Journal of Leukocyte Biology Myeloid cells, immune suppression, tumor immunology
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(Journal of Leukocyte Biology. 2001;70:39-45.)
© 2001 by Society for Leukocyte Biology

Effects of macrophage-CSF on pulmonary-macrophage repopulation after bone marrow transplantation

Tanja Bernier*, Thomas Tschernig{dagger}, Reinhard Pabst{dagger}, Olaf Macke*, Christiane Steinmueller* and Andreas Emmendörffer*

* Department of Immunobiology, Fraunhofer Institute of Toxicology and Aerosol Research, 30625 Hannover, and
{dagger} Department of Functional and Applied Anatomy, Hannover Medical School, 30623 Hannover, Germany

Correspondence: Dr. Thomas Tschernig, Department of Functional and Applied Anatomy, Hannover Medical School, Carl-Neuberg-Str. 1, 30623 Hannover, Germany. E-mail: tschernig.thomas{at}mh-hannover.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pulmonary infections are important causes of morbidity and mortality in immunosuppressed patients after transplantation. After experimental irradiation and syngeneic bone marrow transplantation in mice, macrophages show reduced repopulation in the lung compared with that in other tissues. Macrophages are major microbicidal immune effector cells in host pulmonary defense. Therefore, we examined the role of locally applied cytokines for macrophage repopulation in the lung. An accelerated repopulation of macrophages in the lung was observed after intranasal application of macrophage-colony stimulating factor (M-CSF), but this effect was not enhanced by a combination of M-CSF with interleukin (IL)-3. Local proliferation contributed to this effect. Macrophages in the lung tissue of M-CSF-treated mice displayed greater secretion of IL-6, whereas M-CSF treatment did not enhance the gene expression of other macrophage-specific chemokines. The role of M-CSF treatment was determined in pulmonary murine cytomegalovirus infection using an irradiation/reconstitution model. The M-CSF treatment had no effect on virus load in the lung tissue. However, phosphate-buffered saline-treated mice seemed to develop stronger inflammation after viral infection than M-CSF-treated mice. We conclude that local M-CSF treatment modulates cellular inflammation in the lung during immunosuppression.

Key Words: BMT • M-CSF • MCMV


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bone marrow transplantation (BMT) is widely used as a therapeutic option for many hematologic neoplasms and solid tumors. The posttransplant period is still difficult because of opportunistic infections in these immunocompromised patients. Pulmonary complications in bone marrow transplant recipients are a major cause of morbidity and mortality [1 ]. After syngeneic BMT in BALB/c mice, macrophages show reduced repopulation in the lung compared with that in other tissues after BMT [2 ]. Nakata et al. [3 ] reported that repopulation of the lung with alveolar macrophages (AMs) is completed just 90 days after BMT. Macrophages are involved in all stages of the immune response. They are important in bacterial, fungal, and viral infections, because most pathogens ultimately are eliminated by activated phagocytes. Previous studies have shown that pulmonary macrophages play a central role in regulating immune responses in the lung [4 , 5 ].

Macrophage-colony stimulating factor (M-CSF) stimulates the proliferation, activation, and differentiation of macrophage precursor cells [6 7 8 ] and promotes the activation and proliferation of mature mouse macrophages. In addition to regulating the proliferation and differentiation of progenitor cells in bone marrow, M-CSF has been reported to influence various functions of mature mononuclear phagocytes [9 10 11 ] and to be a potent chemoattractant [12 ]. It could also be demonstrated that the overexpression of M-CSF in a transgenic mouse model showed a protective effect against Listeria monocytogenes infections in the liver [13 ], and It has been shown that AMs proliferate in vitro in the presence of M-CSF, thus contributing to self-renewal of the pulmonary-macrophage population [14 , 15 ]. Another colony stimulating factor (CSF) that stimulates macrophage proliferation is interleukin (IL)-3 (also called "multi-CSF"). IL-3 stimulates the proliferation not only of macrophage precursors but also of granulocyte precursors, erythroid cells, eosinophils, and megakaryocytes.

