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The Journal of Immunology, 2008, 180, 4892 -4900
Copyright © 2008 by The American Association of Immunologists, Inc.
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Relative Levels of M-CSF and GM-CSF Influence the Specific Generation of Macrophage Populations during Infection with Mycobacterium tuberculosis1

David M. Higgins, Joaquin Sanchez-Campillo2, Adrian G. Rosas-Taraco, Jonathan R. Higgins, Eric J. Lee, Ian M. Orme and Mercedes Gonzalez-Juarrero3

Department of Microbiology, Immunology and Pathology, Mycobacteria Research Laboratories, Colorado State University, Fort Collins, CO 80523


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Members of the CSF cytokine family play important roles in macrophage recruitment and activation. However, the role of M-CSF in pulmonary infection with Mycobacterium tuberculosis is not clear. In this study, we show the lungs of mice infected with M. tuberculosis displayed a progressive decrease in M-CSF in contrast to increasing levels of GM-CSF. Restoring pulmonary M-CSF levels during infection resulted in a significant decrease in the presence of foamy macrophages and increased expression of CCR7 and MHC class II, specifically on alveolar macrophages. In response to M-CSF, alveolar macrophages also increased their T cell-stimulating capacity and expression of DEC-205. These studies show that the levels of expression of M-CSF and GM-CSF participate in the progression of macrophages into foamy cells and that these cytokines are important factors in the differentiation and regulation of expression of dendritic cell-associated markers on alveolar macrophages. In addition, these studies demonstrate that M-CSF may have a role in the adaptive immune response to infection with M. tuberculosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tuberculosis (TB)4 remains a major health problem worldwide and along with malaria and AIDS is the most frequent cause of death due to an infectious disease (1). It is estimated that tuberculosis is responsible for 2 million deaths a year and is a significant economic burden to the developing and developed world alike (2). This problem is aggravated today by the increased appearance of MDR-TB and XDR-TB strains (3). Thus, TB is a major health risk worldwide and a better understanding of the mechanisms involved in immunity to tuberculosis is vital to the discovery of novel treatments and vaccines against this disease.

In susceptible humans, Mycobacterium tuberculosis is a respiratory pathogen (2). To this end, the ability to deliver treatments directly to the lungs offers potential for disease treatment and is amendable to basic research. In this regard, the recent development of noninvasive procedures to administer molecules by aerosol directly to the lungs of small animals has greatly advanced research of various diseases, including tuberculosis (4, 5, 6). Specific advantages to this procedure include the ability to transiently modulate the lung environment during M. tuberculosis infection.

M. tuberculosis is typically transmitted by inhalation of aerosolized droplets to the lungs. Thus, alveolar macrophages (AMs) are the first cells in the lungs to encounter the bacillus and are believed to be important in the establishment of an immune response and to the pathogenesis of M. tuberculosis (7). Although once thought to be terminally differentiated, previous studies on AMs have demonstrated the importance of the cytokines M-CSF and GM-CSF in driving proliferation and differentiation of AMs (8, 9). However, only a few studies have addressed the role that M-CSF and GM-CSF may play in altering the phenotype and characteristics of AMs during M. tuberculosis infection (10, 11). In this regard, previous reports have shown that osteopetrotic mice, which are M-CSF deficient, have decreased numbers of AMs and increased susceptibility to infection by certain viruses and intracellular bacteria including M. tuberculosis (12, 13, 14). In the case of the latter, it was previously demonstrated that osteopetrotic M-CSF-deficient mice succumb to infection with M. tuberculosis within 4 wk (15). In contrast to M-CSF, the role of GM-CSF in this infection is much clearer. We recently showed that AMs and dendritic cells (DCs) in GM-CSF-deficient mice are defective in their ability to phagocytose and kill M. tuberculosis, have reduced granuloma formation, and fail to express an adequate TH1 response required to control the bacterial load (10).

