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GFP transgenic mice show dynamics of lung macrophages
Experimental Cell Research 310 (2005) 409 – 416 www.elsevier.com/locate/yexcr
Research Article
GFP transgenic mice show dynamics of lung macrophages Martin Grundy, Charles L. Sentman* Department of Microbiology and Immunology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, USA Received 25 July 2005, revised version received 12 August 2005, accepted 17 August 2005 Available online 19 September 2005
Abstract The dynamics of tissue macrophages are poorly understood. We have developed a model where only lung macrophages express high levels of enhanced green fluorescent protein (EGFP) and are easily identified and followed by confocal microscopy. The EGFP+ cells had the morphology of macrophages and express CD11c, CD11b, and F4/80, but not NK1.1 or CD3. The F4/80+EGFP+ cells were found exclusively in the lung and not in lymph nodes, spleen, blood, liver, intestine, or uterus. These EGFP+ cells are phagocytic and can be activated to migrate within the lung in response to LPS stimulation. In this study, we describe a new model system that allows the specific study of macrophages in the lung. D 2005 Elsevier Inc. All rights reserved. Keywords: Lung macrophage; Green fluorescent protein; Cellular migration; Transgenic mice; CD2; LPS
Introduction Macrophages are long-lived cells found in almost every tissue in the body and have important roles in tissue maintenance, reorganization, and innate immunity. Macrophages act as sentinels of the immune system and have the ability to rapidly recognize and destroy invading microorganisms. In addition, macrophages can produce a large array of cytokines that help initiate a local immune response, and they can be activated by cytokines, such as IFN-g, from other leukocytes to up-regulate their phagocytic and killing activity [1]. Macrophages are believed to arise from monocytes that migrate from the blood and differentiate in tissues to become macrophages [2– 4]. Due to the unique function and cell components of each tissue, macrophages become specialized to perform specific functions in different tissues, while they retain their basic phagocytic and innate immune functions [2– 4]. Pulmonary macrophages are believed to play an important role in the pathogenesis of diseases such as chronic obstructive pulmonary disease (COPD), asthma, and infectious diseases of the lung [5– 9]. Pulmonary diseases are * Corresponding author. Fax: +1 603 650 6223. E-mail address: [email protected] (C.L. Sentman). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.08.007
rapidly rising with significant morbidity and mortality [8– 10]. Pulmonary macrophages have an important role in clearing debris from the alveolar spaces. The dynamics of macrophages within tissues are poorly understood, and the ability to study macrophages within a particular tissue in the absence of infiltrating monocyte/macrophages is difficult. Cell and tissue specific transgenes have been very valuable to study individual cell types within a complex tissue microenvironment [11– 13]. In this paper, we describe a new model for examining the biology and function of pulmonary macrophages based on the selective expression of EGFP in these cells. We have used a modified CD2 promoter to drive EGFP expression and found that pulmonary macrophages selectively express EGFP at a high level. This selective high expression of EGFP allows the identification and study of pulmonary macrophages in the context of the lung environment as they respond to various local challenges.
Materials and methods Transgenic mice Founder transgenic animals bearing the CD2-EGFP DNA construct in C57BL/6 mice were produced at the University
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of Virginia Transgenic Mouse Core Facility (Charlottesville, VA), as previously described [14], and at the Karolinska Institute (Stockholm, Sweden). Out of a total of 44 different potential founders, 16 were deemed positive for the presence of the EGFP construct by PCR. Following analysis of blood lymphocytes for green fluorescence by flow cytometry, three of the higher EGFP expressing strains were selected for further analysis and breeding. As one of these lines was derived from a Virginia founder, it is tentatively referred to here as the ‘‘Va’’ line. The other two lines, derived from Swedish founders, are called Sw739 and Sw740. In addition to continued breeding with C57BL/6J (wild type, wt) mice, the founders were also crossed with, and then F1 progeny backcrossed with, B6.129S7-Rag1tm1Mom (Rag1 / ) mice (Stock # 002216, Jackson Laboratories, Bar Harbor, ME). Mice used in experiments were between 6 and 12 weeks of age. Littermates that were B6 mice or Rag1 / mice served as negative control animals. Mice were typed for EGFP and Rag1 / status by flow cytometry using blood cells as previously described [15]. All experiments were conducted according to protocols approved by Dartmouth College’s Institutional Animal Care and Use Committee. Sample preparation and flow cytometry After sacrifice, tissues were removed and washed briefly with PBS/heparin. Prior to removal, lungs