Despite the overall beneficial effects of CSF on mature mononuclear phagocytes, there might also be drawbacks. For example, the retrovirus HIV might replicate more easily in CSF-stimulated mononuclear phagocytes in vitro [16 , 17 ]. Similar effects were demonstrated for influenza A virus after granulocyte macrophage-CSF (GM-CSF) stimulation of human monocytes for in vitro studies, in which de novo virus protein synthesis was enhanced, more virus particles were released, and host cells were killed at a higher rate [18 ]. A significant pathogen in immunocompromised patients after BMT is human cytomegalovirus (HCMV) [19 ]. Healthy individuals carry HCMV DNA in the absence of any symptoms of its disease. Macrophages are the reservoir for latent HCMV disease [20 ], yet HCMV is known to be disseminated by macrophages during primary infection [21 , 22 ].

The aim of this study was to modulate and enhance the repopulation of macrophages in the lung in an immunosuppression model. BALB/c mice that had undergone irradiation and BMT are a well-established model for immunosuppression [2 ]; therefore, these subjects were treated intranasally with M-CSF and IL-3. To clarify the mechanism of repopulation of macrophages in the lung, we investigated the chemoattractant activity and the number of proliferating macrophages in this tissue. The activation status of the pulmonary cells was also determined by measuring protein expression levels of inflammatory cytokines. Furthermore, we studied whether murine cytomegalovirus (MCMV) infection in vivo is affected by M-CSF stimulation of pulmonary macrophages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Female BALB/c mice (4 weeks of age) were obtained from Charles River (Sulzfeld, Germany). The animals were housed under conventional breeding conditions and analyzed monthly for bacterial or viral infections.

BMT and treatment procedures
Irradiation and BMT were performed as previously described [2 ]. Described in brief, 6-week-old female BALB/c mice were used as bone marrow recipients. They were irradiated by a 60Co source in plastic boxes beneath a 5-mm-thick plastic cover. The lethal irradiation dose was 8 Gy. One hour after irradiation, the mice were injected intravenously with 5 x 106 bone marrow cells from BALB/c donors. The bone marrow had been pretreated with monoclonal anti-Thy 1.2 antibody. Mice received antibiotics in drinking water on the first day after BMT (1% penicillin/streptomycin). Five days after transplantation, one group of mice were treated intranasally for 4 days with 1 x 104 or 5 x 104 U of recombinant murine M-CSF; another group was treated with 1 x 104 M-CSF + 2 x 105 U of IL-3; another group received 2 x 105 U of IL-3 (PharMingen, Hamburg, Germany), and one control group received only phosphate-buffered saline (PBS). Mice were killed 12 h or 3 days after the end of the M-CSF treatment.

M-CSF
Recombinant murine M-CSF was produced in Escherichia coli and isolated by affinity chromatography as previous described by Krautwald and Baccarini [23 ]. Specific activity was tested in a proliferation bioassay (data not shown) (1 µg {cong} 1.9 x 104 U). The treatment dose was chosen after several in vitro and in vivo studies with different doses. The absence of lipopolysaccharide (LPS) was determined by heating experiments; M-CSF was heated for 10 min at 95°C, and the activity was again determined by bioassay. After this procedure no M-CSF activity was found. Because LPS is not destroyed during 10 min at 95°C, contamination with LPS was unlikely.

Preparation of the samples for immunohistological staining
Mice were killed by CO2 at almost the same time in the morning. The bronchoalveolar space was filled via an intratracheal catheter with 1 mL of a 1:4 OCT/PBS (Miles Inc., Elkhart, IN) solution. Finally, the lungs were removed in sections, snap frozen in liquid nitrogen, and stored at -70°C.

