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Abstract |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Pleural mononuclear phagocytes (PleMP) were isolated from normal rats by pleural lavage and compared with autologous peritoneal (PerMP) and bronchoalveolar mononuclear phagocytes (BAMP) isolated by peritoneal and bronchoalveolar lavage, respectively. The phagocytic activity of PleMP, PerMP, and BAMP, evaluated by testing their ability to ingest latex beads, was lower for PleMP and PerMP than for BAMP. The phenotype of PleMP, PerMP, and BAMP was characterized by immunocytochemical staining with a panel of monoclonal antibodies (mAbs). As expected, PleMP, PerMP, and BAMP did not react with OX19, OX33, ED5, MOM/3F12/F2, and anticytokeratin mAbs, specific for T lymphocytes, B lymphocytes, dendritic cells, granulocytes, and epithelial/mesothelial cells, respectively. Moreover, PleMP and PerMP populations were highly enriched with OX6-, OX42-, ED7-, and ED8-positive MP, whereas BAMP population was enriched with ED1- and ED9-positive cells. To test the ability of PleMP, PerMP, and BAMP to function as accessory cells (AC), mitomycin C-treated MP were used as stimulatory cells in mixed leukocyte reaction experiments, using allogeneic T cells as responders. 3HdTR incorporation by T cells was assessed as an index of AC function. PleMP and PerMP were more potent AC than BAMP. Moreover, when cultured together with autologous pulmonary interstitial dendritic cells, PleMP and PerMP exerted a more potent ability to stimulate T-cell proliferation than did BAMP. To investigate the capacity of MP to function as bactericidal and fungicidal cells, we tested their ability to kill Escherichia coli and Cryptococcus neoformans, respectively. PleMP and PerMP were less potent bactericidal and fungicidal cells than BAMP. The results of this study demonstrate that PleMP isolated from normal rat pleural space are functionally and phenotypically different from BAMP but similar to PerMP, and suggest that these cells might play an important role in cell-mediated immune reactions in the pleural space.
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Introduction |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Mononuclear phagocytes (MP) originate in bone marrow from stem cells. The monoblast and promonocyte give rise to monocytes, which remain in bone marrow very briefly and then enter blood circulation (1, 2). Monocytes ultimately migrate into the tissues, where maturation and differentiation of macrophages begin in response to environmental stimuli. Despite their probable origin from a common bone marrow progenitor population (3), macrophages display considerable heterogeneity (4). This diversity is expressed by differences in morphology, biochemistry, secretory products, function, and surface phenotype (5). These characteristics determine the overall phenotypic appearance of macrophages, which differs depending on their location in different organs. The expression of several selected rat macrophage markers, identified with monoclonal antibodies (mAbs), confirms the diversity of these cells in different tissues (9).
Although the phenotype and functional features of peripheral blood, bronchoalveolar, and peritoneal MP have been investigated (5), little is known about the phenotype and the cell biology of pleural MP (7). Several roles could be postulated for these cells. Pleural MP are likely to be importantly involved as effector cells in local defense against tumors, microbes, and environmental pollutants. Second, pleural MP might produce cytokines that are involved in the generation of inflammatory responses. Finally, these cells might function as accessory cells for immune responses in the pleural space.
The aims of the present study were (1) to develop a methodology of pleural lavage in rats and recover a virtually pure population of pleural mononuclear phagocytes (PleMP), and (2) to determine the phenotype and functional capabilities of PleMP in comparison with bronchoalveolar mononuclear phagocytes (BAMP) and with peritoneal mononuclear phagocytes (PerMP).
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Materials and Methods |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Animals
Male Brown Norway and Wistar rats (250 g) were purchased from Charles River Laboratories (Calco, Italy) and maintained under routine laboratory conditions.