Given the significance of these CSFs in the proliferation and differentiation of myeloid cells in the lungs and the importance of these cells in immunity, this study evaluated the role of M-CSF during pulmonary infection with M. tuberculosis and compared it to GM-CSF. The results of this study indicate that the relative levels of M-CSF and GM-CSF over the course of this infection influence the expression of DC markers on AMs and promote the formation of highly vacuolated foamy macrophages (M{phi}s), which are characteristic of granuloma formation. Also, the results of this study suggest that M-CSF may have a role in the promotion of the adaptive immune response by increasing the expression of CCR7 and MHC class II.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Specific pathogen-free female C57BL/6 and female BALB/c mice, 6–8 wk old, were purchased from The Jackson Laboratory. Mice were maintained in the biosafety level 3 biohazard facility at Colorado State University and were given sterile water, mouse chow, bedding, and enrichment for the duration of the experiments. The specific pathogen-free nature of the mouse colonies at these facilities was demonstrated by testing sentinel animals. These were shown to be negative for 12 known mouse pathogens. All experimental protocols used in this study were approved by the Animal Care and Use Committee of Colorado State University.

Experimental infections

Mice were infected by low-dose aerosol challenge with M. tuberculosis strain Erdman using a Glas-Col (Terre Haute) aerosol generator calibrated to deliver 50–100 bacteria into the lungs. To confirm success of bacterial deposition in the lungs, 24 h after challenge the lungs from five mice were used to determine the number of bacteria deposited. Bacterial counts in the lung (n = 5) at each time point of the study were determined as previously described (10). Briefly, bacterial loads in lungs of infected mice were determined by plating serial dilutions of the organ homogenates on nutrient 7H11 agar and counting CFU after 3 wk incubation at 37°C. Lungs from mice (n = 5) in the same groups were harvested for histological, cell population and ELISA analysis. The results shown in this study are representative of two experiments.

Intratracheal administration of cytokines

Intratracheal administration of cytokines was performed by previously reported methods (4, 5) and adapted for this study. Specifically, mice were anesthetized by i.p. injection of 100 mg/kg body weight ketamine and 10 mg/kg xylazine. The mice were suspended by their upper teeth on a platform slanted at a 45° angle. The tongue was gently pulled out with padded tweezers and a small laryngoscope was used to visualize the trachea. An intratracheal microsprayer (Penn-Century) was inserted into the trachea and used to deliver 1 µg of rM-CSF or rGM-CSF (both from PeproTech) in 50 µl of PBS. Control mice were dosed with 50 µl of PBS. The microsprayer was immediately withdrawn and the mouse was removed from the platform. Mice were monitored until they recovered from anesthesia.

Lung cell digestion

Mice were euthanized by CO2 asphyxiation, the thoracic cavity was opened and the lungs were perfused with an ice-cold solution containing PBS and heparin (50 U/ml; Sigma-Aldrich) through the pulmonary artery. The left lobe of the lung and the draining lymph nodes were dissected and incubated with complete RPMI 1640 (cRPMI) containing collagenase XI (0.7 mg/ml; Sigma-Aldrich) and type IV bovine pancreatic DNase (30 µg/ml; Sigma-Aldrich) for 30 min at 37°C. The digested lungs were further disrupted by gently pushing the tissue through a cell strainer (BD Biosciences). RBC were lysed with Gey’s solution, washed, and resuspended in cRPMI. Total cell numbers per lung were determined using a hemocytometer.

Immunohistochemistry and histological analysis

Lobes from the lungs of each group of mice were placed in a histology cassette, incubated in zinc fixative (BD Pharmingen), and then embedded in paraffin. Paraffin-embedded blocks were cut in sections 5- to 7-µm thick and the paraffin was removed from the tissue sections using EZ-DeWax solution (BioGenex). Sections were then washed and endogenous peroxidase was blocked with a solution of methanol containing 0.3% H2O2 for 20 min and 3% BSA in PBS for 30 min. Following this step, each section was incubated overnight at 4°C with a primary Ab for IFN-{gamma} (Invitrogen Life Technologies), MHC class II (eBioscience), or TGF-β (Santa Cruz Biotechnology). The slides were then incubated with the secondary Ab for 30 min. Thereafter, the specific Ab-binding reaction was amplified using the Tyramide Signal Amplification system (PerkinElmer Life and Analytical Sciences). After the amplification step, the slides were washed and incubated again for 5 min with 3,3'-diaminobenzidine (DakoCytomation). Finally, the slides were counterstained by immersing the sections in hematoxylin (DakoCytomation) and mounted for microscopic observation.