Immunohistological staining
Whole lungs of each animal were cut at -20°C using a cryostat, and sections (5 µm thick) were fixed in acetone (10 min at -20°C). The alkaline phosphatase anti-alkaline phosphatase (APAAP) technique [24 ] was used to identify the phenotype of a subset of macrophages (BM8; Biomedicals AG, Augst, Switzerland) [25 ]. The slides were incubated with the primary antibodies for 30 min at room temperature (21°C). After washing with Tris-buffered saline (TBS)-Tween (0.05% Tween 20; Serva, Heidelberg, Germany) and incubating with a bridging antibody (rabbit anti-rat, 30 min; Dako, Hamburg, Germany), the APAAP complex (rat, 30 min; Dako) was applied. To increase the staining intensity, incubation with the bridging antibody and addition of the APAAP complex were repeated once. Fast blue (Sigma, Munich, Germany) served as the substrate for alkaline phosphatase. Positive and negative controls produced the expected results. All sections were counterstained with hemalaun and mounted in glycergel (Dako).

Isolation of AMs
AMs were isolated using a bronchoalveolar lavage (BAL) technique modified by the method of Kobzik et al. [26 ]. In brief, the lung was flushed with 1 mL of ice-cold Mg2- and Ca2-free PBS containing 0.4 mM EDTA. The lavage procedure was repeated eight times under moderate massage of the lung. Differential cell counts were performed on cytospin preparations stained with May-Grünwald (5 min) and Giemsa (1:20 in distilled water for 5 min) to determine the percentage of macrophages in the lavage fluid.

Isolation of lung interstitial cells
Macrophages from the lung interstitium (IMs) were harvested by the method of Holt et al. [27 ]. Described briefly, the lavaged and perfused lungs were minced with a tissue chopper and incubated under moderate agitation for 60 min in complete medium containing collagenase (100 U/mL; Worthington type 1; Bayer Diagnostic, Munich, Germany) and DNase (50 U/mL; Sigma). The cell suspension was pressed through a sterile steel sieve, washed in cold RPMI 1640 medium, and layered on a discontinuous Percoll gradient. The macrophage percentage was determined by esterase staining of cytospots.

In vivo detection of cell proliferation
Mice received intraperitoneal injections with 0.5 mL of 16 mM bromodeoxyuridine (BRDU) 12 h before preparation of lung digest cells. Cytospots were fixed 10 min with cold isopropanole and incubated 30 min at 70°C in formamide-sodium citrate. Detection of in vivo-incorporated BRDU in proliferating cells was performed by standard immunohistological staining using 10% normal goat serum to block nonspecific binding sites, anti-BRDU primary antibodies (Becton Dickinson, Heidelberg, Germany), and alkaline phosphatase-conjugated secondary antibodies (Dianova, Hamburg, Germany). Fast red (Sigma, Deisenhofen, Germany) served as substrate for the alkaline phosphatase. All sections were counterstained with hematoxylin (Sigma). Slides stained without primary antibody served as negative controls. Controls using an isotype-matched irrelevant antibody yielded negative results.

Cytokine and chemokine expression analysis
Total RNA from the lung tissues of M-CSF-treated and PBS-treated mice was isolated by guanidine thiocyanate-phenol-chloroform extraction [28 ]. Cytokine RNA levels were analyzed by RNase protection assay using a RiboQuant multiprobe kit (PharMingen), following the manufacturer’s directions. A 20-µg sample of the total RNA was hybridized overnight to a 32P-labeled probe set (mCK5) and a custom probe set [including IL-4, tumor necrosis factor (TNF){alpha}, IL-1{alpha}, M-CSF, IL-1 Ra, IL-2, IL-6, interferon (IFN){gamma}, L32, and glyceraldehyde 3-phosphate dehydrogenase]. Free probe and other single-stranded RNAs were digested with RNase. Protected mRNAs were purified and resolved on a 5% denaturating polyacrylamide gel and quantified by instant imaging. RNA loading was standardized against the protected fragments of the housekeeping gene L32.

IL-6 bioassay
IL-6 levels in supernatant of macrophage cultures, as decribed previously [5 ], were determined by their ability to stimulate the proliferation of IL-6-dependent B hybridoma cells (7TD1) as measured by the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide [29 ]. RIL-6 was used as a standard (PharMingen).

