascular endothelial cells also
increased from basal levels to peak at Day 7 of infection in
both mouse strains (Figures 4B, 5E, and 5F) and then decreased to background by Day 35 of infection. VCAM-1
expression on infected SCID and C.B-17 mice was significantly increased above that seen in naive mice at Days 5, 7, 14, and 28 of infection (SCID: Day 5 P < 0.05, Days 7, 14, and 28 P < 0.006; C.B-17: Day 5 P < 0.05, Day 7 P < 0.0001, Day 14 P < 0.006, Day 28 P < 0.05). When infected SCID and C.B-17 mice were compared with saline-inoculated C.B-17 mice, a significantly elevated level of
expression of VCAM-1 was observed at Days 5 (P < 0.05),
7 (P < 0.0005), 14 (P < 0.0001), 28, and 35 (SCID: P 
0.003; C.B-17: P = 0.05) in the infected mice. However, expression of VCAM-1 on C.B-17 and SCID mice did not
differ significantly.
ICAM-1, which was constitutively expressed at very high levels on both vascular endothelial and alveolar epithelial cells in both mouse strains (Figure 5G) gradually increased as the infection progressed during the first 5 d. In C.B-17 mice, the level of expression continued to increase to maximal levels at Days 7 and 14 (Figures 4C, 5H, and 5I) and then slightly decreased. ICAM-1 expression in SCID mice was observed to plateau from Day 5 onward. Statistical comparison of C.B-17 mice with saline-inoculated C.B-17 mice showed a significant increase in the expression of ICAM-1 in infected C.B-17 mice at Days 7 (P < 0.006) and 14 (P < 0.001). SCID mice lungs were also significantly different from saline-inoculated C.B-17 mice at Days 7 and 14 (P < 0.05). When the staining score for ICAM-1 in naive mice was compared with that seen in SCID mice throughout the infection, staining in SCID mice was significantly different only at Day 14 (P < 0.05), whereas a significant difference was observed at Days 7 and 14 in C.B-17 mouse lungs (P < 0.006), even though there was no significant difference between SCID and C.B-17 mice at either time.
Expression of E-selectin, VCAM-1, and ICAM-1 on Epithelial Cells and Recruited Monocytes in Cne-Infected Lungs
E-selectin, VCAM-1, and ICAM-1 expression on epithelial cells and infiltrating leukocytes was also assessed. Interestingly, macrophages in both strains of mice at Day 7 stained faintly for E-selectin (Figures 5B and 5C) when
the mAb 9A9 was used. When a control IgG of the same
isotype was used at the same concentration as 9A9, no
staining was observed on infiltrating leukocytes. However, E-selectin staining was not observed when another anti-E-selectin mAb, 10E9.6, was used. Both mAbs gave similar vascular endothelial cell staining (not shown). A murine
macrophage cell line (RAW 264.7) stimulated with lipopolysaccharide/interferon-
(IFN-) showed very faint
cell-surface staining with the 9A9 mAb, but no staining
with 10E9.6 mAb. At Day 7 of infection, VCAM-1 mAb stained epithelial cells at infected foci in both C.B-17 and
SCID mice lungs (Figures 5E and 5F). In some areas, macrophages were also stained. Macrophages and epithelial
cells at sites of leukocyte infiltration in both C.B-17 and
SCID mice also expressed high levels of ICAM-1 (Figures
5H and 5I).