To quantify the presence of foamy cells in the lungs, cytospins were made using 100 µl of the lung cell suspension described above. Slides were fixed with cold methanol and stained with H&E. Using a field of x20, 50 photographs of the cytospins were taken at random. The number of foamy cells in each frame was counted and the average of all frames was determined by two separate researchers.

ELISA and NO determination

The diaphragmatic lobe of the lungs of each mouse was placed into 1 ml of sterile saline and thereafter was homogenized. The samples were then screened in triplicate by ELISA following the manufacturer’s protocol for GM-CSF, M-CSF, total TGF-β, and IFN-{gamma} ELISA kits (R&D Systems). Total TGF-β was determined by first treating with 1 N HCL for 15 min at room temperature and neutralizing with an equal volume of 1 N NaOH before ELISA analysis. For NO determination, lung cell suspensions were cultured at a concentration of 5 x 106 cells/well and infected with 5 x 105 bacteria/well. After 5 days of culture. Greiss Reagent (Sigma-Aldrich) was used to measure NO levels by a colorimeter.

Flow cytometric analysis of cell surface markers

Cell suspension from each individual mouse was incubated with mAbs labeled with FITC, PE, PerCP, PE-Cy7, Alexa Fluor 430, Alexa Fluor 350, or allophycocyanin at 4°C for 30 min in the dark. After washing the cells with PBS containing 0.1% sodium azide (Sigma-Aldrich), the cells were incubated with mAbs against CD11c, CD11b, CCR7, DC-SIGN, MHC class II, or DEC-205 molecules and rat IgG2a, IgG2b, IgG1,or hamster IgG were used in this study. The Abs for DEC-205 and MHC II were purified and donated by Dr. J. Spencer (Colorado State University, Fort Collins, CO) and conjugated to Alexa 430 and Alexa 350, respectively, using an Alexa Fluor Protein Labeling Kit (Invitrogen Life Technologies). The remaining mAbs were purchased from BD Pharmingen or eBioscience as direct conjugates to FITC, PE, PerCP, PE-Cy7, or allophycocyanin. Data acquisition and analysis for this study were done using a LSRII (BD Biosciences) and FACSDiva software, respectively. Analyses were performed with an acquisition of at least 100,000 total events in the cell gate. Live/dead cell staining was performed using LIVE/DEAD cell stain kits with a blue-fluorescent reactive dye (Invitrogen Life Technologies) on appropriate samples to confirm live cells were gated in analysis.

Bronchoalveolar lavage (BAL) and CFSE staining of cultured cells

Mice were euthanized by CO2 asphyxiation. A small incision was made into the trachea and the flexible piece of an 18-gauge catheter (Terumo) attached to a 3-ml syringe was inserted into the trachea. Mice (n = 5) were lavaged three times with 1-ml aliquots of cRPMI medium and the subsequent samples were pooled. The pooled cells were washed and resuspended in 5 ml of cold PBS with 5 µM CFSE (Molecular Probes) and incubated at 37°C for 15 min. The cells were washed and resuspended in 5 ml of cRPMI medium and incubated at 37°C for 30 min. Cells were then washed and resuspended in 1 ml of cRPMI medium and total cell numbers were counted using a hemocytometer. Cells were divided equally into four groups in a 24-well plate containing only cRPMI medium or with 20 ng/ml of each cytokine. After culture for 5 days at 37°C and 5% CO2, the data were collected and analyzed by flow cytometry as described above. Additionally, samples were observed using an inverted Olympus microscope.

Mixed lymphocyte reaction

BALB/c CD4+ cells were used as responder cells in a MLR assay. Spleens were harvested from BALB/c mice and single-cell suspensions were made by gently pushing the tissue through a cell strainer (BD Biosciences). RBC were lysed with Gey’s solution, washed, and resuspended in cRPMI medium. CD4+ cells were sorted using CD4 microbeads (Miltenyi Biotec) and then counted using a hemocytometer. Sorted CD4+ cells were stained with 5 µM CFSE. These CFSE-stained CD4+ cells and BAL cells from C57BL/6 mice cultured in cytokines were incubated together in a 1:5 ratio. After 5 days at 37°C and 5% CO2, the cells were incubated with PE-conjugated CD4 Ab. Data acquisition and analysis for CFSE expression of CD4+ cells was done using a LSRII and FACSDiva software, respectively.