were perfused with 10 ml PBS/heparin via the right side of the heart until the lobes of the lungs became white. After perfusion, the lobes of the lungs were separated from the heart, major vessels, airways, and any connective tissue. Liver samples were perfused by intra-lobal injection with PBS/heparin. Lungs and liver were finely chopped with a scalpel and then subjected to digestion with collagenase (1 mg/ml) and DNAse (0.1 mg/ml) for 1 h at 37-C. Cells were dispersed from spleens and lymph nodes using forceps and the blunt end of a plastic syringe. Cells were washed with staining buffer (PBS containing 1% heat-inactivated fetal calf serum) and filtered through a 100 Am cell strainer (BD Biosciences, Bedford, MA). Viable cells were isolated using Histopaque1083 (Sigma-Aldrich, St. Louis, MO) gradients and then washed extensively in staining buffer. Fc receptors on cells were blocked with mouse mAb to mouse CD16/CD32 (Caltag Laboratories, Burlingame, CA) and total mouse gamma globulin (Jackson ImmunoResearch, West Grove, PA). Lymphocytes from blood, spleens, lymph nodes, lungs, and liver were also stained with some or all of the following: PE or APC-conjugated F4/80 (both clone BM8, eBioscience, San Diego, CA), APC-conjugated anti-CD11c (clone N418, eBioscience), PE-conjugated anti-CD11b (clone M1/70, BD Biosciences), APC-conjugated anti-CD86 (clone GL1, eBioscience), APC-conjugated anti-CD80 (clone 16-10A1, eBioscience), APC-conjugated anti-NK1.1 (clone PK136, eBioscience), PE-conjugated anti-CD8a (clone OX-8, BD Biosciences), PE-conjugated anti-CD3e (clone 145-2C11,
BD Biosciences), and PE-conjugated anti-CD19 (clone 1D3, BD Biosciences). Cell acquisition was performed with a dual-laser flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and data analyzed using CellQuest version 3.3 software (BD Biosciences). Confocal microscopic analysis Whole tissue pieces were placed on a microscope slide and examined for the presence and distribution of EGFP+ cells using a model LSM 510 META confocal microscope and version 3.2 of the supporting LSM 510 META software (Carl Zeiss, Thornwood, NY) that allowed single images, tile images, or image stacks to be collected. Small (approximately 2 mm3) samples of tissues were used for antibody staining. Prior to antibody staining, Fc receptors on cells were blocked with anti-CD16/CD32 and mouse gamma globulin for 2 h at 4-C. Staining was performed overnight in the dark at 4-C with APC-conjugated mAbs against NK1.1, F4/80, CD11c, CD80, or CD86. Following o/n incubation, samples were subjected to three 20-min washes in staining buffer at 4-C. Confocal images were obtained with the pinhole set to give an optical slice of 1.5 to 3 Am and using either a 10 or 20 plan apochromatic objective or a 40 oil immersion objective lens.
Results To develop a model system to study NK cell localization and function within various tissues in vivo, we have used the CD2 promoter to drive the expression of enhanced green fluorescent protein (EGFP) in transgenic mice. This promoter has been shown to give good expression of transgenes on T cells and NK cells [14,16]. To create an NK cell specific expression, these mice were bred to Rag1-deficient mice. Rag1 / mice lack T and B cells and can be reconstituted with normal T and B cells to create a mouse, where only NK cells express EGFP. When we examined these CD2EGFP.Rag1 / mice for the presence of EGFP+ cells in the lung, we observed a very bright EGFP+ cell population (Fig. 1). Lung tissue has some autofluorescence that allows the observation of airways and bronchioles. In addition, the EGFP transgenic mice had cells in the lung that expressed high levels of EGFP, making them easy to observe. These EGFP+ cells were evenly distributed throughout the lung tissue and had a mean diameter of 11.7 Am (T1.9 Am) and a mean cell density of 264/mm2 (T48/mm2) in the tissue. The cells were generally round, and some had cellular protrusions (Fig. 1i insert). The cell size, cell morphology, and distribution suggested that these cells were not lymphocytes. In contrast, lung NK cells (as identified by NK1.1 expression) expressed EGFP at a lower level (rendering them difficult to observe by EGFP alone using a confocal microscope) and had a mean diameter of 7.0 Am (T0.1 Am). Rag1 / EGFP transgenic mice had a similar number and distribution of these large EGFP+ cells as Rag gene expressing EGFP+
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Fig. 1. EGFP expression in lung cells of CD2-EGFP transgenic mice. Fresh lung pieces were analyzed by confocal microscopy for expression of EGFP+ cells. Lung tissue was taken from wild type (Rag1+) mice (a, c, e, g) or from Rag1 / mice (b, d, f, h) isolated from control non-transgenic littermates (a, b) or from three different lines of EGFP transgenic mice: Va mice (c, d), Sw740 (e, f), or Sw739 (g, h). (i) A composite image of part of the left lobe of the lung from Va.Rag1 / mice showing the wide distribution of EGFP+ cells and a large airway with smaller airway branches. All images were taken at using a 20 objective. The insert in panel (i) is an image obtained using a 40 objective and shows the morphology of the EGFP+ cells.
