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Morphologic Detection and Functional Assessment of Reconstituted Normal Alveolar Macrophages in the Lungs of SCID Mice¹

Division of Pulmonary, Allergy, Critical Care and Occupational Medicine, Department of Medicine, Indiana University School of Medicine, Indianapolis, IN 46202

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alveolar macrophages (AMs) from immunocompetent animals were isolated from bronchoalveolar lavage and labeled with the fluorescent marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). These AMs were administered intratracheally into mechanically ventilated SCID mice. From 1 to 28 days later, the recipient mice underwent bronchoalveolar lavage to isolate their AMs. To determine whether reconstituted AMs were still immunocompetent, the recovered AMs were assayed for their ability to phagocytose fluorescein-labeled zymosan-coated beads. After incubation with the beads, samples were assayed using a fluorescent-activated cell sorter to identify DiI-labeled reconstituted AMs, unlabeled resident AMs, and the proportion of these two groups undergoing phagocytosis. DiI-labeled AMs accounted for 50% of all returned AMs. Additionally, the reconstituted AMs from normal BALB/c mice retained phagocytic activity compared with AMs from immunodeficient SCID mice. Reconstituted AMs demonstrated enhanced phagocytic activity compared with resident SCID AMs for up to 28 days following reconstitution. These results indicate that immunocompetent AMs can be successfully reconstituted into an immunodeficient host to partially restore alveolar host defense.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Alveolar macrophages (AMs)³ represent the first line of host defense within the lung (1, 2). AMs are critical to the primary immune response and recruit and modulate the subsequent immune response. For example, AMs are responsible for the initial phagocytosis and clearance of invading microorganisms as well as recruitment of inflammatory and effector cells (1). Physical clearance of microorganisms by AMs occurs by phagocytosis and the generation of reactive oxygen species and reactive nitrogen intermediates (3).

AM depletion or dysfunction significantly contributes to host susceptibility to invading pathogens (4, 5). Depletion of AMs by agents such as dichloromethylene diphosphonate results in a much greater risk of pulmonary infection (1, 6, 7, 8). Additionally, AM dysfunction induced by drugs such as corticosteroid or cytotoxic agents also increases risk of opportunistic pulmonary infection (9, 10, 11).

During immunodeficiency the important role of critical components in the immune system to resist infection is classically demonstrated by reconstituting normal immune cells into the immunodeficient host and demonstrating whether restoration of immunity affords protection. As an example, the critical role of CD4⁺ lymphocytes in defense against Pneumocystis carinii organisms was demonstrated by reconstitution of CD4⁺ cells into SCID mice (12, 13). Although AMs are thought to be important in host defense, similar reconstitution studies using AMs have not been conducted.

We have recently demonstrated that genetically engineered J774A.1 macrophages can be delivered into the lungs of recipient SCID mice (14). In the current study, we test the hypothesis that AMs from a normal host would restore alveolar host defense in an immunodeficient mouse. To test the hypothesis, we labeled normal immunocompetent AMs with the membrane marker 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI) (15, 16, 17) and reconstituted the labeled AMs into the lungs of SCID mice. DiI labeling was retained by the donor AMs and represented a marker for the reconstituted AMs in both tissue and bronchoalveolar lavage (BAL) as well as by flow cytometry analysis. Reconstituted normal AMs retained functional activity during the 28 days of the experiment. Apparently, AMs independent of other immune cells can partially restore alveolar host defense. These studies suggest that reconstituted AMs are easily identified as being widely distributed in the lungs and retain normal function and activity within the lungs of the recipient immunodeficient mice.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Isolation and labeling of immunocompetent AMs