Cell proliferation in the MLR was used as the parameter to determine the capacity of cytokine-cultured BAL cells to stimulate naive CD4+ T cells. Analysis of cell division determined as loss of CFSE fluorescence was restricted to CD4+ cells, excluding doublets. In a histogram gated on CD4-CFSE cells, each peak was defined as a single round of cell division. Determination of mitotic events and precursor frequencies was determined by previously reported methods (16). Briefly, the number of CD4-CFSE division precursors was extrapolated from the sum of the number of daughter cells under each division peak divided by (2n), where n = number of rounds of cell division. The estimated number of mitotic events was determined as the sum of individual mitotic events under each peak.

Phagocytic assays

BAL cells cultured as described above were washed and resuspended in 1 ml of cRPMI medium. TNF-{alpha} (1 ng/ml) was added to the medium for 24 h. Thereafter, cells were counted using a hemocytometer and 1-µm latex beads labeled with YO dye (Polysciences) were added into the culture at a ratio of 20 beads to 1 cell. After a 24-h incubation at 37°C and 5% CO2, the cells were washed twice with cRPMI medium and the number of beads per cell was determined using fluorescent microscopy. The number of beads per cell was observed by two separate researchers.

Statistical analysis

The results presented in this publication are representative of two or three experiments. The data are expressed as the mean values (n = 5) from duplicate or triplicate assays. A parametric method, the Student t test was used to assess statistical significance between groups of data. Values of p < 0.05 (*) were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Lungs of mice chronically infected with M. tuberculosis had a progressive loss of M-CSF and increased levels of GM-CSF

In a first study, lung homogenates from M. tuberculosis-infected mice were analyzed by ELISA for levels of M-CSF and GM-CSF on 0, 21, 35, and 60 days of the infection. The levels of GM-CSF in the lungs of infected mice showed a 2-fold increase from 0 to 60 days, whereas the levels of M-CSF decreased 3-fold after 35 days and remained low thereafter (Fig. 1A). The ratio of GM-CSF to M-CSF increased as the infection progressed and remained 2-fold higher 60 days after challenge (Fig. 1B).


Figure 1
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  		FIGURE 1. Decreased levels of M-CSF and increased levels of GM-CSF in M. tuberculosis-infected mice. A, Levels of M-CSF ({blacksquare}) and GM-CSF ({blacktriangleup}) by ELISA of homogenate in the lungs of mice infected with a low dose of M. tuberculosis. The data represent the mean ± SD. B, Ratio of M-CSF to GM-CSF during M. tuberculosis infection. C, Levels of M-CSF in lung homogenate by ELISA after 2 wk of M-CSF treatment in the lungs of mice 35 days after challenge. Samples collect 48 h after last treatment. The data represent the mean ± SD.

 
Intratracheal delivery of rM-CSF increased pulmonary M-CSF levels in M. tuberculosis-infected mice and was associated with a reduction in the number of foamy M{phi}s

To determine the effect of restoring M-CSF levels during infection with M. tuberculosis, rM-CSF was delivered to mice every 48 h beginning 21 days after challenge. After 2 wk of treatment, the lungs were harvested for ELISA as described above. After treatment, M-CSF levels were increased ~2-fold in M-CSF-treated mice when compared with control mice receiving PBS treatment (Fig. 1C).

The lung pathology in these mice was examined and showed that M-CSF-treated mice had many perivascular and peribronchial infiltrates containing mainly lymphocytes and M{phi}s. Surrounding the perivascular and peribronchial infiltrates were areas of alveolitis of different sizes (Fig. 2A). In control mice, these areas consisted of small clusters of epithelioid M{phi}s interspaced with many lymphocytes and few neutrophils (Fig. 2A). Also, the periphery of these areas of alveolitis in control mice was infiltrated with numerous vacuolated foamy M{phi}s (Fig. 2A). In contrast, M-CSF-treated mice had a significant reduction in the presence of foamy M{phi}s (Fig. 2A) and areas of alveolitis in these mice were mainly formed by clusters of M{phi}s. Additionally, M-CSF-treated mice had fewer perivascular infiltrates and areas of alveolitis than control mice.