transgenic mice indicating that the EGFP+ cells were not T cells and that their presence in the lung was not dependent on factors derived from T or B cells (Fig. 1). To determine whether this EGFP expression was due to a random insertion of the transgene into a unique site that resulted in this unexpected EGFP expression in lung cells, we analyzed three independently derived transgenic mice (called Va, Sw740 and Sw739). The Va transgenic line had the highest expression levels, and similar patterns of EGFP+ cells were found within the lung of all three transgenic mice both on a Rag1+ and Rag1 / background. The size and distribution of these EGFP+ cells suggested that they were likely to be macrophages or dendritic cells. Flow cytometric analysis of lung leukocytes demonstrated that these cells were CD11c+, F4/80+, NK1.1 , and CD3
(Fig. 2). These EGFP+ cells expressed moderate levels of CD80 and low levels of CD86. Most of the EGFP+ cells were CD11c+, CD11b , although a small subset of EGFP+ cells were CD11c+, CD11b+(Fig. 3). The CD11c+, F4/80+, and EGFP+ cells were readily found in the bronchoalveolar lavage (data not shown). These data and the morphological analysis by confocal microscopy support the idea that the majority of these cells are lung macrophages, although some may be dendritic cells. As expected using the CD2 promoter, NK1.1+ cells are also EGFP+, but the EGFP expression in these lung NK cells was quite low compared to the expression found on the lung macrophages. Thus, it was easy to distinguish these two cell types based on cell size and EGFP expression. To characterize these EGFP+ cells in more detail, we used confocal microscopy and staining of live tissue cells.
Fig. 2. EGFP+ lung cells express CD11c, F4/80, CD80 and CD86. Single cell suspensions of lung tissue were stained for different cell surface markers and analyzed by flow cytometry to determine the phenotype of the EGFP expressing cells. Cells were isolated from Va.Rag1 / mice (top row) or Rag1 / nontransgenic littermates (bottom row). The expression of different cell surface molecules is presented on the y axis and EGFP expression on the x axis. Data are representative of five independent experiments.
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Fig. 3. EGFP+ lung cells express CD11c but not CD11b. Lung cells from Va.Rag1 / mice were analyzed for CD11c and CD11b (a), and subgating was used to determine the EGFP expression in CD11c+, CD11b cells (b); CD11c+, CD11b+ cells (c); and CD11c , CD11b+ cells (d). Analysis of CD11c and EGFP expression (e) demonstrated that 87% of CD11chi cells express EGFP.
Perfused lungs were dissected into small (2 mm3 approximately) pieces and incubated with APC-conjugated antibodies overnight at 4-C. Non-specific cell staining was blocked by inclusion of unlabeled mouse IgG antibodies. Tissue samples were analyzed by confocal microscopy (Fig. 4). This method does not use tissue fixatives or enzymes that may destroy antibody epitopes. This type of method has been employed on human tissue samples to allow analysis of cells within their tissue microenvironments [17]. We determined that the EGFP+ cells expressed both CD11c and F4/80 but not NK1.1 (Fig. 4). These data were consistent with our findings using isolated single cells from perfused and enzyme-digested lung tissue. These data support the idea that this transgenic model led to expression of EGFP on lung macrophages. To determine whether these EGFP+ cells have functional characteristics of macrophages, we tested their ability to rapidly adhere to plastic and to phagocytose latex beads. After 1-h adherence to plastic, we found an increase in the percentage of EGFP+ cells in the adherent population compared with the non-adherent population (Fig. 5). In other experiments, we observed that the EGFP+ cells were able to take up latex beads within 10 min (data not shown). These functional assays were consistent with the EGFP+ cell population containing primarily macrophages.
Although the level of EGFP expression in these lung macrophages was rather surprising, we only observed EGFP expression in CD11c+ or F4/80+ cells in the lung. We have examined EGFP expression in spleen, lymph node, blood, liver, and uterus and have not found elevated EGFP expression in CD11c+ or F4/80+ cells compared with nontransgenic mice (Fig. 6). Although macrophages and other large cells have a higher level of autofluorescence compared to lymphocytes, we observed no increase in the FL1 channel of CD11c+ or F4/80+ cells compared with cells from nontransgenic littermates. However, we did observe EGFP+ cells in these other tissues both using flow cytometry and confocal microscopy. The EGFP+ cells in other tissues were all NK cells (NK1.1+, CD3 ) in the Rag1 / mice and NK cells and T cells (CD3+) in Rag+ transgenic mice. In this EGFP model, the only macrophages that express EGFP were found in the lung. Blood monocytes did not express EGFP, so the EGFP+ cells that we observed were from within the lung tissue. We did not observe the presence of CD11c+, EGFP+ or F4/80+, EGFP+ cells in the draining lymph nodes from the lung, suggesting that these EGFP+ cells in the lung are resident cells and not able to migrate to lymph nodes, even after a tumor challenge (data not shown). This resident phenotype is consistent with the EGFP+ cells being lung macrophages and not dendritic cells.