Isolation of PleMP, PerMP, and BAMP
Pleural, peritoneal, and bronchoalveolar lavages (BAL) were performed using Brown Norway rats to isolate enriched MP populations from these three compartments. Briefly, rats were anesthetized by intramuscular injection of xilazine (10 mg/kg) and ketamine (90 mg/kg) and the peritoneal cavity was lavaged 10 times by slowly instilling and withdrawing 8 ml of warm (37°C) washing buffer (WB) (phosphate-buffered saline + 10% fetal calf serum [FCS] + 100 ng/ml penicillin + 100 ng/ml streptomycin + 10 U/ml heparin + 20 U/ml DNase) (all from GIBCO, Paisley, UK). For pleural lavage, the abdomen was opened and the pleural cavity was lavaged through the diaphragm using the same technique described for peritoneal lavage. Finally, lungs and trachea were excised en masse and lavaged 10 times by slowly instilling and withdrawing 5 ml of warm (37°C) Ca2+/Mg2+-free Hanks' balanced salt solution (HBSS) (GIBCO).
Peritoneal and pleural lavage fluids were centrifuged (300 × g, 10 min) in 50-ml tubes (Sterilin, Teddington, UK), and the recovered cells were washed three times in HBSS. Peritoneal and pleural MP were obtained by centrifugation on a discontinuous (55% and 30%) Percoll gradient (Sigma Chemical Co., St. Louis, MO) using 15-ml conical tubes (Sterilin); Percoll 55% and 30% (P = 1.070 g/ml and P = 1.039 g/ml, respectively) solutions were made by diluting with WB and assumed as 100% Percoll solution made with 95 parts of Percoll and 5 parts of WB (12). Briefly, cells were resuspended in 5 ml of 55% Percoll solution and put on the bottom of the tube; 3 ml of 30% Percoll solution were gently layered on the top of the cell suspension and tubes were centrifuged (360 × g) for 30 min. The low-density fractions were recovered from the interface of the gradients, washed three times in HBSS, and resuspended in complete medium (CM) (RPMI 1640 + 10% FCS + 25 mM N-2-hydroxyethylpiperazine-N'-ethane sulfonic acid + 2 mM glutamine + 100 ng/ml penicillin + 100 ng/ml streptomycin) (all from GIBCO). These cells were allowed to adhere to plastic dishes (Sterilin) at 37°C, 5% CO2. After overnight incubation, plates were rinsed five times with HBSS and adherent cells were collected by gently scraping the plates with a rubber policeman. Pleural and peritoneal low-density-adherent cell fractions contained 100% mononuclear cells. These cells will hereafter be referred to as PleMP and PerMP, respectively.
BAL fluid was centrifuged (300 × g, 10 min), and cells were recovered from the bottom of the tubes, washed three times in HBSS, and resuspended in CM. Although > 90% of these cells were MP, they underwent the same purification procedures described for PleMP and PerMP populations (i.e., centrifugation on Percoll gradient followed by adherence). The recovered cells were washed three times in HBSS and resuspended in CM. These cells will hereafter be referred to as BAMP.
PleMP, PerMP, and BAMP were composed of > 95% MP, as confirmed by nonspecific esterase staining.
Characterization of PleMP, PerMP, and BAMP with mAbs
The following mAbs were used in this study: OX-6 specific
for Ia antigen, expressed on dendritic cells, B lymphocytes,
and some MP; OX19 specific for a determinant expressed
on all thymocytes and peripheral T cells, expressed on
T lymphocytes; OX33 specific for a high-molecular-weight
band of the leukocyte common antigen, expressed on B lymphocytes; anticytokeratin specific for cytokeratin 13, expressed on epithelial and mesothelial cells; MOM/3F12/F2 of unknown specificity, expressed on granulocytes; OX-42
specific for complement receptor type 3, expressed on MP,
granulocytes, and dendritic cells; ED7 and ED8, both specific for 
2-integrin subfamily, expressed on MP, granulocytes, and dendritic cells; ED1 and ED9 of unknown specificity, expressed on "resident" MP; and ED5 of unknown
specificity, expressed on dendritic cells (all from Serotec,
Oxford, UK).