Figure 2
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  		FIGURE 2. Histological changes and reduction in number of foamy M{phi}s in infected mice treated with M-CSF. A, H&E staining of lung sections from M-CSF-treated and control M. tuberculosis infected mice at 10 and 40x. Arrows indicate foamy cells. B, Cytospin analysis for presence of foamy cells. Pictured is a representation of a foamy cell in one field. Data represented as mean of 50 fields per sample ± SD. C, Bacterial load of M-CSF treated and control mice 35 days after challenge. The data represent the mean ± SD.

 
To quantitate the difference in foamy M{phi} numbers between the two groups, lung cell suspensions from M-CSF-treated and control mice were analyzed on cytospin slides and these cells were counted. Cell suspensions from lungs of M-CSF-treated mice had 3-fold fewer numbers of foamy M{phi}s per field when compared with control mice (Fig. 2B). However, after M-CSF treatment, the bacterial load between both groups of mice was similar (Fig. 2C).

Higher levels of M-CSF modify the distribution and quantity of MHC class II, TGF-β, and IFN-{gamma}

With the intention of further defining the observed differences between M-CSF-treated and control mice demonstrated above, MHC class II Ag, a marker of activated myeloid cells critical to Ag presentation and T cell activation was assessed by immunohistochemistry. The M{phi}s in M-CSF-treated mice were positive for surface expression of MHC class II (Fig. 3A). In contrast, foamy M{phi}s had low expression of MHC class II (Fig. 3A).


Figure 3
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  		FIGURE 3. Immunohistochemistry and ELISA of lungs from infected mice treated with M-CSF. Sections of lungs from M-CSF-treated and control mice were analyzed after 35 days after challenge (A), MHC class II staining (B), TGF-β staining (C), and IFN-{gamma} staining (D). The level of IFN-{gamma} was analyzed by ELISA. The data represent the mean ± SD. E, The level of total TGF-β was analyzed by ELISA. The data represent the mean ± SD. F, Nitrite level analyzed by Greiss reaction on lung cell suspensions from infected mice cultured for 5 days. The data represent the mean ± SD.

 
The expression of TGF-β in lung cells during M. tuberculosis infection has not been determined and therefore TGF-β was assessed by ELISA and immunohistochemistry. By ELISA the levels of TGF-β remained similar between the two groups of treated mice (Fig. 3E). However, when lung tissue sections from both groups of mice were stained with Abs recognizing TGF-β, the most notable difference was the expression of TGF-β in M{phi}s found in M-CSF-treated mice (Fig. 3A). In contrast, foamy M{phi}s did not express any TGF-β (Fig. 3A).

We also determined the effect of M-CSF treatment on lung IFN-{gamma} levels in the same manner. The level of IFN-{gamma} in M-CSF-treated mice decreased significantly, to less than half the level of control mice on day 35 after infection (Fig. 3D). In addition, this study also evaluated the distribution of IFN-{gamma} and found that expression of IFN-{gamma} was colocalized in groups of M{phi}s located in the areas of alveolitis in M-CSF-treated mice (Fig. 3A).

M{phi}s have the capacity for high antimicrobial activity and therefore the levels of NO were determined using the Greiss reaction on lung cell suspensions from M-CSF-treated and control mice. The levels of NO were similar in both M-CSF- treated and control mice (Fig. 3D).

Increased expression of DC-associated cell surface markers as a result of M-CSF instillation

After flow cytometric analysis CD11c+CD11bmid cells (R1) were defined as AMs, CD11c+CD11b+ cells (R2) were defined as DCs, and CD11cmidCD11bmid cells (R3) were defined as M{phi}s based on previous studies (17, 18) (Fig. 4A). These AM, DC, and M{phi} regions were analyzed by staining for CCR7, MHC class II, and DEC-205 cell surface Ags. Cells in region R2 (DCs) had the highest expression of CCR7 and MHC class II compared with the other regions (Fig. 4C). However, only cells in region R1 (AM) increased levels of expression of both CCR7 and MHC class II in response to M-CSF treatment. Cells in region R2 (DC) increased the levels of expression of only CCR7 after M-CSF treatment. Additionally, there were elevated levels of expression of DEC-205 on cells in region R1 (AM) and region R3 (MØ), although the data were not statistically significant (Fig. 4C).