Normal immunocompetent AMs were isolated from 8- to 10-wk-old BALB/c mice (Harlan Sprague Dawley, Indianapolis, IN) by BAL (18). Mice were sacrificed using CO₂ inhalation. Following death, their trachea were exposed via a midline neck incision using blunt dissection. A 20-gauge angiocath (BD Biosciences, Sandy, UT) was inserted into the trachea and a lavage was performed with a 1-ml syringe. Lavage fluid consisted of HBSS (Life Technologies, Grand Island, NY) with 0.6 mM ethylenediaminetetraacetic acid, 100 U/ml penicillin, and 100 µg/ml streptomycin. Mice were lavaged 10 times with 0.8 ml per lavage. The cell suspension was pooled and centrifuged (1400 x g x 5 min) and the supernatant discarded. Trace amounts of RBCs were lysed by resuspending the pellet in a 0.01 M potassium bicarbonate and 0.15 M ammonium chloride solution. The AMs were again centrifuged (1400 x g x 5 min) and resuspended in DMEM plus 100 U/ml penicillin and 100 µg/ml streptomycin, and the number of AMs was enumerated with a hemacytometer (Reichert, Buffalo, NY). AM viability was >98% using trypan blue exclusion. Cellular composition was determined by cytopreparation smears prepared in a Cytospin 2 (Thermo Shandon, Pittsburgh, PA). Cytopreparation smear slides were stained with Hema III stain (Biochemical Sciences, Swedesboro, NJ) and cellular differential was performed. Only cellular suspensions that contained >98% AMs were used for reconstitution studies.

AMs were labeled by incubating with the membrane marker DiI (Molecular Probes, Eugene, OR) (17) at a concentration of 10 µM of DiI at 37°C x 10 min. The suspension was then washed three times to remove unincorporated DiI and the final pellet of AMs was resuspended in the appropriate volume of HBSS.

Reconstitution of AMs

AMs were intratracheally (i.t.) administered into SCID mice (19). Recipient BALB/c SCID mice (Harlan Sprague Dawley) were anesthetized with 0.15 ml of an anesthetic mixture consisting of 10 mg/ml ketamine (Bedford Laboratories, Bedford, OH), 0.11 mg/ml atropine (Butler, Columbus, OH), and 2 mg/ml acepromazine (Butler). When the mice had reached a sufficient level of anesthesia, a midline neck incision was made and the trachea was exposed using blunt dissection. The mice experienced no discernible pain. A small incision was made between tracheal rings and a 20-gauge angiocath was inserted into the trachea. DiI-labeled AMs at a concentration of 5 x 10⁴, 5 x 10⁵, or 2.5 x 10⁶ in 50 µl HBSS were directly administered into the lungs via the tracheal catheter. The cell suspension was followed by 200 µl of air to clear the angiocath and upper respiratory tract. To ensure even distribution of the AMs, the mice were mechanically ventilated as previously described from our laboratory (19). The mouse was maintained on the ventilator (Analytic Specialties, St. Louis, MO) that was directly connected to the airway catheter. The ventilator was set at a maximum peak inspiratory pressure of 4 cm H₂O, a respiratory rate of 150 breaths/min, and an inspiratory to expiratory ratio of 1:2. This resulted in a tidal volume of 150–250 µl/breath. Mice were ventilated for 10 min and then allowed to recover. To determine viability of donor AMs, DiI-labeled AMs were assessed for trypan blue exclusion as described above. Lung sections and cytopreparation smears of BAL were morphologically assessed using phase and epifluorescent microscopy (Olympus BX60FS fluorescent microscope; Melville, NY) as well as confocal microscopy (Zeiss LSM-510 confocal microscope; Thornwood, NY).

Distribution of AMs in the lungs

To determine the distribution of the i.t.-delivered AMs, the mice were sacrificed 24 h later, the thoracic cavity was opened, and the lungs were perfused with 2 ml HBSS plus heparin via the right ventricle. The right lobe was then removed and the three sections (upper, middle, lower) were separated and average weight was determined. Each section was then placed into 1 ml of HBSS and homogenized with a Tissue Tearor (Dremel, Racine, WI). Fluorescence was determined using 100-µl samples in triplicate using cytofluor (model 2350, Fluorescent Measurement Systems; Millipore, Bedford, MA) with an excitation of 530 nm and an emission of 590 nm. Lung tissue of similar mass without any reconstituted cells was used as a background. Data are presented as an evenness index as previously described (19). The evenness index is the ratio of ((fluorescence/gram wet mass_piece)/(fluorescence/gram wet mass_whole)) x 100.