Cytocentrifuge preparations of PleMP, PerMP, and BAMP were made, fixed in cold acetone (5 min), and allowed to air-dry for 2 h. Immunocytochemical stainings were made by using the alkaline phosphatase antialkaline phosphatase (APAAP) method, as previously described in detail (13). The alkaline phosphatase reaction product was visualized by using as substrate a Naphtol AS-BI-fast red (Sigma) solution. For each specimen, a minimum of 300 cells per slide were counted with a ×40 objective. Irrelevant mAbs of the same isotype were used as controls.
Latex Bead Ingestion by PleMP, PerMP, and BAMP
PleMP, PerMP, and BAMP (10 × 106 cells/ml) were resuspended in HBSS + 20% FCS with 25 ml of a 1% latex bead suspension (diameter 1.09 mm; Sigma). The cells (5 × 106) were incubated (37°C, 5% CO2) for 1, 2, and 24 h, respectively. At the end of incubation, cells were washed three times in HBSS (200 g, 10 min) to remove the noningested latex particles. Cytocentrifuge preparations were made and stained with Diff-Quik (Merz-Dade, Dudingen, Switzerland). For each specimen, a minimum of 300 cells per slide were counted with a ×40 objective. Data are expressed as percentages of phagocytic cells.
Mixed Leukocyte Reaction (MLR)
We first isolated peripheral blood T cells to be used as responders in MLR experiments. Wistar rats were anesthesized by intraperitoneal injection of urethane (750 mg). The chest was opened, and blood was drawn into sterile syringes containing heparin sodium (1,000 U/ml) via cardiac puncture. Blood was diluted 1:6 with 0.9% saline and centrifuged on a Ficoll-Hypaque density gradient (400 × g, 25 min). Mononuclear cells were recovered from the interface of the gradient, washed three times in HBSS, resuspended in CM, and counted. Lymphocyte-enriched fractions were obtained from mononuclear cells by fractionation on a one-step Percoll (Pharmacia) gradient centrifugation, as previously described (14). To purify the T-cell population, lymphocyte-enriched fractions were resuspended in CM and passed twice through nylon wool columns (1 h, 37°C, 5% CO2) (14). The recovered T-cell fraction was > 95% OX19-positive.
PleMP, PerMP, and BAMP, were incubated (37°C, 5% CO2) with mitomycin C (25 mg/ml, Sigma). After 20 min, cells were washed three times in CM and resuspended at a concentration of 5 × 105 cells/ml. Purified T lymphocytes (5 × 105/well) were cultured alone or with various numbers of PleMP, PerMP, and BAMP. Cultures were performed in quadruplicate in round-bottomed microtiter plates (Sterilin) in 0.2 ml of CM.
In additional MLR experiments, PleMP, PerMP, and BAMP (5,000/well) were incubated with allogeneic T lymphocytes (100,000/well) in the presence and in the absence of autologous interstitial dendritic cells (DC) (1,000/well) isolated as previously described (12). Cultures were performed in triplicate in round-bottomed microtiter plates (Sterilin) in 0.2 ml of CM.
Cultures were incubated for 6 d (37°C, 5% CO2) and, 18 h before termination, 0.5 mCi/well of [3H]thymidine (3HTdR, 5.0 Ci/mM; Amersham Corp., Bucks, UK) were added to the cultures. Cells were collected with an automated cell harvester (Flow Laboratories, Oslo, Norway) and counted in a scintillation counter (LS 1801; Beckman Instruments, Irvine, CA). Data are expressed as mean counts per minute of quadruplicate and triplicate cultures, respectively.
Ingestion and Killing of E. coli by PleMP, PerMP, and BAMP
E. coli was kindly provided by Dr. M. Menozzi (Ospedale
V. Cervello, Palermo, Italy). Bacteria were stored at
20°C in tryptose agar (Sclavo, Siena, Italy). In preparation for experiments, E. coli were cultured in tryptose agar
for 24 h (37°C), washed twice with HBSS, and resuspended in HBSS at the concentration of 2.5 × 108/ml for
inoculation.