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Materials and Methods
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Discussion
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Using an in vivo murine Cne pulmonary infection model, the current studies have shown that E-selectin, VCAM-1, and ICAM-1 expression on vascular endothelial cells were upregulated in C.B-17 and SCID mice and that this expression was similar, both qualitatively and quantitatively. Expression of these adhesion molecules peaked at Days 5 to 7 of infection prior to maximal influx of infiltrating leukocytes. There was a trend for ICAM-1 expression to be lower in SCID mice than in C.B-17 mice at Days 7 and 14 of infection, but this was not significant. Only C.B-17 mice showed ICAM-1 staining that was significantly higher than that of naive mice at Day 7, whereas both strains were significantly increased on Day 14. When compared with saline-inoculated mice, both strains were significantly increased at Days 7 and 14. Overall, the results of this study suggest that the expression of E-selectin, VCAM-1, and ICAM-1 throughout the infection was independent of T cells and likely involved signals generated by Cne-lung cell interactions. However, although adhesion molecule expression did not differ significantly between the two strains, the inflammatory response was significantly lower at Day 14 in SCID mouse lungs than in C.B-17 mice, when resolution of infection is known to begin. Specifically, recruitment of monocytes into infected SCID mice lungs was suboptimal, suggesting that immune T cells were necessary, in combination with vascular expression of appropriate adhesion molecules, for efficient recruitment. The degree of inflammation in both C.B-17 and SCID mice lungs declined after Day 14 of infection and was associated with decreased staining for both E-selectin and VCAM-1. In C.B-17 mice, resolution of infection was associated with decreasing numbers of granulomas. However, in SCID mouse lungs after Day 14, although organisms did not decrease and were scattered throughout the lungs, relatively few inflammatory cells were present. Worthy of discussion is why adhesion molecule expression decreased in SCID mice if the presence of Cne, and not T-cell immunity, were a signal for their initial upregulation.
The results presented here showed that vascular endothelial adhesion molecules were upregulated in both C.B-17 and SCID mice and that T cells were not required to
upregulate their expression. The proinflammatory cytokines, interleukin (IL)-1 and tumor necrosis factor-
(TNF-), upregulate these vascular adhesion molecules (10). Human monocytes and bronchoalveolar macrophages
secrete TNF- in response to cryptococcal stimulation
(19). This observation suggests that the interaction between Cne and macrophages within the lung could provide
sufficient levels of TNF- to upregulate vascular adhesion
molecules. TNF- secretion by bronchoalveolar lavage
cells from C.B-17 mice has been detected as early as Day 3, prior to the onset of a detectable immune response during a Cne pulmonary infection (K. A. Hoag, M. F. Lipscomb, A. A. Izzo, and N. Street, unpublished observations) and thus could be responsible for the induction of
vascular adhesion molecules prior to the expression of T
cell-mediated mechanisms.
T cells play an important role in recruitment of leukocytes to inflammatory sites, and the absence of T cells may explain the inefficient recruitment of macrophages into SCID mouse infected lungs. Nevertheless, the ability of leukocytes in SCID mice to respond to chemokines was not formally tested, and it is possible that these responses were aberrant. Furthermore, it is also possible that monocytes were recruited but the decreased monocyte numbers in SCID mouse lungs were the result of decreased retention or increased apoptosis. However, we favor the concept that the absence of T cells and their secreted factors were the cause for inefficient recruitment. Several T-cell products are candidates for recruitment of monocytes. MCP-1, which is produced by lymphocytes, monocytes, endothelial cells, and smooth muscle cells, was shown to be an important mediator for the recruitment of monocytes into Cne-infected lungs (18). The evidence was that treatment of infected mice with anti-MCP-1 Ab prevented recruitment of monocytes into the lungs and prevented resolution of infection. In this study, CD4+ T-cell migration into infected lungs was also inhibited by antibody treatment. RANTES, a chemokine produced by tissue macrophages and lymphocytes, has been shown to be a chemoattractant for memory T cells and monocytes. Devergne and coworkers (20) showed that RANTES was strongly expressed in lymphoid tissues associated with granulomatous reactions in cases of tuberculosis and sarcoidosis. Therefore, the production of RANTES during pulmonary infection by activated lymphocytes and macrophages in C.B-17 mice could also be important for recruitment of monocytes into infected sites. MCP-1 and RANTES have also been shown to increase the expression of CD11b/CD18 and CD11c/CD18 on the surface of monocytes, and to enhance the binding to unstimulated and IL-1-stimulated endothelium (19). Therefore, the production of chemokines during Cne infection not only serves to attract migrating leukocytes to infected foci, but also functions to activate recruited cells and to maintain adhesion molecule expression. The absence of T cells in SCID mice would therefore contribute to a reduction in these cytokines even in the face of a continually high level of infection.