Retention of DiI label by AMs

To determine whether the DiI label is retained by the AMs, we conducted two different experiments. In the first experiment, we assessed retention of the DiI labeling in vivo by DiI labeling donor AMs that have a unique cell surface Ag not present on cells in the recipient mouse. DiI-labeled AMs from normal BALB/c mice positive for H₂D surface Ag and negative for H₂K Ag (H₂D⁺/H₂K^-) were i.t. administered into normal C3H mice whose resident AMs are negative for H₂D but positive for H₂K surface Ag (H₂D^-/H₂K⁺). BALs were collected at days 1, 3, 7, 14, and 28 from the C3H mice and incubated with FITC-conjugated anti-mouse H₂D (clone no. 34-2-12)/anti-mouse H₂K (clone no. AF3-12.1) mAbs to the MHC class I surface Ags (BD Biosciences, San Diego, CA) for 1 h at 37°C to identify the AM surface Ags (H₂D or H₂K). After incubation with the FITC-conjugated anti-mouse Ab, the AMs were washed and centrifuged (500 x g for 10 min) to remove unbound Ab. The fraction of AMs labeled in each cell population was then determined by FACS analysis. The cells from the BAL were also incubated with an appropriate control isotype mAb (IgG1 and IgG2a subtype) (BD Biosciences).

In a second experiment, we assessed AM retention of the DiI label in vitro. BALB/c mice AMs were labeled with DiI and were coincubated with unlabeled AMs in a 100-mm culture plate (BD Biosciences, Lincoln Park, NJ). The identical cells were assessed and photographed up to 2 wk to examine retention of the DiI label.

Assessment of AM function in vitro

Attachment/phagocytosis of zymosan-coated beads by AMs was used as a measure of AM function. DiI-labeled AMs from normal BALB/c mice (5 x 10⁵/50 µl) were i.t. administered into BALB/c SCID mice as described above. Control studies were conducted with i.t. administration of SCID AMs into SCID mice and normal AMs in normal BALB/c mice. Recipient mice were sacrificed at 1, 3, 7, or 28 days after reconstitution and were lavaged as described earlier. Each lavage was kept separate and resuspended in a final volume of 1 ml DMEM plus 10% FBS and 100 U/ml penicillin and 100 µg/ml streptomycin. AMs were counted on a hemacytometer. Two cytopreparation smears were prepared. One cytopreparation slide was stained with Hema III stain for a cellular differential, while the other was viewed under fluorescence microscopy using an Olympus BX60F5 microscope with an Optronics International DEI-750 digital output visualization system (Goleta, CA).

AMs were incubated for 1 h with fluorescein-labeled zymosan-coated beads (Molecular Probes, Eugene, OR). Fluorescent patterns for DiI-labeled AMs and fluorescein-labeled zymosan-coated beads were then determined using a FACScan flow cytometer (BD Biosciences). The FACS separates the experimental sample into four groups: unlabeled (resident) AMs with or without zymosan-coated beads, DiI labeled (donor) AMs with or without zymosan-coated beads. The percentage of total cells in each group was determined. Phagocytosis was expressed as percentage of AMs (unlabeled or labeled with DiI) with zymosan-coated beads/total number of respective AMs that are unlabeled or labeled with DiI. For reference, the following groups were individually assayed by FACS: freshly isolated AMs, freshly labeled DiI AMs, and zymosan-coated beads.