MCMV preparation of infection
The Smith strain of MCMV was kindly supplied by Prof. U. H. Koszinowski, (Ludwig-Maximillians-University of Munich, Munich, Germany). The virus was maintained by salivary gland passage in BALB/c mice [30 ]. The virus was propagated on a large scale by infecting BALB/c mouse embryo fibroblast (MEF) cells. The infected MEF cells were clarified by centrifugation at 600 g for 5 min. The supernatant was divided into aliquots and frozen at -80°C until use. An aliquot was subjected to analysis for a plaque-forming unit (PFU) assay. A single inoculum pool was used for all experiments. BMT- and M-CSF- or PBS-treated mice were infected with 108 PFU of MCMV intranasally and were killed 1 week after infection (Fig. 1 ).



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  		Figure 1. Schedule of the study design.

 
Plaque assay
Virus loads in lungs were determined with a slightly altered version of a plaque assay on MEF cells described by Chalmer et al. [31 ]. Organ homogenates were diluted 10-fold serially, and 200 µL of each dilution were plated in duplicate on confluent monolayers of MEF cells. After 1 h at 37°C, monolayers were washed with PBS and then overlaid with 1 mL of Dulbecco’s modified Eagle’s meduim-0.6% agar with 4% fetal calf serum. Cells were incubated for 5 days (37°C in 5% CO2). On day 6, cells were fixed with 3.7% formaldehyde for 24 h. Dulbecco’s modified Eagle’s medium-agar and formaldehyde were removed, and cells were stained with 1% methylene blue for 30 s.

Statistics
For calculation of statistics such as means and standard errors, Excel 5.0 for Windows 3.1 (Microsoft Corp., Redmond, WA) was used along with SAS software 7.0 (Statistical Analysis System Institute, Heidelberg, Germany) for the levels of significance. The differences between group means of PBS- and M-CSF-treated animals were analyzed using the Mann-Whitney U-test. Results have been presented as mean values ±SE or SD.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Accelerated repopulation of macrophages in the lung after M-CSF treatment
Local M-CSF treatment after irradiation and BMT resulted in a significantly increased number of macrophages in lung tissue but not in the BAL, in comparison with cell numbers in the lung tissue of PBS-treated mice (Fig. 2 ). Unspecific growth factor IL-3 alone only slightly increased the number of macrophages in lung tissue, and the effect of a combination of M-CSF and IL-3 on the number of macrophages was not significantly different from the effect of M-CSF alone. A higher dose of M-CSF also did not affect the number of macrophages in the BAL; i.e., the macrophage count in this tissue was in the same range as that in tissue at the lower dose (Fig. 3 ).



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  		Figure 2. Enhanced numbers of macrophages in lung tissue and BAL after irradiation, BMT, and cytokine treatment. The numbers of esterase-positive macrophages after M-CSF (104 U), IL-3, and combined treatments were counted 12 h after the end of each treatment in tissue and BAL and compared with the number of macrophages of PBS-treated mice. Both M-CSF and IL-3 increased the number of macrophages in lung tissue compared with that in lung tissue of PBS-treated mice. Values are means and SD of the numbers of macrophages from four animals per group (*, P<0.05, in comparison with the PBS-treated group using the Mann-Whitney U-test). The experiment was repeated twice with similar results.

 


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  		Figure 3. Effect of M-CSF treatment on numbers of macrophages in lung tissue and BAL. After irradiation and BMT, mice were treated with PBS (closed boxes) or two different doses of M-CSF [1 x 104 U (hatched boxes) and 5 x 104 U (open boxes)], and macrophages from the lung tissue and BAL were isolated and counted after 3 days. The numbers of macrophages in lung tissue increased after M-CSF treatment, whereas those in the BAL did not.. Data are means and SD of the numbers of macrophages from four animals per group (*, P<0.05 in comparison with the PBS-treated group using the Mann-Whitney U-test). The experiment was repeated once with similar results.

 
Similar results were obtained by histological analysis. An infiltration of BM8+ macrophages was detected by immunohistological staining in the M-CSF-treated lung, whereas only a few macrophages were found in PBS-treated control mice after BMT (Fig. 4A and B ).