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Fig. 4. CD11c+ and F4/80+ cells express EGFP in lung tissue. Pieces of whole lung from Va.Rag1 / mice were stained with APC-conjugated antibodies against NK1.1, CD11c, or F4/80 and analyzed by confocal microscopy. Data are shown as images for EGFP channel alone, APC channel alone, or a combined image. Samples stained with control antibody conjugated with APC did not show any APC staining (not shown). All samples were done in duplicate and representative images are shown. Similar data were observed in 4 independent experiments using lung tissue taken from both Va.Rag1 / and Sw740.Rag1 / mice.
This transgenic EGFP model can be used to study macrophage activation and movement within the lung. Pieces of lung tissue were cultured with 1 Ag/ml LPS or media only, and the movement of macrophages was monitored (Fig. 7). Following overnight culture, tissue pieces cultured with LPS demonstrated a striking movement of macrophages towards the edge of the tissue. These findings are consistent with LPS inducing local chemokines in lung tissue that recruit macrophages towards the stimulus.
These data indicate that the EGFP+ macrophages are not stationary cells but have the ability to actively move within the lung to respond to stimuli.
Discussion In this study, we report on a new mouse model with specific and high expression of EGFP in lung macrophages.
Fig. 5. EGFP+ lung cells adhere to plastic. Single cell suspensions of Va.Rag1 / lung cells were placed in tissue culture plates for 1 h. Total cells (a), plastic non-adherent cells (b), and plastic adherent cells (c) were analyzed for EGFP expression by flow cytometry. The percent EGFP+ cells is shown. Data shown are representative of 3 experiments, and all samples were done in duplicate.
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Fig. 6. EGFP+ macrophages are only found in lung tissue. Tissues were analyzed for the expression of EGFP on the F4/80+ cells, as indicated. Tissues were taken from Va.Rag1 / mice or non-transgenic Rag1 / littermates. Only in lungs were F4/80+ cells observed that had high EGFP expression compared to non-transgenic littermates. Data are representative of 3 to 6 experiments for each tissue.
This is unique in that no other macrophage and monocytes express EGFP in this particular model. Although surprising that a CD2 promoter led to high EGFP expression in lung macrophages, there have been reports of low CD2 expres-
sion on lung macrophages and dendritic cells [18,19]. If the cell surface expression of CD2 is rather low, then why is there a high level of EGFP expression? One possible reason is that cell surface proteins may be turned over more rapidly.
Fig. 7. Lung macrophages migrate in tissue in presence of LPS. Tissue pieces of Va.Rag1 / lung tissue were incubated for 1 h or overnight with vehicle or LPS (1 Ag/ml). Samples were done in duplicate, and images are representative of data from two independent experiments.
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Upon binding to ligands, cell surface molecules may be removed from the cell surface so the steady state expression of these molecules can be quite low. However, EGFP may accumulate inside cells over time and lead to quite high expression levels. The EGFP+ lung cells express CD11c and F4/80. These markers have often been used to identify dendritic cells [20,21] and macrophages [22,23], respectively. This raises the issue of what type of cell expressed EGFP in the lungs of these transgenic mice? Both monocytederived DCs and macrophages are derived from blood monocytes so it is not surprising that they share many characteristics. Activated DCs can express F4/80 [24], and activated macrophages [25] and pulmonary macrophages can express CD11c [26]. The EGFP+ cells are mostly negative for CD11b, readily adherent to plastic, and phagocytic, so we conclude that most, if not all, of these cells are macrophages. Some EGFP+ cells may be dendritic cells or able to differentiate into dendritic cells, however, we did not observe long dendrites on these cells in the tissue nor did we find CD11c+, NK1.1 cells in the draining lymph nodes. This suggested that the EGFP+ cells did not move into lymph nodes and were unlikely to be dendritic cells. It is particularly interesting that blood monocytes in this model do not express EGFP. Monocytes differentiate into macrophages as they move from the blood into tissues. Macrophages in various tissues have unique characteristics and specialized functions for those tissues [3,4]. The signals that induce EGFP expression in these cells may involve interactions of monocytes with other cells within the lung or due to environmental factors present in the lung. However, macrophages at other mucosal sites (intestine and uterus) did not express EGFP, suggesting that unique cells or lung specific environmental stimuli account for the induction of EGFP expression in lung macrophages. This macrophage EGFP model may be useful for studying the function of lung macrophages during infection or in response to other physiological challenges. We have been able to observe these macrophages as they interact with tumor metastasis and in response to LPS exposure. The EGFP is found within the cytoplasm of these cells, so it reveals the changes in cell shape and dynamics as the macrophages become activated. In some cases, we have observed an increase in the amount of dendrites and lamellapodia on the lung macrophages. In summary, we describe a new transgenic mouse model that will allow the greater identification and study of pulmonary macrophages within the lung.
Acknowledgments We would like to thank Klaus Ley of the University of Virginia for providing the ‘‘Va’’ line of transgenic EGFP
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mice. Also, thanks to Ken Orndorff, Gary Ward and Alice Givan for technical assistance with flow cytometry and confocal microscopy. This work was supported by grants from the Cancer Research Institute and the National Institutes of Health (CA101748).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 08.007.