Assessment of AM function in vivo

AMs from normal BALB/c mice (5 x 10⁵/50 µl) were i.t. administered into SCID mice as described. Seven days later, mice were again placed on mechanical ventilation and received i.t. administration of 2 x 10⁶ zymosan-coated beads (Molecular Probes) labeled with FITC in 50 µl. One hour later, a BAL was performed and the BAL was divided into two aliquots. From one aliquot of BAL, duplicate cytopreparation smears were prepared. One was stained with Hema III for a cellular differential, while the other was viewed under fluorescence microscopy as described. Attachment/phagocytosis of the zymosan-coated beads by the BAL cells was demonstrated by phase and fluorescent microscopy. The other aliquot of BAL was analyzed by FACS as described above to assess the attachment/phagocytosis of zymosan-coated beads by DiI-labeled reconstituted AMs and resident AMs in SCID mice.

Statistical analysis

All experiments were performed in triplicate and each experiment was repeated three times. The difference between the control and experimental data were compared using Student’s t test or ANOVA with paired comparisons. The difference was considered statistically significant when p < 0.05 (20).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
To determine the optimal number of AMs for reconstitution, various concentrations of DiI-labeled AMs were i.t. delivered into SCID mice. After 7 days, these mice were sacrificed and a BAL was performed. The percent of AMs labeled with DiI was determined by flow cytometry (Fig. 1). Administration of 5 x 10⁵ DiI-labeled AMs resulted in approximately one-half of the returned BAL cells representing donor AMs. This concentration of administered AMs was used for the remainder of the experiments.

View larger version (14K): [in this window] [in a new window]   FIGURE 1. Determination of the optimal number of AMs for reconstitution in SCID mice. AMs obtained by BAL of BALB/c mice were incubated with 10 µM DiI for 10 min and were i.t. administered to mechanically ventilated SCID mice. After 7 days, the mice were sacrificed and a BAL was performed. The percentage of recovered DiI-labeled AMs was determined with flow cytometry. Results are expressed as mean ± SEM of three experiments performed in triplicate with significance as p < 0.05.

View larger version (55K): [in this window] [in a new window]   FIGURE 2. Morphologic evidence of DiI-labeled AMs in a recipient mouse lung. DiI-labeled normal AMs (5 x 105) or normal saline were administered i.t. into SCID mice. After 1 day, the reconstituted and normal mice were sacrificed and the lungs were viewed with confocal microscopy. A, Lung section from a control mouse, showing the autofluorescence of the alveolar structures and a resident AM. B, Lung section from a reconstituted mouse revealing red-orange fluorescent DiI-labeled donor AMs on the alveolar walls.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. Distribution of reconstituted AMs in the recipient mouse lung. SCID mice were reconstituted with 5 x 105 DiI-labeled immunocompetent AMs. After 24 h, the mice were sacrificed and the right lungs were divided into three sections. Each section was homogenized and the fluorescence was determined. The evenness index was calculated as the ratio of ((fluorescence/gram wet masspiece)/(fluorescence/gram wet masswhole)) x 100. The lower lobe had a significantly greater evenness index (*, p < 0.05). Results are expressed as mean ± SEM of three experiments performed in triplicate.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Retention of DiI label by AMs in vivo. DiI-labeled AMs from BALB/c mice (H2D+ and H2K-) were i.t. administered into normal C3H mice (H2D-and H2K+). BAL of the recipient mice were performed at 1, 3, 7, 14, and 28 days and the BAL cells were incubated with FITC-labeled mAbs to H2D and H2K for 1 h at 37°C and analyzed by flow cytometry. The data indicate that the DiI label was retained by the donor H2D+ AMs for up to 28 days with no evidence of transfer of the DiI label to the resident H2K+ AMs in vivo (Fig. 4A). The percentage of AMs positive for H2D (DiI-labeled) and for H2K (unlabeled) remained the same for the full 28 days of the experiment (Fig. 4B). Results are expressed as mean ± SEM of three experiments performed in triplicate.