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  		Figure 4. Detection of macrophages in lung tissue 3 days after M-CSF treatment and after CMV infection. In immunohistologically stained lungs, different numbers of BM8+ macrophages of (a) PBS-, (b) M-CSF (104 U)-, (c) PBS- and CMV-, and (d) M-CSF (104 U)- and CMV-treated mice were found after irradiation and BMT. M-CSF treatment resulted in a higher repopulation of macrophages in the lung after irradiation and BMT (b) but in combination with CMV infection did not lead to a pronounced inflammatory reaction (d). Similar findings were detected in at least 5 animals per group. br, bronchus; v, vessel._art>

 
To investigate the effect of M-CSF treatment on other cell populations, a cellular profile of BAL was performed by differential cell counts. Although the portion of lymphocytes remained constant at 1% for both M-CSF concentrations (1 x 104 and 5 x 104 U) tested, the portion of granulocytes increased significantly (from 3 to 11%) at the highest M-CSF dose but was not significantly increased (from 3 to 4.5%) at the low concentration.

Proliferation of pulmonary cells after M-CSF treatment
The numbers of proliferating cells in BAL and tissue were analyzed by in situ BRDU staining. A massive increase in BRDU+ cells was found in the lung tissue after M-CSF treatment, compared with that of the PBS control, which was even greater after combining M-CSF with IL-3 (Fig. 5 ). The number of BRDU+ BAL cells did not increase significantly after either M-CSF or IL-3 treatment or a combination of the two, in comparison with cells after PBS treatment.



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  		Figure 5. Enhanced number of proliferating pulmonary cells after irradiation, BMT, and cytokine treatments. The relative numbers of BRDU-positive cells in tissue and BAL 3 days after M-CSF (104 U), IL-3, and combined treatment compared with PBS treatment are shown. Both cytokines enhanced the numbers of proliferating pulmonary cells. Data are means and SD of the numbers of BRDU-positive cells from four animals per group (*, P<0.05 in comparison with the PBS-treated group, using the Mann-Whitney U-test). The experiment was repeated twice with similar results.

 
Cytokine and chemokine expression in lung tissue
The expression of cytokines and chemokines in lung tissue was analyzed by an RNase protection assay for the following chemokines: regulated on activation, normal T expressed and secreted, eotaxin, macrophage-inflammatory protein (MIP)-1ß, MIP-1{alpha}, MIP-2, mouse IP-10, and monocyte chemoattractant protein-1. All chemokines were expressed at a detectable level, but the expression of the investigated chemokines was not increased (data not shown), whether mice were treated with a dose of 1 x 104 U of M-CSF or 5 x 104 U of M-CSF, compared with PBS-treated mice.

M-CSF not only stimulates the proliferation and chemoattractive activity of macrophages, but is also a potent inducer of macrophage cytokine production. Therefore, the activation state of macrophages in lung tissue and the expression of TNF{alpha} and IL-1{alpha} were determined. TNF{alpha} and IL-1{alpha} gene expression tended to increase in M-CSF-treated mice in comparison with that in PBS-treated control mice (Fig. 6 ), but the differences were not significant.



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  		Figure 6. Gene expression in lung tissue after irradiation, BMT, and PBS or M-CSF (1x104 or 5x104 U, respectively) treatment. RNA expression was quantified by instant imaging of an RNase protection assay with 20 µg of RNA from the lung tissue of five animals per group [closed columns, PBS treated; hatched columns, M-CSF treated (1x104 U); open columns, M-CSF treated (5x104 U). Values are relative expression of IL-1{alpha} and TNF{alpha} after PBS and M-CSF treatment, given as means and SE. The experiment was repeated twice with similar results.

 
IL-6 production ex vivo
To analyze the activation state of macrophages at the protein level, IL-6 bioassays were carried out with culture supernatants of ex vivo-cultivated AMs isolated by BAL and IMs harvested by lung tissue digestion. AMs showed a dose-dependent increase in IL-6 production after M-CSF treatment, compared with IL-6 production of cells from PBS-treated mice. IMs also showed a significant increase of IL-6 at both M-CSF concentrations in comparison with PBS-treated mice, but there was no significant difference between the two treatment doses (Fig. 7 ).