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Research Article
GFP transgenic mice show dynamics of lung macrophages Martin Grundy, Charles L. Sentman* Department of Microbiology and Immunology, Dartmouth Medical School, One Medical Center Drive, Lebanon, NH 03756, USA Received 25 July 2005, revised version received 12 August 2005, accepted 17 August 2005 Available online 19 September 2005
Abstract The dynamics of tissue macrophages are poorly understood. We have developed a model where only lung macrophages express high levels of enhanced green fluorescent protein (EGFP) and are easily identified and followed by confocal microscopy. The EGFP+ cells had the morphology of macrophages and express CD11c, CD11b, and F4/80, but not NK1.1 or CD3. The F4/80+EGFP+ cells were found exclusively in the lung and not in lymph nodes, spleen, blood, liver, intestine, or uterus. These EGFP+ cells are phagocytic and can be activated to migrate within the lung in response to LPS stimulation. In this study, we describe a new model system that allows the specific study of macrophages in the lung. D 2005 Elsevier Inc. All rights reserved. Keywords: Lung macrophage; Green fluorescent protein; Cellular migration; Transgenic mice; CD2; LPS
Introduction Macrophages are long-lived cells found in almost every tissue in the body and have important roles in tissue maintenance, reorganization, and innate immunity. Macrophages act as sentinels of the immune system and have the ability to rapidly recognize and destroy invading microorganisms. In addition, macrophages can produce a large array of cytokines that help initiate a local immune response, and they can be activated by cytokines, such as IFN-g, from other leukocytes to up-regulate their phagocytic and killing activity [1]. Macrophages are believed to arise from monocytes that migrate from the blood and differentiate in tissues to become macrophages [2– 4]. Due to the unique function and cell components of each tissue, macrophages become specialized to perform specific functions in different tissues, while they retain their basic phagocytic and innate immune functions [2– 4]. Pulmonary macrophages are believed to play an important role in the pathogenesis of diseases such as chronic obstructive pulmonary disease (COPD), asthma, and infectious diseases of the lung [5– 9]. Pulmonary diseases are * Corresponding author. Fax: +1 603 650 6223. E-mail address: [email protected] (C.L. Sentman). 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2005.08.007
rapidly rising with significant morbidity and mortality [8– 10]. Pulmonary macrophages have an important role in clearing debris from the alveolar spaces. The dynamics of macrophages within tissues are poorly understood, and the ability to study macrophages within a particular tissue in the absence of infiltrating monocyte/macrophages is difficult. Cell and tissue specific transgenes have been very valuable to study individual cell types within a complex tissue microenvironment [11– 13]. In this paper, we describe a new model for examining the biology and function of pulmonary macrophages based on the selective expression of EGFP in these cells. We have used a modified CD2 promoter to drive EGFP expression and found that pulmonary macrophages selectively express EGFP at a high level. This selective high expression of EGFP allows the identification and study of pulmonary macrophages in the context of the lung environment as they respond to various local challenges.
Materials and methods Transgenic mice Founder transgenic animals bearing the CD2-EGFP DNA construct in C57BL/6 mice were produced at the University
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of Virginia Transgenic Mouse Core Facility (Charlottesville, VA), as previously described [14], and at the Karolinska Institute (Stockholm, Sweden). Out of a total of 44 different potential founders, 16 were deemed positive for the presence of the EGFP construct by PCR. Following analysis of blood lymphocytes for green fluorescence by flow cytometry, three of the higher EGFP expressing strains were selected for further analysis and breeding. As one of these lines was derived from a Virginia founder, it is tentatively referred to here as the ‘‘Va’’ line. The other two lines, derived from Swedish founders, are called Sw739 and Sw740. In addition to continued breeding with C57BL/6J (wild type, wt) mice, the founders were also crossed with, and then F1 progeny backcrossed with, B6.129S7-Rag1tm1Mom (Rag1 / ) mice (Stock # 002216, Jackson Laboratories, Bar Harbor, ME). Mice used in experiments were between 6 and 12 weeks of age. Littermates that were B6 mice or Rag1 / mice served as negative control animals. Mice were typed for EGFP and Rag1 / status by flow cytometry using blood cells as previously described [15]. All experiments were conducted according to protocols approved by Dartmouth College’s Institutional Animal Care and Use Committee. Sample preparation and flow cytometry After sacrifice, tissues were removed and washed briefly with PBS/heparin. Prior to removal, lungs were perfused with 10 ml PBS/heparin via the right side