Both C.B-17 and SCID mice were initially able to upregulate the expression of vascular endothelial adhesion
molecules. If macrophages interacting with the progressively increasing numbers of Cne caused upregulation as a
result of TNF- secretion, why cell adhesion molecule expression decreased in SCID mice after Day 14 is unclear.
Furthermore, total inflammation declined despite continued infection (see Figure 2). The absence of T cells in SCID mice resulted not only in reduced numbers of macrophages, but without IFN- secreting T cells, those recruited were not activated and the organism's growth
could not be controlled. Under these circumstances, Cne
disseminated to extrapulmonary sites (5), subsequently resulting in the development of high titers of cryptococcal
polysaccharides such as glucuronoxylomannan (GXM) in
the blood (21). A possible explanation for the lack of continued recruitment is that GXM is known to bind to neutrophils and cause shedding of L-selectin, which reduces
their capacity to migrate into inflamed tissue (22), and this
could also be true for monocytes. An explanation for the
loss of adhesion molecules with progressive Cne growth in
SCID mice relates to the observation that high levels of
GXM also decrease TNF- production by monocytes in
vitro in a dose-dependent manner (23). Overproduction of
GXM could consequently downregulate TNF--induced
endothelial cell adhesion molecule expression. In summary, progressively increasing numbers of Cne in the
SCID would contribute at least two GXM-dependent
mechanisms to inhibit additional recruitment of leukocytes in infected lung tissue. Then, over time, the number
of inflammatory cells from Days 14 to 35 of infection
would gradually decrease, reflecting apoptosis, a lack of
retention within the lung, or both.
In the current study, immunohistochemical analysis revealed that E-selectin, VCAM-1, and ICAM-1 were also expressed on infiltrating inflammatory cells. E-selectin has been shown to be shed during infection, such as in cases of septic shock (24), and it is possible that E-selectin detected on the activated macrophages was bound to ligands on these macrophages. The finding that one mAb stained stimulated macrophages, but the other did not, suggests that in Cne-infected lungs, 9A9 detected an epitope from shed E-selectin, whereas 10E9.6 could not. Perhaps the latter epitope was lost or underwent configurational changes when E-selectin was shed. Alternatively, binding to the macrophage receptor might hide the relevant epitope. It is also possible that tissue macrophages express a cross- reacting epitope that is not E-selectin but is uniquely detected by 9A9 but not by 10E9.6. VCAM-1 expression on leukocytes was described by Philipp (25), who found VCAM-1 expression on inflammatory cells of the monocyte/macrophage lineage in rejected human corneal allografts. On the other hand, Kitani and associates (26) showed that soluble VCAM-1 was bound to lymphocytes in the synovial fluid of rheumatoid arthritis patients. A similar phenomenon may occur in the lungs of infected mice where soluble VCAM-1 binds to infiltrating cells. ICAM-1 expression on macrophages has been demonstrated previously (11). Epithelial cells have been shown to express VCAM-1 (27), which could be upregulated in vitro by proinflammatory cytokines; and ICAM-1, which also increases on lung epithelial cells, was reported in the lungs of asthmatic patients, although basal levels were also seen in normal subjects (28).
This study has shown that E-selectin, VCAM-1, and ICAM-1 are all upregulated on vascular endothelial cells during pulmonary Cne infection. It will be important to determine which of these adhesion molecules are important for macrophage recruitment in this model.
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Footnotes |
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Address correspondence to: Dr. Mary F. Lipscomb, University of New Mexico, School of Medicine, Department of Pathology, 915 Stanford Drive NE, Albuquerque, NM 87131-5301.
(Received in original form December 23, 1997 and in revised form March 2, 1998).
Abbreviations: Cryptococcus neoformans, Cne; glucuronoxylomannan, GXM; hematoxylin and eosin, H&E; intercellular adhesion molecule-1, ICAM-1; monoclonal antibody, mAb; monocyte chemotactic protein-1, MCP-1; tumor necrosis factor-, TNF-; vascular cell adhesion molecule-1,
VCAM-1.