Bactericidal activity was determined following the methodology described by Esposito and colleagues (15) with minor modifications. Briefly, PleMP, PerMP, and BAMP were resuspended in HBSS at the concentration of 3 × 106/ml. Eight hundred microliters of cell suspension and 100 µl of FCS were plated in 35-mm petri dishes (Sterilin) and incubated 5 min at 37°C, 5% CO2. At the end of incubation, 100 µl of E. coli suspension (see previous description) were added to each culture and plates were incubated (37°C) for 1, 2, and 24 h, respectively. At the end of incubation, supernatants were removed and plates were washed three times with HBSS to remove extracellular organisms. Seventy-five percent decreased proportions of MP and bacteria were plated in eight-well chamber slides (Sterilin), and the same steps were followed as for petri dishes.
Phagocytosis was quantitated by reading the number of MP containing bacteria and the number of organisms within each positive cell directly from Diff-Quik-stained chamber slides. Two hundred consecutive cells were evaluated under ×100 oil immersion magnification. Data were recorded as the percentage of MP with bacteria and as the mean number of bacteria per MP containing organisms. Results are expressed as phagocytic index (PI) by the following formula:
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Bacterial killing was assessed by determining the number of viable intracellular bacteria. Briefly, 1 ml of cold, sterile water was added to the dishes and cells were disrupted with a rubber policeman and by a 10-min incubation on ice. At the end of incubation, 1 ml of 2× HBSS was added to each plate and the number of viable bacteria was determined by plating undiluted as well as serial 10-fold dilutions on blood agar plates (Sclavo), which were incubated (37°C) for 18 h. The number of colony-forming units (CFU) was then quantitated, and the data were recorded as the mean value; results are expressed as CFU/ml of cell lysate by the following formula:
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Ingestion and Killing of Cryptococcus neoformans by PleMP, PerMP, and BAMP
C. neoformans H99/C3D was kindly provided by Drs. G. Huffnagle and G. H. Chen (University of Michigan, Ann Arbor, MI). This clone, originally isolated from human cerebrospinal fluid, was chosen because in in vitro systems it does not increase capsule size in response to physiologic concentrations of CO2 (16). H99/C3D was stored on Sabouraud's slants (Difco, Detroit, MI) and passaged to fresh slants monthly. H99/C3D was prepared for inoculation by resuspending a slant in Sabouraud's broth (Difco) and incubating for 24 h at 35°C. C. neoformans H99/C3D was then prepared for inoculation by washing three times in complete medium and resuspending at the concentration of 104/ml in medium. CFU were verified by making serial 10-fold dilutions and plating on Sabouraud's agar.
PleMP, PerMP, and BAMP were resuspended in complete medium and plated in flat-bottomed microtiter plates (Sterilin). Cultures were infected with C. neoformans by adding to each well 50 µl of fungal suspension (see previous discussion), and incubated for various lengths of time. Macrophage cultures were lysed with 0.1% Triton X-100 (Bio-Rad, Richmond, CA) and agitated to resuspend the cryptococcus. Serial dilutions were placed on Sabouraud's agar for quantitative determination. All conditions were conducted in triplicate. Preliminary experiments verified that 0.1% Triton X-100 had no effect on the viability of C. neoformans.
CFU were determined in triplicate for each experiment and are expressed as mean CFU/ml of culture medium ± SD as previously described (17). In detail, the percent change in cryptococcal growth compared with control was calculated as
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where MP denotes CFU determined in wells containing
MP, whereas No MP indicates CFU in wells without MP;
To indicates measurement of CFU at initial inoculation
and Tx indicates measurement of CFU at various intervals
after inoculation. Therefore, a value of 0% change indicates that there was no difference in cryptococcal growth
compared with No MP, and 100% represents complete
stasis of cryptococcal growth.
Statistics
Data are expressed as mean counts ± SD; comparisons were made by using Student's t test for paired data.