Figure 4
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  		FIGURE 4. M-CSF increases expression of DC-associated cell surface markers in lungs of infected mice. The lungs of M-CSF-treated and control mice were harvested for analysis by flow cytometry on 35 days after challenge with M. tuberculosis. A, Gated regions correlate with R1 (AMs), R2 (DCs), and R3 (M{phi}). B, CCR7, MHC class II, and DEC-205 surface Ags were analyzed in gated regions from a CD11c/CD11b plot. The data represent mean fluorescence intensity (MFI) x 103 ± SD. C, Total cells in R1, R2, and R3. The data represent the mean ± SD.

 
Cells in region R1 (AM) and region R2 (DC) showed little or no difference, respectively, in cell numbers after M-CSF treatment (Fig. 4B). However, in region R3 numbers of M{phi}s were 2-fold higher in M-CSF-treated mice (Fig. 4B). Also, in both M-CSF-treated and control mice, AMs increased expression of CD11b after infection (data not shown).

M-CSF is not a significant proliferation factor for AMs when compared with GM-CSF

The results presented above demonstrated that M-CSF causes differentiation of AMs and proliferation of other M{phi} populations. To further study the proliferation of AMs in response to these cytokines, BAL cells from naive or infected mice were cultured in medium alone or with rM-CSF, rGM-CSF, or both cytokines. The effect of each cytokine on AM proliferation was measured by loss of fluorescence of CFSE after every round of cell division. AMs cultured in medium had no proliferation levels after 5 days of culture and most of these cells died. AMs cultured in M-CSF had low levels of proliferation, whereas AMs that were cultured with GM-CSF had much higher levels of proliferation (Fig. 5A).


Figure 5
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  		FIGURE 5. In vitro culture of AMs with M-CSF or GM-CSF causes maturation or proliferation. BAL cells were collected from C57BL/6 mice, pooled, and cultured. Cells were stained on day 1 of culture with CFSE and harvested on day 5 for analysis by flow cytometry. A, Loss of expression of CFSE was determined on BAL cells and represented by shaded lines. Open bold lines represent culture with medium alone. B, Cultured naive AMs were analyzed by microscopy. C, Representative plots of CD11c and CD11b expression in AMs. Numbers in upper right corner are percentage in each quadrant.

 
By microscopy, naive AMs cultured in M-CSF contained central nuclei, large cytoplasmic areas, and an elongated, spindle-like shape. Naive AMs cultured in GM-CSF were small nonadherent cells with lateral nuclei location and small cytoplasmic areas (Fig. 5B). By flow cytometric analysis, BAL cells before culture were found to be CD11c+CD11b–. However, after culture, these cells increased their expression of CD11b and were CD11cmidCD11bmid (Fig. 5C).

M-CSF differentiates AMs toward a DC-like phenotype

Based on the morphological differences observed, the results presented above suggest that M-CSF causes AMs to differentiate toward DC-like cells. Thus, we compared changes in AM expression levels of CCR7, MHC class II, and DEC-205 surface markers by flow cytometry. Similar to results found in vivo, naive AMs cultured in M-CSF had a >2.5-fold increase in levels of CCR7, MHC class II, and DEC-205, whereas AMs cultured in GM-CSF showed no significant change in levels of these cell surface markers (Fig. 6A).


Figure 6
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  		FIGURE 6. M-CSF differentiates AMs into DC-like cells. BAL cells were collected from C57BL/6 mice, pooled, and cultured. After 5 days of culture, the cells were analyzed or used to stimulate naive CD4+ T cells. A, Percentage of culture expressing CCR7, MHC class II, or DEC-205. B, Cultured BAL cells with intracellular latex beads. Data represent the mean beads per cell of 50 cells per group ± SEM. Image representative of cell with fluorescent beads, arrows indicate beads. C, Representative plots of CD4+ cells and CFSE fluorescence after 3 days of stimulation with cultured BAL cells. Numbers in upper left corner are the estimated number of mitotic events. The data represent the mean x 105 ± SD.

 
When the expression of these Ags were studied in AM cultures derived from infected mice, CCR7 and DEC-205 Ags were elevated and MHC class II was decreased 2-fold, when comparing naive to infected AM cultures. However, only M-CSF increased levels of CCR7 expression (Fig. 6A).