of the heart until the lobes of the lungs became white. After perfusion, the lobes of the lungs were separated from the heart, major vessels, airways, and any connective tissue. Liver samples were perfused by intra-lobal injection with PBS/heparin. Lungs and liver were finely chopped with a scalpel and then subjected to digestion with collagenase (1 mg/ml) and DNAse (0.1 mg/ml) for 1 h at 37-C. Cells were dispersed from spleens and lymph nodes using forceps and the blunt end of a plastic syringe. Cells were washed with staining buffer (PBS containing 1% heat-inactivated fetal calf serum) and filtered through a 100 Am cell strainer (BD Biosciences, Bedford, MA). Viable cells were isolated using Histopaque1083 (Sigma-Aldrich, St. Louis, MO) gradients and then washed extensively in staining buffer. Fc receptors on cells were blocked with mouse mAb to mouse CD16/CD32 (Caltag Laboratories, Burlingame, CA) and total mouse gamma globulin (Jackson ImmunoResearch, West Grove, PA). Lymphocytes from blood, spleens, lymph nodes, lungs, and liver were also stained with some or all of the following: PE or APC-conjugated F4/80 (both clone BM8, eBioscience, San Diego, CA), APC-conjugated anti-CD11c (clone N418, eBioscience), PE-conjugated anti-CD11b (clone M1/70, BD Biosciences), APC-conjugated anti-CD86 (clone GL1, eBioscience), APC-conjugated anti-CD80 (clone 16-10A1, eBioscience), APC-conjugated anti-NK1.1 (clone PK136, eBioscience), PE-conjugated anti-CD8a (clone OX-8, BD Biosciences), PE-conjugated anti-CD3e (clone 145-2C11,
BD Biosciences), and PE-conjugated anti-CD19 (clone 1D3, BD Biosciences). Cell acquisition was performed with a dual-laser flow cytometer (FACSCalibur; BD Biosciences, San Jose, CA) and data analyzed using CellQuest version 3.3 software (BD Biosciences). Confocal microscopic analysis Whole tissue pieces were placed on a microscope slide and examined for the presence and distribution of EGFP+ cells using a model LSM 510 META confocal microscope and version 3.2 of the supporting LSM 510 META software (Carl Zeiss, Thornwood, NY) that allowed single images, tile images, or image stacks to be collected. Small (approximately 2 mm3) samples of tissues were used for antibody staining. Prior to antibody staining, Fc receptors on cells were blocked with anti-CD16/CD32 and mouse gamma globulin for 2 h at 4-C. Staining was performed overnight in the dark at 4-C with APC-conjugated mAbs against NK1.1, F4/80, CD11c, CD80, or CD86. Following o/n incubation, samples were subjected to three 20-min washes in staining buffer at 4-C. Confocal images were obtained with the pinhole set to give an optical slice of 1.5 to 3 Am and using either a 10 or 20 plan apochromatic objective or a 40 oil immersion objective lens.
Results To develop a model system to study NK cell localization and function within various tissues in vivo, we have used the CD2 promoter to drive the expression of enhanced green fluorescent protein (EGFP) in transgenic mice. This promoter has been shown to give good expression of transgenes on T cells and NK cells [14,16]. To create an NK cell specific expression, these mice were bred to Rag1-deficient mice. Rag1 / mice lack T and B cells and can be reconstituted with normal T and B cells to create a mouse, where only NK cells express EGFP. When we examined these CD2EGFP.Rag1 / mice for the presence of EGFP+ cells in the lung, we observed a very bright EGFP+ cell population (Fig. 1). Lung tissue has some autofluorescence that allows the observation of airways and bronchioles. In addition, the EGFP transgenic mice had cells in the lung that expressed high levels of EGFP, making them easy to observe. These EGFP+ cells were evenly distributed throughout the lung tissue and had a mean diameter of 11.7 Am (T1.9 Am) and a mean cell density of 264/mm2 (T48/mm2) in the tissue. The cells were generally round, and some had cellular protrusions (Fig. 1i insert). The cell size, cell morphology, and distribution suggested that these cells were not lymphocytes. In contrast, lung NK cells (as identified by NK1.1 expression) expressed EGFP at a lower level (rendering them difficult to observe by EGFP alone using a confocal microscope) and had a mean diameter of 7.0 Am (T0.1 Am). Rag1 / EGFP transgenic mice had a similar number and distribution of these large EGFP+ cells as Rag gene expressing EGFP+
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Fig. 1. EGFP expression in lung cells of CD2-EGFP transgenic mice. Fresh lung pieces were analyzed by confocal microscopy for expression of EGFP+ cells. Lung tissue was taken from wild type (Rag1+) mice (a, c, e, g) or from Rag1 / mice (b, d, f, h) isolated from control non-transgenic littermates (a, b) or from three different lines of EGFP transgenic mice: Va mice (c, d), Sw740 (e, f), or Sw739 (g, h). (i) A composite image of part of the left lobe of the lung from Va.Rag1 / mice showing the wide distribution of EGFP+ cells and a large airway with smaller airway branches. All images were taken at using a 20 objective. The insert in panel (i) is an image obtained using a 40 objective and shows the morphology of the EGFP+ cells.