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  		Figure 7. Influence of M-CSF treatment after irradiation and BMT on IL-6 levels. AMs and IMs were isolated from mice after irradiation, BMT and subsequent treatment with M-CSF [1 x 104 (hatched columns) and 5 x 104 units (open columns)] or PBS (closed columns). Culture supernatants were assayed in an IL-6 bioassay. There was a significant dose-dependent increase of IL-6 production in AMs and a significant increase in IMs. Data are means and SD of the IL-6 levels of three animals per group (*, P<0.05 in comparison with the PBS-treated group, using the Mann-Whitney U-test).

 
MCMV infection after BMT and cytokine treatment
To investigate the effect of the M-CSF treatment after BMT on pulmonary MCMV infection, mice were infected with 108 PFU of MCMV intranasally after the M-CSF/PBS treatment. The virus load in the right lobe of the lung was determined 1 week after infection by a plaque assay with lung homogenates, and no differences in the amount of virus load in the lung tissue of M-CSF-treated mice after BMT were found compared with virus in the lung tissue of PBS-treated mice after BMT (data not shown).

Intranasal cytomegalovirus (CMV) infection in the lung resulted in severe pneumonitis with an infiltration of mononuclear inflammatory cells. The number of macrophages in the lung tissue was analyzed by histological staining of tissue from infected M-CSF/PBS-treated mice that also received the macrophage-specific antibody BM8. The macrophage level in the M-CSF-pretreated, MCMV-infected mice did not differ from that of M-CSF-treated mice without MCMV infection. A massive macrophage infiltration was found in the lungs of PBS-pretreated mice but not in M-CSF-pretreated mice (Fig. 4C and 4D) , indicating a more severe inflammatory reaction in nontreated mice than in M-CSF-treated animals.

The expression of IL-1{alpha}, TNF{alpha}, and IFN{gamma} in the lungs was investigated by RNase protection assay. The relative expression rates of IL-1{alpha} and TNF{alpha} were slightly but not significantly higher in PBS-pretreated MCMV-infected mice compared with M-CSF-pretreated mice, while the relative expression of IFN{gamma} showed the opposite trend (Fig. 8 ).



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  		Figure 8. Gene expression in lung tissue after irradiation, BMT, PBS/M-CSF (5x104 U), and CMV infection. RNA expression of TNF{alpha}, IL-1{alpha}, and IFN{gamma} is shown as a result of quantification of an RNase protection assay of RNA of the lung tissues from five animals per group. In PBS-treated mice (closed columns), TNF{alpha} and IL-1{alpha} are more highly expressed than in M-CSF-treated mice (open columns) after MCMV infection, but it was the reverse with the relative expression of IFN{gamma}. Data are means +SE.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
After irradiation and BMT of BALB/c mice, a pronounced increase could be demonstrated in the number of macrophages in lung tissue after local M-CSF treatment. However, this effect could not be enhanced by additional IL-3 treatment. An important aspect is the M-CSF dose. No granulocyte infiltration was detected at a treatment dose of 104 U of M-CSF, but at higher doses, the macrophage system should be activated by the increased secretion of IL-6. The expression of inflammatory cytokines IL-1{alpha} and TNF{alpha} also tended to be enhanced and seemed to confirm the activation status of the cells through M-CSF treatment. After MCMV infection, M-CSF treatment showed no effect on virus load, but we found pronounced inflammation in PBS-treated control mice.