Evidence for retained function of reconstituted AMs

Reconstituted and resident AMs obtained by BAL at specific time points following reconstitution were incubated with fluorescein-labeled zymosan-coated beads and attachment/phagocytosis of the beads was determined by flow cytometry (Fig. 5). Additionally, freshly isolated AMs were incubated with zymosan-coated beads to compare the functional activity of fresh cells with those of reconstituted and resident AMs. At each time point, the reconstituted AMs demonstrated greater attachment/phagocytosis of the beads than did the resident SCID AMs (p < 0.05; Fig. 6A). As a control experiment, SCID AMs were i.t. administered into resident SCID mice and there were no differences in functional activity between the two SCID AM populations (Fig. 6B). Similarly, reconstitution of normal immunocompetent BALB/c AMs into normal BALB/c mice showed no differences in functional activity between the populations (Fig. 6C).

View larger version (39K): [in this window] [in a new window]   FIGURE 5. Flow cytometric analysis of attachment/phagocytosis of zymosan-coated beads by reconstituted AMs and resident SCID AMs. SCID mice were reconstituted with 5 x 105 DiI-labeled normal AMs. After 1 day, a BAL was performed and the cell suspension was incubated with fluorescein-labeled zymosan-coated beads for 1 h. Flow cytometry of the cell suspension revealed four groupings: A, DiI-labeled AMs without zymosan-coated beads; B, DiI-labeled AMs with zymosan-coated beads; C, resident SCID AMs without DiI label and without zymosan-coated bead; D, resident SCID AMs without DiI label but with zymosan-coated beads. The results demonstrate that reconstituted normal AMs are more functionally active compared with resident SCID AMs.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. Attachment/phagocytosis by reconstituted and resident AMs in vitro. A, SCID mice were reconstituted with 5 x 105 DiI-labeled immunocompetent AMs. After 1, 3, 7, or 28 days, the mice were sacrificed and BAL was performed. The recovered AMs were incubated with fluorescein-labeled zymosan-coated beads for 1 h. Attachment/phagocytosis by reconstituted normal AMs was significantly greater than occurred with the resident SCID AMs (*, p < 0.05). B, As a control experiment, SCID AMs were i.t. delivered into recipient SCID mice. There were no significant differences in functional activity between the two populations of SCID AMs. C, As a control experiment, BALB/c AMs were i.t. delivered into recipient BALB/c mice. Again, there were no significant differences in functional activity between the two populations of normal AMs. These results also suggest that the DiI labeling itself does not activate the donor AMs above baseline levels of the resident population. Results are expressed as mean ± SEM of three experiments performed in triplicate.

View larger version (68K): [in this window] [in a new window]   FIGURE 7. Morphologic evidence of attachment and phagocytosis by reconstituted and resident AMs. A, SCID mice were reconstituted with 5 x 105 DiI-labeled immunocompetent AMs. After 7 days, the animals were challenged with i.t. delivery of zymosan-coated beads. One hour later, the mice were sacrificed and a BAL was performed. Cytopreparation smears were prepared and were viewed using phase and fluorescent microscopy (40x) with the following results: a, Represents a phase image of the BAL. b, Reveals the green fluorescence of the zymosan-coated beads. c, Reveals red-orange fluorescence of the reconstituted DiI-labeled AMs. d, Shows the merged view of the three previous images. The zymosan-coated beads are predominantly associated with the DiI-labeled reconstituted AMs. B, Confocal microscopy was used to verify internalization of the fluorescein-labeled zymosan-coated beads by the AMs. The Z plane imaging of the cells (a–d) reveals the internalization of the beads by the AMs.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

AMs have many roles in host defense (25). Immunosuppression likely compromises the effectiveness of AMs regardless of cause. To assess the importance of AMs in health and disease, two approaches have traditionally been used: 1) in vitro assessment of AM function, or 2) in vivo AM depletion and assessment of effect on host defense. The former method suffers from the limitation of all in vitro studies as they may not reflect the in vivo condition; whereas, the latter method, using agents such as dichloromethylene diphosphonate, not only depletes the number of AMs, but also induces nonspecific effects such as marked inflammation (6). Use of reconstitution studies to restore AM function in vivo have not been reported. Thus, the definitive role of AMs in response to infection or potentially injurious particles remains surprisingly circumstantial.