transgenic mice indicating that the EGFP+ cells were not T cells and that their presence in the lung was not dependent on factors derived from T or B cells (Fig. 1). To determine whether this EGFP expression was due to a random insertion of the transgene into a unique site that resulted in this unexpected EGFP expression in lung cells, we analyzed three independently derived transgenic mice (called Va, Sw740 and Sw739). The Va transgenic line had the highest expression levels, and similar patterns of EGFP+ cells were found within the lung of all three transgenic mice both on a Rag1+ and Rag1 / background. The size and distribution of these EGFP+ cells suggested that they were likely to be macrophages or dendritic cells. Flow cytometric analysis of lung leukocytes demonstrated that these cells were CD11c+, F4/80+, NK1.1 , and CD3
(Fig. 2). These EGFP+ cells expressed moderate levels of CD80 and low levels of CD86. Most of the EGFP+ cells were CD11c+, CD11b , although a small subset of EGFP+ cells were CD11c+, CD11b+(Fig. 3). The CD11c+, F4/80+, and EGFP+ cells were readily found in the bronchoalveolar lavage (data not shown). These data and the morphological analysis by confocal microscopy support the idea that the majority of these cells are lung macrophages, although some may be dendritic cells. As expected using the CD2 promoter, NK1.1+ cells are also EGFP+, but the EGFP expression in these lung NK cells was quite low compared to the expression found on the lung macrophages. Thus, it was easy to distinguish these two cell types based on cell size and EGFP expression. To characterize these EGFP+ cells in more detail, we used confocal microscopy and staining of live tissue cells.
Fig. 2. EGFP+ lung cells express CD11c, F4/80, CD80 and CD86. Single cell suspensions of lung tissue were stained for different cell surface markers and analyzed by flow cytometry to determine the phenotype of the EGFP expressing cells. Cells were isolated from Va.Rag1 / mice (top row) or Rag1 / nontransgenic littermates (bottom row). The expression of different cell surface molecules is presented on the y axis and EGFP expression on the x axis. Data are representative of five independent experiments.
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Fig. 3. EGFP+ lung cells express CD11c but not CD11b. Lung cells from Va.Rag1 / mice were analyzed for CD11c and CD11b (a), and subgating was used to determine the EGFP expression in CD11c+, CD11b cells (b); CD11c+, CD11b+ cells (c); and CD11c , CD11b+ cells (d). Analysis of CD11c and EGFP expression (e) demonstrated that 87% of CD11chi cells express EGFP.
Perfused lungs were dissected into small (2 mm3 approximately) pieces and incubated with APC-conjugated antibodies overnight at 4-C. Non-specific cell staining was blocked by inclusion of unlabeled mouse IgG antibodies. Tissue samples were analyzed by confocal microscopy (Fig. 4). This method does not use tissue fixatives or enzymes that may destroy antibody epitopes. This type of method has been employed on human tissue samples to allow analysis of cells within their tissue microenvironments [17]. We determined that the EGFP+ cells expressed both CD11c and F4/80 but not NK1.1 (Fig. 4). These data were consistent with our findings using isolated single cells from perfused and enzyme-digested lung tissue. These data support the idea that this transgenic model led to expression of EGFP on lung macrophages. To determine whether these EGFP+ cells have functional characteristics of macrophages, we tested their ability to rapidly adhere to plastic and to phagocytose latex beads. After 1-h adherence to plastic, we found an increase in the percentage of EGFP+ cells in the adherent population compared with the non-adherent population (Fig. 5). In other experiments, we observed that the EGFP+ cells were able to take up latex beads within 10 min (data not shown). These functional assays were consistent with the EGFP+ cell population containing primarily macrophages.
Although the level of EGFP expression in these lung macrophages was rather surprising, we only observed EGFP expression in CD11c+ or F4/80+ cells in the lung. We have examined EGFP expression in spleen, lymph node, blood, liver, and uterus and have not found elevated EGFP expression in CD11c+ or F4/80+ cells compared with nontransgenic mice (Fig. 6). Although macrophages and other large cells have a higher level of autofluorescence compared to lymphocytes, we observed no increase in the FL1 channel of CD11c+ or F4/80+ cells compared with cells from nontransgenic littermates. However, we did observe EGFP+ cells in these other tissues both using flow cytometry and confocal microscopy. The EGFP+ cells in other tissues were all NK cells (NK1.1+, CD3 ) in the Rag1 / mice and NK cells and T cells (CD3+) in Rag+ transgenic mice. In this EGFP model, the only macrophages that express EGFP were found in the lung. Blood monocytes did not express EGFP, so the EGFP+ cells that we observed were from within the lung tissue. We did not observe the presence of CD11c+, EGFP+ or F4/80+, EGFP+ cells in the draining lymph nodes from the lung, suggesting that these EGFP+ cells in the lung are resident cells and not able to migrate to lymph nodes, even after a tumor challenge (data not shown). This resident phenotype is consistent with the EGFP+ cells being lung macrophages and not dendritic cells.