The repopulation of macrophages to the lung after M-CSF treatment might be explained by the following mechanisms: on one hand, M-CSF could stimulate the existing recipient macrophages for increased in situ proliferation; on the other hand, M-CSF might stimulate, directly or indirectly, cells to produce macrophage-specific chemoattractant proteins or function as a direct chemoattractant itself [12 , 32 ]. In the study reported here, no increased expression of common macrophage chemokines was found, so that an autocrine repopulation mechanism could be excluded. An increased number of proliferating pulmonary cells was found in the lung tissue after M-CSF treatment. Previous studies have demonstrated that AMs and IMs have the potential for self-renewal by local proliferation [15 ], but a recent study proved that an AM population is completely replaced by donor AMs within 90 days after allo-BMT [3 ]. The increased repopulation of macrophages in lungs seems to be a parallel effect of a direct chemoattractive activity of M-CSF, leading to increased recruitment of circulating monocytes to the lung and stimulation of local proliferation of the recipient pulmonary macrophages. The enhanced number of macrophages in lung tissue consists predominantly of IMs, which are located in this tissue. The number of AMs located in the BAL does not significantly increase after M-CSF treatment. In normal lung tissue, AMs are supposed to be a developmental end point of blood monocytes, whereas IMs may represent a more precursorlike stage of the macrophage lineage [33 34 35 ], which may explain a higher proliferation potential for IMs compared with AMs.

Another effect of M-CSF on pulmonary macrophages after local application is cell activation, which could be either a direct or indirect result of treatment. We detected increased secretion of IL-6 ex vivo by AMs and IMs isolated from M-CSF-treated mice. In contrast to proliferation results, IMs were less activated than AMs. Franke-Ullmann et al. [4 ] reported that, if IMs and AMs are activated at the same level, IMs isolated from normal mice are better equipped for immunoregulatory and accessory functions in vitro. It was shown that LPS-preactivated IMs secreted much more IL-6 and IL-1 compared with AMs in vitro. The more pronounced activation of AMs in the present study could be explained by activation of these cells; M-CSF was applied intranasally, leading to a concentration gradient between the alveolar space and pulmonary tissue. These results confirm a locally restricted M-CSF effect. A tendency toward increased TNF{alpha} and IL-1{alpha} gene expression in M-CSF-treated mice was detected by an RNase protection assay of RNA in lung tissue. This increased activation of pulmonary macrophages might lead to enhanced immunoregulatory efficiency in pulmonary host defense.

In previous in vitro studies by our group (Heike Höflich, unpublished data), M-CSF treatment of macrophages increased viral infection. To investigate whether local M-CSF treatment in vivo aggravates viral infection, irradiated BMT and PBS/M-CSF-treated mice were infected intranasally with MCMV. It was found that the virus load in M-CSF and PBS-treated mice does not differ after CMV infection. In contrast to our in vitro data and that of others [16 17 ], which show an increased virus yield in macrophages after M-CSF treatment, the M-CSF treatment in our in vivo model has no influence on virus replication. Surprisingly we found a massive influx of macrophages in the lung in PBS-treated mice but not in M-CSF-treated mice. The cytokine expression of TNF{alpha} and IL-1{alpha} was slightly decreased in M-CSF mice compared with PBS-treated mice, while the IFN{gamma} level showed the opposite picture. The increased number of macrophages in M-CSF-treated mice prior to the CMV infection could be a reason for the decreased inflammatory reaction in lungs after CMV infection. Taken together, the M-CSF treatment seems to reduce the severity of virus-induced inflammatory reaction. As proof of this principle, the overexpression of M-CSF in a transgenic-mouse model was effective as a prophylaxis by increasing the resistance against Listeria monocytogenes infection in the liver [13 ].

In conclusion, these data demonstrate that intranasally applied prophylactic M-CSF treatment after irradiation and BMT led to an accelerated repopulation of macrophages in the lung. This result could be of interest in developing new approaches to protect immunocompromised individuals against respiratory infections.


   ACKNOWLEDGEMENTS
 
This study was supported by the following grants: InSan I 1 0498-V-3800, BMBF (German Federal Ministry of Education and Science) 01KI 9307, and BMBF 01 KE 8910.

The authors thank Karin Westermann and Dagmar Stelte for excellent technical help and Sheila Fryk for correction of the English.

Received July 5, 2000; revised February 12, 2001; accepted April 16, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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