Acknowledgments: This work was supported by NIH Grant AI21951. The authors thank Barbara Forrister, Kevin Hudenko, and Kenneth Olejar, for excellent technical assistance; and Mike Grady for photographic assistance. They also thank Drs. C. Richard Lyons, Julie Wilder, and Barbara Masten for many discussions and critical review of the manuscript.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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1. Patterson, T. F., and V. T. Andriole. 1989. Current concepts in cryptococcosis. Eur. J. Clin. Microbiol. Infect. Dis. 8: 457-465 [Medline].
2. Kozel, T. R. 1993. Cryptococcosis. In Fungal Infections and Immune Responses. J. W. Murphy, H. Friedman, and M. Bendinelli, editors. Plenum Press, New York. 277-302.
3.
Mitchell, T. G., and
J. R. Perfect.
1995.
Cryptococcosis in the era of AIDS
100 years after the discovery of Cryptococcus neoformans.
Clin. Micro.
Rev.
8:
515-548
[Abstract].
4. Mody, C. H., M. F. Lipscomb, N. E. Street, and G. B. Toews. 1990. Depletion of CD4+ (L3T4+) lymphocytes in vivo impairs murine host defense to Cryptococcus neoformans. J. Immunol. 144: 1472-1477 [Abstract].
5.
Huffnagle, G. B.,
J. L. Yates, and
M. F. Lipscomb.
1991.
Immunity to a pulmonary Cryptococcus neoformans infection requires both CD4+ and
CD8+ T cells.
J. Exp. Med.
173:
793-800
6.
Hill, J. O., and
A. G. Harmsen.
1991.
Intrapulmonary growth and dissemination of an avirulent strain of Cryptococcus neoformans in mice depleted
of CD4+ or CD8+ T cells.
J. Exp. Med.
173:
755-758
7. Huffnagle, G. B., M. F. Lipscomb, J. A. Lovchik, K. A. Hoag, and N. E. Street. 1994. The role of CD4+ and CD8+ T cells in the protective inflammatory response to a pulmonary cryptococcal infection. J. Leuk. Biol. 55: 35-42 [Abstract].
8. Lovchik, J. A., C. R. Lyons, and M. F. Lipscomb. 1995. A role for gamma interferon-induced nitric oxide in pulmonary clearance of Cryptococcus neoformans. Am. J. Respir. Cell Mol. Biol. 13: 116-124 [Abstract].
9. Bevilacqua, M. P., and R. M. Nelson. 1993. Selectins. J. Clin. Invest. 91: 379-387 .
10.
Luscinskas, F. W.,
H. Ding, and
A. H. Lichtman.
1995.
P-selectin and vascular cell adhesion molecule 1 mediate rolling and arrest, respectively, of
CD4+ T lymphocytes on tumor necrosis factor -activated vascular endothelium under flow.
J. Exp. Med.
181:
1179-1186
11. Springer, T. A.. 1994. Traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm. Cell 76: 301-314 [Medline].
12. Meerschaert, J., and M. B. Furie. 1994. Monocytes use either CD11/CD18 or VLA-4 to migrate across human endothelium in vitro. J. Immunol. 152: 1915-1926 [Abstract].
13.
Issekutz, T. B..
1995.
In vivo blood monocyte migration to acute inflammatory reactions, IL-1, TNF-, IFN-, and C5a utilize LFA-1, Mac-1, and
VLA-4: the relative importance of each integrin.
J. Immunol
154:
6533-6540
[Abstract].
14. Leeuwenberg, J. F. M., T. M. A. A. Jeunhomme, and W. A. Buurman. 1992. Role of ELAM-1 in adhesion of monocytes to activated human endothelial cells. Scand. J. Immunol. 35: 335-341 [Medline].
15.
Luscinskas, F. W.,
H. Ding,
P. Tan,
D. Cumming,
T. F. Tedder, and
M. E. Gerritsen.
1996.
L- and P-selectin, but not CD49d (VLA-4) integrins, mediated monocyte initial attachment to TNF--activated vascular endothelium under flow in vitro.