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Results |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Cellular Composition of Lavages
Cellular composition of pleural and peritoneal lavages and BAL was evaluated by performing Diff-Quik and nonspecific esterase stainings. As expected, BAL contained a virtually pure population of MP. In contrast, both pleural and peritoneal lavages were composed by the majority of MP as well as by low percentages of lymphocytes, granulocytes, and mesothelial cells (Table 1).
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Isolation and Phenotypic Characterization of PleMP, PerMP, and BAMP
PleMP, PerMP, and BAMP were phenotypically characterized by stainings with mAbs. They did not react with OX19, OX33, ED5, MOM/3F12/F2, and anticytokeratin mAbs, expressed on T lymphocytes, B lymphocytes, dendritic cells, mesothelial cells, and granulocytes, respectively; they were > 95% nonspecific esterase positive and appeared at Diff-Quik stainings as macrophagelike cells, confirming that PleMP, PerMP, and BAMP populations were almost entirely composed of MP.
A panel of mAbs specific for MP was used to characterize the phenotype of PleMP, PerMP, and BAMP. Nearly 80% of PleMP and PerMP stained positively with anti-Ia mAb, whereas, as expected, only a minority of BAMP reacted with the anti-OX6 mAb specific for Ia antigen. Additionally, both PleMP and PerMP populations were enriched by OX42-positive, ED7-positive, and ED8-positive cells, whereas BAMP population was highly enriched by ED9-positive MP. Moreover, all three MP populations were enriched by ED1-positive cells (Table 2).
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Phagocytic Capacity of PleMP, PerMP, and BAMP
We compared the phagocytic ability of MP isolated from the three compartments. PleMP, PerMP, and BAMP were incubated with latex particles, and the percentages of latex-positive cells were evaluated at different time points. Approximately 80% of BAMP were latex positive after 1 h, whereas only 50% of PleMP and PerMP had ingested latex beads after the same incubation period. The percentages of latex-positive cells after 2 h were 82% for BAMP and 50% and 57% for PleMP and PerMP, respectively. In contrast, after 24 h incubation, nearly 90% of PleMP, PerMP, and BAMP had ingested latex particles (Figure 1).
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Accessory Activity of PleMP, PerMP, and BAMP
The finding that, similarly to PerMP but differently from BAMP, the majority of PleMP express Ia antigen prompted us to evaluate comparatively their accessory ability (Figure 2). Accordingly, although BAMP exerted a weak accessory activity, PleMP and PerMP were potent accessory cells. No difference was detected between PleMP and PerMP. Moreover, because previous studies (12) have demonstrated that interstitial pulmonary MP, but not BAMP, are able to improve DC ability to stimulate T-lymphocyte proliferation, we sought to compare the ability of the three MP populations to augment the immunostimulatory activity of DC isolated from rat pulmonary interstitium. As expected, pulmonary interstitial DC were potent accessory cells and their accessory function was enhanced by the three MP populations. Interestingly, the ability of PleMP and PerMP to enhance the accessory activity of DC was significantly higher than the corresponding ability of BAMP (Figure 3).
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Bactericidal and Fungicidal Activity of PleMP, PerMP, and BAMP
Because MP can kill phagocyted microorganisms, we tested the ability of PleMP, PerMP, and BAMP to kill bacteria and fungi. The three MP populations were incubated with E. coli and C. neoformans, respectively, and bactericidal and fungicidal activities were evaluated as described (see MATERIALS AND METHODS for details) at different time points.
At all time points, PleMP exerted similar killing activity against E. coli to that of PerMP. At 1 and 2 h, the bactericidal activity of both PleMP and PerMP was lower than the bactericidal activity of BAMP; these differences were statistically significant at 1 h, but not at 2 h. In contrast, after 24 h no difference was detected among the three MP populations (Figure 4).
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As regards fungicidal function, PleMP and PerMP exerted negligible killing activity against C. neoformans at all time points. In marked contrast, BAMP were efficient fungicidal cells after 2 and 24 h (Figure 5).