A mouse has 6–8 million alveoli (26, 27) and it has been reported that there are 6 million AMs (27). In our study, a ratio of 1:10 donor AMs to the estimated number of alveoli using 5 x 10⁵ DiI-labeled AMs resulted in nearly 50% of returned AMs by BAL to be reconstituted AMs. When the ratio of instilled reconstituted AMs to alveoli was increased to nearly 1:1 (or 5 x 10⁶ AMs), almost all cells returned in the lavage were reconstituted normal BALB/c AMs. Likewise, when only 5 x 10⁴ (AMs-alveolar ratio of 1:100) AMs were reconstituted, almost all returned cells were resident SCID AMs. If prior morphometric assessment of endogenous AM numbers are accurate, these data suggest that resident AMs may be more adherent to alveolar walls than reconstituted AMs and therefore be less accessible to retrieval by BAL. Nonetheless, the concentration of 5 x 10⁵ AMs consistently proved to provide a good basis of comparison between reconstituted AMs and resident AMs.

DiI-labeled AMs were easily detectable within the alveolar environment as alveolar walls are identified by their autofluorescence. Similar to another study from our laboratory using fluorescent beads (19), a greater number of AMs were present in the caudal portion of the lung following i.t. administration. Although the percentage of AMs in the caudal portion of the lung was greater, there were still significant numbers of reconstituted AMs in the cephalad portion of the lung.

Most importantly, reconstituted AMs retained their ability to effectively phagocytose even when in the local alveolar environment of the immunosuppressed host. This was verified by both in vitro and in vivo measures of phagocytosis. Both reconstituted and resident AMs were isolated via BAL and were challenged with zymosan-coated beads. Using flow cytometry, it was found that a greater percentage of reconstituted cells had attached and phagocytosed the zymosan-coated beads. The reconstituted AMs retained this ability up to 28 days postreconstitution. The function of reconstituted AMs was similar to that of freshly isolated AMs from immunocompetent mice. This was not due to some artifact related to the in vitro incubation of AMs with the zymosan-coated beads in vitro, as AM function was also determined in vivo. SCID mice that were reconstituted with DiI-labeled AMs were subsequently challenged in vivo with the i.t. administration of zymosan-coated beads. The reconstituted AMs demonstrated significantly greater phagocytic activity in vivo when compared with the resident AMs from the SCID mice. It did not appear that the reconstituted normal AMs affected the baseline activity of the resident SCID AMs.

A surprising feature of these studies was the retention of superior AM function by the reconstituted AMs up to 28 days following reconstitution. We assumed that in the absence of proinflammatory cytokines in the immunosuppressed environment of the alveoli in SCID mice there would be a decline in AM function by 28 days. We do not have a clear explanation for this observation. It is possible that because SCID mice are "leaky" and retain some B and T cell function, the process of reconstitution is such that the reconstituted AMs are stimulated to a sufficient degree to retain near normal AM function for the period of this study.

In summary, we have developed a new method to effectively deliver and track normal AMs in the lungs of a recipient immunodeficient mouse. DiI labeling permits easy detection of the reconstituted AMs and provides comparison to resident AMs in the SCID mice. The DiI labeling of the donor AMs is fastidious and is easily identified weeks following reconstitution. This finding is consistent with the work of others who demonstrated that DiI labeling can track cells in vivo for months (28, 29). Reconstituted AMs retained viability and function as throughout the course of the experiments. This study provides a new approach to assess the role of AMs in both normal and diseased conditions.

	Footnotes

 
¹ This study was supported by National Institutes of Health Grants RO1 NIH AI48455 and RO1 NIH HL61285.

² Address correspondence and reprint requests to Dr. William J. Martin II at the current address: College of Medicine, University of Cincinnati, P. O. Box 670555, Cincinnati, OH 45267-0555.

³ Abbreviations used in this paper: AM, alveolar macrophage; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; BAL, bronchoalveolar lavage; i.t., intratracheal.

Received for publication January 10, 2002. Accepted for publication August 5, 2002.

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