J. Immunol.
156:
326-335
.
16.
Lukacs, N. W.,
R. M. Strieter,
V. M. Elner,
H. L. Evanoff,
M. Burdick, and
S. L. Kunkel.
1994.
Intercellular adhesion molecule-1 mediates expression
of monocyte derived MIP-1 during monocyte-endothelial cell interaction.
Blood
83:
1174-1178
17. Furie, M. B., and G. J. Randolph. 1995. Chemokines and tissue injury. Am. J. Pathol. 146: 1287-1301 [Abstract].
18. Huffnagle, G. B., R. M. Strieter, T. J. Standiford, R. A. McDonald, M. D. Burdick, S. L. Kunkel, and G. B. Toews. 1995. The role of monocyte chemotactic protein-1 (MCP-1) in the recruitment of monocytes and CD4+ T cells during a pulmonary Cryptococcus neoformans infection. J. Immunol. 155: 4790-4797 [Abstract].
19.
Vaddi, K., and
R. C. Newton.
1994.
Regulation of monocyte integrin expression by -family chemokines.
J. Immunol.
153:
4721-4732
[Abstract].
20.
Devergne, O.,
A. Marfaing-Koka,
T. T. Schall,
M.-B. Leger-Ravet,
M. Sadick,
M. Peuchmaur,
M.-C. Crevon,
T. Kim,
P. Galanaud, and
D. Emilie.
1994.
Production of the RANTES chemokine in delayed-type hypersensitivity reactions: involvement of macrophages and endothelial cells.
J.
Exp. Med.
179:
1689-1694
21. Diamond, R. D., and J. E. Bennet. 1974. Prognostic factors in cryptococcal meningitis: a study of 111 cases. Ann. Intern. Med. 80: 176-181 .
22. Dong, Z. M., and J. W. Murphy. 1996. Cryptococcal polysaccharides induce L-selectin shedding and tumor necrosis factor receptor loss from the surface of human neutrophils. J. Clin. Invest. 87: 689-698 .
23.
Vecchiarelli, A.,
C. Retini,
D. Pietrella,
C. Monari,
C. Tascini,
T. Beccari, and
T. R. Kozel.
1995.
Downregulation by cryptococcal polysaccharide of
tumor necrosis factor alpha and interleukin-1 secretion from human monocytes.
Infect. Immun.
63:
2919-2923
[Abstract].
24. Newman, W., L. D. Deall, C. W. Carson, G. G. Hunder, N. Graben, Z. I. Randhawa, T. V. Gopal, J. Wiener-Kronish, and M. A. Matthay. 1993. Soluble E-selectin is found in supernatants of activated endothelial cells and is elevated in the serum of patients with septic shock. J. Immunol. 150: 644-654 [Abstract].
25. Philipp, W.. 1994. Leukocyte adhesion molecules in rejected corneal allografts. Graefes Arch. Clin. Exp. Ophthalmol. 232: 87-95 [Medline].
26. Kitani, A., N. Nakashima, T. Matsuda, B. Xu, S. Yu, T. Nakamura, and T. Matsuyama. 1996. T cells bound by vascular cell adhesion molecule-1/ CD106 in synovial fluid in rheumatoid arthritis: inhibitory role of soluble vascular cell adhesion molecule-1 in T cell activation. J. Immunol. 156: 2300-2308 [Abstract].
27. Wuthrich, R. P., T. A. Jenkins, and T. L. Snyder. 1993. Regulation of cytokine-stimulated vascular cell adhesion molecule-1 expression in renal tubular epithelial cells. Transplantation 55: 172-177 [Medline].
28. Montefort, S., W. R. Roche, P. H. Howarth, C. Djukanovic, C. Gratziou, M. Carroll, L. Smith, K. M. Britten, D. Haskard, T. H. Lee, and S. T. Holgate. 1992. Intercellular adhesion molecule-1 (ICAM-1) and endothelial leucocyte adhesion molecule-1 (ELAM-1) expression in the bronchial mucosa of normal and asthmatic subjects. Eur. Respir. J. 5: 815-823 [Abstract].
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