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Discussion |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The aim of our study was to characterize MP isolated from the pleural space both from a phenotypic and functional point of view. We chose to compare them with PerMP and with BAMP because these MP are present in a "closed" compartment anatomically similar to the pleural space and in an open district of the respiratory apparatus continuously exposed to the outside environment, respectively. In previous studies by our group on human PleMP, these cells were isolated from effusions; because the development of pleural effusions is a pathologic event, a functional and phenotypic characterization of normal PleMP was missing (18). In the present study, we used an animal model to characterize PleMP in the absence of ongoing inflammatory processes.
Although the methodology for isolation of PerMP and BAMP had been described previously (12), we developed a novel methodology to isolate a pure PleMP population. The first step of the latter methodology was pleural lavage. Interestingly, the cell recoveries of pleural and peritoneal lavages were similar to each other, with a majority of MP and lower percentages of lymphocytes, granulocytes, and mesothelial cells. Cells recovered from pleural and peritoneal lavages and BAL were further purified by a discontinuous Percoll gradient followed by adherence.
We first comparatively tested the expression of some
MP related markers on PleMP, PerMP, and BAMP. Interestingly, PleMP and PerMP expressed similar markers,
whereas the phenotype of BAMP was different (Table 2).
OX6 is reactive with a constant region determinant of major histocompatibility class Ia antigen (20) and is essential
for antigen-presenting function. ED7, ED8, and OX42 are
adhesion molecules belonging to the 2-integrin subfamily (CD11b/CD18) and play a crucial role in cell-mediated immune reactions (CMIR) by mediating cell-cell and cell-
matrix interactions. The finding that high percentages of
PleMP and PerMP, but not of BAMP, express these molecules (Table 2) supports the hypothesis that PleMP and
PerMP play a different role in immunologic defense against noxae than does BAMP. Moreover, because ED7, ED8,
and OX42 markers are expressed in peripheral blood
monocytes (21), our findings suggest that in pleural and
peritoneal cavities PleMP and PerMP undergo a turnover
more rapidly than BAMP as a result of the involvement of
these three molecules in mediating transmigration through
the endothelium. In this regard, it has been demonstrated
that, during inflammation, the accumulation of MP in the
pleural space is mainly related to an influx of monocytes
from the blood circulation, and that this phenomenon is
related to the release of chemotactic mediators by pleural
structural cells and not by inflammatory cells (22, 23). Alternatively, ED7, ED8, and OX42 molecules might mediate the adhesion of MP to the mesothelium, promoting the
presence of a pool of "marginated MP" to guarantee a local self-maintaining turnover of MP in the serous cavities.
In this regard, it has been demonstrated that, under normal conditions, the PerMP population is maintained for at
least 49 days without replacement by recruited cells from
the blood (24), which supports the concept of self-maintenance of the resident macrophage population in the peritoneal cavity.
The finding that PleMP and PerMP express surface molecules in a similar fashion supports the hypothesis that a pool of marginated macrophages might also be present in the pleural compartment and that the mobilization of these "marginated cells" might contribute, at least in part, to the physiologic "self-maintaining turnover" of pleural MP. The ED1 mAb recognizes an antigen distributed in almost all MP (21). Our data confirm that high percentages of ED1-positive cells are present in BAMP and PerMP populations (21, 25), and demonstrate that PleMP express this molecule in a similar manner. In vivo, all monocytes express the ED9 antigen, whereas some mature MP lose the expression of this marker during differentiation processes. The differential loss of this antigen in vivo might be due to a local deficiency in microenvironmental stimuli (21). Our findings that both PleMP and PerMP similarly express the ED9 marker in low percentages, whereas 75% of BAMP are ED9 positive cells, confirm the concept that PleMP and PerMP phenotypically resemble each other but differ from BAMP.