AMs cultured in GM-CSF show greater capacity to phagocytose latex particles

As shown above, M-CSF differentiated AMs toward DC-like cells based on their morphology and phenotype. AMs and DCs differ in their ability to phagocytose particles; therefore, this study evaluated whether M-CSF modified the phagocytic capacity of AMs. BAL cells obtained from naive mice were tested for their ability to phagocytose latex beads. AMs cultured in GM-CSF had a 3-fold increase in the number of ingested beads per cell than controls and M-CSF did not increase the phagocytic capacity of AMs (Fig. 6B).

AMs cultured in M-CSF had a greater capacity to stimulate naive CD4+ T cells in a MLR

The phenotypes, morphology, and phagocytic function of AMs cultured in M-CSF or GM-CSF indicated that AMs cultured in M-CSF differentiated more toward DC-like cells. Thus, a MLR was performed to determine the ability of these cultured AMs to stimulate naive CD4+ T cells. CD4+ T cells stimulated by naive AMs cultured with M-CSF had a 3-fold increase in the number of mitotic events when compared with AMs cultured with GM-CSF (Fig. 6C). However, in cultures of AMs obtained from infected mice, there was only a slight increase in the number of mitotic events in AMs cultured in M-CSF when compared with controls (Fig. 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
The cytokine environment is an essential component in lung homeostasis; and in this regard the cytokines M-CSF and GM-CSF are known to be essential factors in the lungs in the differentiation of myeloid cells and the immune response (12, 14, 19). Although previous reports have studied the role of the GM-CSF in pulmonary infections with M. tuberculosis, the role of M-CSF in this regard is less clear. The results presented in this study demonstrated that there is a significant divergence in the levels of pulmonary M-CSF and GM-CSF during the course of infection with M. tuberculosis in mice. Using intrapulmonary installation of M-CSF, we determined that the divergence of these cytokines coincides with the alveolar infiltration of myeloid cells, many of them with a foamy appearance. The main effect of M-CSF treatment was a reduction in the number of these foamy M{phi}s and increased expression of DC-associated cell surface Ags specifically on AMs. When we studied these AMs in vitro, we demonstrated that these cells possess the plasticity to alter their phenotype and function depending on the local environment.

Previous studies from our group indicated that foamy M{phi}s express DEC-205, which is a marker expressed in high levels on DCs (20). Based on these results, it was tempting to speculate that these foamy M{phi}s originate from DCs. However, in the present study, we demonstrated that depending on the cytokine environment, AMs have the ability to resemble DCs by up-regulating CD11b, CCR7, MHC class II, and DEC-205 markers. These results demonstrate the complexity in defining discrete AM and DC populations in the lungs. More specifically, although CD11c, MHC class II, DEC-205, and DC-SIGN are markers associated with DCs, using these markers to define DCs in the lungs is very challenging because AMs express these markers to varying degrees as demonstrated here and elsewhere (17, 20, 21). For instance, in the present study, we demonstrated that AMs, in the presence of M-CSF, can up-regulate CCR7 and can increase their T cell stimulating activity, both known characteristics of DCs.

CCR7 is a chemokine receptor essential in the transport of DCs to lymph nodes and participates in the development of an adaptive immune response to M. tuberculosis (18, 22). This study demonstrated M-CSF up-regulates CCR7 on AMs, suggesting an increased capacity to migrate out of the lungs to lymph nodes. This is consistent with the observation that mice deficient in M-CSF did not have bacilli reaching the draining lymph nodes early after infection (15). Taken together, these results suggest that M-CSF may have an important role in the transport of activated APCs to lymph nodes by a CCR7-dependent mechanism during the early stages of the infection. However, as the infection enters into the chronic stage, migration of APCs from the lungs to the lymph nodes is known to be selectively arrested (18) at the same time that pulmonary M-CSF levels decrease. Increasing GM-CSF levels into the chronic stage of the infection results in elevated cell proliferation and phagocytic activity. Taken altogether, we suggest that because the host cannot resolve the infection, the sustained relative divergence in the levels of M-CSF and GM-CSF is an imbalance which results in a subsequent accumulation of foamy cells and increased disease pathology.