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Fig. 4. CD11c+ and F4/80+ cells express EGFP in lung tissue. Pieces of whole lung from Va.Rag1 / mice were stained with APC-conjugated antibodies against NK1.1, CD11c, or F4/80 and analyzed by confocal microscopy. Data are shown as images for EGFP channel alone, APC channel alone, or a combined image. Samples stained with control antibody conjugated with APC did not show any APC staining (not shown). All samples were done in duplicate and representative images are shown. Similar data were observed in 4 independent experiments using lung tissue taken from both Va.Rag1 / and Sw740.Rag1 / mice.
This transgenic EGFP model can be used to study macrophage activation and movement within the lung. Pieces of lung tissue were cultured with 1 Ag/ml LPS or media only, and the movement of macrophages was monitored (Fig. 7). Following overnight culture, tissue pieces cultured with LPS demonstrated a striking movement of macrophages towards the edge of the tissue. These findings are consistent with LPS inducing local chemokines in lung tissue that recruit macrophages towards the stimulus.
These data indicate that the EGFP+ macrophages are not stationary cells but have the ability to actively move within the lung to respond to stimuli.
Discussion In this study, we report on a new mouse model with specific and high expression of EGFP in lung macrophages.
Fig. 5. EGFP+ lung cells adhere to plastic. Single cell suspensions of Va.Rag1 / lung cells were placed in tissue culture plates for 1 h. Total cells (a), plastic non-adherent cells (b), and plastic adherent cells (c) were analyzed for EGFP expression by flow cytometry. The percent EGFP+ cells is shown. Data shown are representative of 3 experiments, and all samples were done in duplicate.
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Fig. 6. EGFP+ macrophages are only found in lung tissue. Tissues were analyzed for the expression of EGFP on the F4/80+ cells, as indicated. Tissues were taken from Va.Rag1 / mice or non-transgenic Rag1 / littermates. Only in lungs were F4/80+ cells observed that had high EGFP expression compared to non-transgenic littermates. Data are representative of 3 to 6 experiments for each tissue.
This is unique in that no other macrophage and monocytes express EGFP in this particular model. Although surprising that a CD2 promoter led to high EGFP expression in lung macrophages, there have been reports of low CD2 expres-
sion on lung macrophages and dendritic cells [18,19]. If the cell surface expression of CD2 is rather low, then why is there a high level of EGFP expression? One possible reason is that cell surface proteins may be turned over more rapidly.
Fig. 7. Lung macrophages migrate in tissue in presence of LPS. Tissue pieces of Va.Rag1 / lung tissue were incubated for 1 h or overnight with vehicle or LPS (1 Ag/ml). Samples were done in duplicate, and images are representative of data from two independent experiments.
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Upon binding to ligands, cell surface molecules may be removed from the cell surface so the steady state expression of these molecules can be quite low. However, EGFP may accumulate inside cells over time and lead to quite high expression levels. The EGFP+ lung cells express CD11c and F4/80. These markers have often been used to identify dendritic cells [20,21] and macrophages [22,23], respectively. This raises the issue of what type of cell expressed EGFP in the lungs of these transgenic mice? Both monocytederived DCs and macrophages are derived from blood monocytes so it is not surprising that they share many characteristics. Activated DCs can express F4/80 [24], and activated macrophages [25] and pulmonary macrophages can express CD11c [26]. The EGFP+ cells are mostly negative for CD11b, readily adherent to plastic, and phagocytic, so we conclude that most, if not all, of these cells are macrophages. Some EGFP+ cells may be dendritic cells or able to differentiate into dendritic cells, however, we did not observe long dendrites on these cells in the tissue nor did we find CD11c+, NK1.1 cells in the draining lymph nodes. This suggested that the EGFP+ cells did not move into lymph nodes and were unlikely to be dendritic cells. It is particularly interesting that blood monocytes in this model do not express EGFP. Monocytes differentiate into macrophages as they move from the blood into tissues. Macrophages in various tissues have unique characteristics and specialized functions for those tissues [3,4]. The signals that induce EGFP expression in these cells may involve interactions of monocytes with other cells within the lung or due to environmental factors present in the lung. However, macrophages at other mucosal sites (intestine and uterus) did not express EGFP, suggesting that unique cells or lung specific environmental stimuli account for the induction of EGFP expression in lung macrophages. This macrophage EGFP model may be useful for studying the function of lung macrophages during infection or in response to other physiological challenges. We have been able to observe these macrophages as they interact with tumor metastasis and in response to LPS exposure. The EGFP is found within the cytoplasm of these cells, so it reveals the changes in cell shape and dynamics as the macrophages become activated. In some cases, we have observed an increase in the amount of dendrites and lamellapodia on the lung macrophages. In summary, we describe a new transgenic mouse model that will allow the greater identification and study of pulmonary macrophages within the lung.
Acknowledgments We would like to thank Klaus Ley of the University of Virginia for providing the ‘‘Va’’ line of transgenic EGFP
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mice. Also, thanks to Ken Orndorff, Gary Ward and Alice Givan for technical assistance with flow cytometry and confocal microscopy. This work was supported by grants from the Cancer Research Institute and the National Institutes of Health (CA101748).
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.yexcr.2005. 08.007.
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