The described differences among the three MP populations in the expression of OX6, OX42, ED7, and ED8 molecules, importantly involved in CMIR, may partly explain our findings that PleMP as well as PerMP exert a potent accessory activity for T lymphocytes (Figure 3), whereas BAMP are weaker AC (26). Moreover, although lymphocytes (Table 1) and DC (27) are physiologically present in both pleural and peritoneal compartments, they are not significantly present in the alveoli. In this context, our findings that PleMP and PerMP, but not BAMP, are strong stimulators of interstitial DC accessory function (Figure 3) again highlights the functional differences among MP from different sites. Because in previous studies it has been demonstrated that pulmonary interstitial MP increase the accessory function of DC by releasing cytokines (interleukin-1, granulocyte macrophage (GM)-CSF) (12), it is conceivable that PleMP and PerMP might produce a different pattern of cytokines than BAMP. Further studies aimed to identify the pattern of soluble factors produced by normal PleMP are necessary to validate this hypothesis. These findings strongly suggest that the essential "tools" requested to initiate a CMIR are physiologically present in the pleural and peritoneal spaces but not in the alveoli. Moreover, the finding that BAMP is virtually the only cell type present in the normal lower respiratory tract suggests that the function of BAMP, continuously exposed to the outside environment, is to maintain the integrity of the lower respiratory tract surface by eliminating noxae by phagocytizing and killing them and not by activating a CMIR, which, generating the activation of inflammatory and immunocompetent cells and the production of inflammatory mediators, might result in damage for the host. Our findings that BAMP exert stronger phagocytic as well as antibactericidal and antifungicidal activities than PleMP and PerMP (Figures 1, 4, and 5), strongly support this hypothesis.
In this regard, recent observations have demonstrated
that cells that normally are as quiescent as BAMP can be
activated by incubation with GM-CSF and IFN-
to increase their antimicrobial activity. This augments the cells'
ability to phagocytize and kill pathogens but not by increasing their ability to stimulate the development of
CMIR (12, 18, 30). On the other hand, the phagocytic activity of PerMP is not affected by incubation with GM-CSF (31). Moreover, in response to the same stimuli, PerMP metabolize arachidonic acid predominantly to cyclooxygenase products, whereas BAMP produce predominantly lipoxygenase metabolites (32). These findings demonstrate that although PerMP and BAMP differentiate
from the same precursor, they acquire the ability to respond differently to the same stimuli. Taken together, our results and these observations strongly support the hypothesis that PleMP, which resembles PerMP, responds to
a stimulus in the same fashion as PerMP, and that the
pleural space, though belonging to the respiratory apparatus, can be considered a "closed" compartment not only
from an anatomic point of view but also from an immunologic point of view. It is conceivable that, in this scenario,
the PleMP is one of the most important immunocompetent cells in the pleural space, one that maintains the integrity of the pleural space mainly by initiating and regulating the development of a local compartmentalized CMIR
rather than by exerting phagocytic activity.
In conclusion, our study shows a phenotypic and functional characterization of PleMP and a comparison of these cells with BAMP and PerMP. To our knowledge, this is the first report providing a characterization of PleMP in physiologic conditions.
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Footnotes |
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Abbreviations: alkaline phosphatase antialkaline phosphatase, APAAP; bronchoalveolar lavage, BAL; bronchoalveolar mononuclear phagocytes, BAMP; cell-mediated immune reaction, CMIR; colony-forming unit, CFU; complete medium, CM; dendritic cells, DC; fetal calf serum, FCS; Hanks' balanced salt solution, HBSS; mixed leukocyte reaction, MLR; monoclonal antibody, mAb; mononuclear phagocytes, MP; peritoneal mononuclear phagocytes, PerMP; pleural mononuclear phagocytes, PleMP; washing buffer, WB.
(Received in original form October 28, 1997 and in revised form April 21, 1998).
Acknowledgments: This work was supported by the Italian National Research Council. The writers thank Dr. M. Menozzi for providing E. coli and for helpful suggestions on bactericidal assays, Drs. G. Haffnagle and G. H. Chen for providing C. neoformans and for helpful suggestions on fungicidal assays, and Dr. F. Di Pietra for veterinary assistance.
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References |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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