High numbers of foamy M{phi}s in the alveolar spaces have also been found in other murine models believed to have an imbalance in the relative levels of M-CSF and GM-CSF due to an overexpression or lack of GM-CSF in the lungs (10, 19, 23). From this study, we demonstrated that restoring the pulmonary levels of M-CSF slowed the formation of foamy cells and instead resulted in an increased lung infiltration of small M{phi}s expressing low levels of MHC class II Ags. These results correlate with other models in which M-CSF has been demonstrated to reduce foam cell formation by enhancing the excretion of cholesterol from M{phi}s (24).

Despite expression of an activated phenotype in both AMs and DCs in M-CSF-treated mice, levels of the Th1 cytokine IFN-{gamma} were reduced when compared with control mice. The above results are the first to suggest that M-CSF may down-regulate IFN-{gamma} in a chronic pulmonary infection with M. tuberculosis, possibly by inducing immunoregulatory characteristics in monocytes which has been demonstrated by others (12). M-CSF treatment did not influence the levels of TGF-β which has been shown to block antimicrobial activity (25). Our results therefore suggest that changes in the levels of expression of pulmonary M-CSF do not influence TGF-β levels or antimicrobial activity by NO during the course of a pulmonary M. tuberculosis infection.

The major cell component in BAL cell suspensions are AMs. Although once considered to be terminally differentiated cells with little ability to proliferate or alter their phenotype, more recent studies and this current study support the hypothesis that AMs have the ability to proliferate in the presence of GM-CSF as well as differentiate in response to M-CSF. This is not in fact a new finding (8) and with newer techniques available, we were able here to further characterize the altered phenotype of AMs stimulated by M-CSF, specifically increased levels of CCR7, MHC class II, and DEC-205. In conjunction with an increase in MHC class II expression, there was an increase in the T cell stimulating activity of M-CSF-treated AMs, which correlates with studies defining MHC class II as a necessary factor for APCs to stimulate T cells in a MLR (26). This result also correlates with previous studies that demonstrate that M-CSF enhances MHC presentation in DCs and increases proliferation of lymphocytes in a model of autoimmune disease (27, 28). An increase in DEC-205 is also associated with enhanced Ag- presenting capacity of APCs (29, 30), further suggesting the role of M-CSF in developing a DC-like phenotype in AMs.

The in vitro studies presented here demonstrated that MHC class II expression is down-regulated on AMs in M. tuberculosis-infected mice, which coincides with studies demonstrating MHC class II down-regulation during this infection (17, 18). This down-regulation could not be rescued by M-CSF, in fact, unlike naive AMs, there was only a slight increase in MHC class II on infected AMs after culture with M-CSF. However, levels of CCR7 did significantly increase in these same cells in response to M-CSF which demonstrated that the M-CSF receptor (CD115) is still functioning. Therefore, the infection is capable of inhibiting the up-regulation of MHC class II by an M-CSF-independent mechanism.

From this study, we concluded that changes in the relative levels of expression of M-CSF and GM-CSF in pulmonary infection with M. tuberculosis promotes the accumulation of foamy M{phi}s. The data suggest that the AM population is more dynamic than once thought and depending on the cytokine environment may resemble a DC-like cell. In the lungs, the distinction between AMs and DCs thus appears to be more complex than comparing similar cell types in other tissues. Additionally, although M-CSF does not contribute to increased antimicrobial activity, it may have a significant role in promoting the adaptive immune response by up-regulating CCR7 to promote migration of APCs to the lymph nodes draining the sites of infection.


   Acknowledgments
 
We thank the Laboratory Animal Resources staff at Colorado State University for their help in the development of the intratracheal procedures and Dr. John Spencer (Department of Microbiology, Immunology and Pathology, Colorado State University) for donating purified DEC-205 and MHC class II Abs.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant AI44072. Back

2 Current address: Departamento de Histologia y Anatomia Patologica Comparadas, Universidad de Murcia, 30071 Murcia, Spain. Back

3 Address correspondence and reprint requests to Dr. Mercedes Gonzalez-Juarrero, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523. E-mail address: malba{at}mail.colostate.edu Back

4 Abbreviations used in this paper: TB, tuberculosis; AM, alveolar macrophage; DC, dendritic cell; BAL, bronchoalveolar lavage; cRPMI, complete RPMI. Back

Received for publication October 18, 2007. Accepted for publication January 21, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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