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The Journal of Immunology, 2004, 173: 4875-4881.
Copyright © 2004 by The American Association of Immunologists

Flt3-Ligand, IL-4, GM-CSF, and Adherence-Mediated Isolation of Murine Lung Dendritic Cells: Assessment of Isolation Technique on Phenotype and Function1

Kena A. Swanson*, Yan Zheng{dagger}, Kathleen M. Heidler{dagger}, Zhen-Du Zhang{dagger}, Tonya J. Webb{dagger} and David S. Wilkes2,*,{dagger}

Departments of * Microbiology and Immunology and {dagger} Medicine, Indiana University School of Medicine, Indianapolis, IN, 46202


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Lung dendritic cells (DCs) are difficult to study due to their limited quantities and the complexities required for isolation. Although many procedures have been used to overcome this challenge, the effects of isolation techniques on lung DCs have not been reported. The current study shows that freshly isolated DCs (CD11c+) have limited ability to induce proliferation in allogeneic T cells, and are immature as indicated by low cell surface expression of costimulatory molecules compared with liver or splenic DCs. DCs isolated after overnight culture or from mice treated with Flt3L are phenotypically mature and potent stimulators of allogeneic T cells. DCs could not be propagated from lung mononuclear cells in response to IL-4 and GM-CSF. Contrary to data reported for nonpulmonary DCs, expression of CCR6 was decreased on mature lung DCs, and only a subset of mature DCs expressed higher levels of CCR7. Absence of CD8{alpha} expression indicates that freshly isolated DCs are myeloid-type, whereas mature DCs induced by overnight culture are both "lymphoid" (CD8{alpha}+) and "myeloid" (CD8{alpha}–). DCs from mice genetically deficient in CD8{alpha} expression were strong simulators of allogeneic T cells which was consistent with data showing that CD8{alpha}– DCs from CD8{alpha}-sufficient mice are better APCs compared with CD8{alpha}+ DCs from the same mice. These data show that freshly isolated lung DCs are phenotypically and functionally distinct, and that the isolation technique alters the biology of these cells. Therefore, lung DC phenotype and function must be interpreted relative to the technique used for isolation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lung dendritic cells (DCs)3 are strategically located within the airway epithelium, and interstitium of the lung where they are poised to sample airborne and hematogenous Ags. Although reported as the most potent APC, lung DCs are difficult to study due to the complexities required to isolate sufficient quantities of cells for biological assays. Many of the techniques used to obtain lung DCs involve adhering cells to a solid phase to enrich before more selective isolation (reviewed in Ref.1). However, adherence induces maturation and potent Ag presentation in DCs isolated from tissues other than the lung. Systemic injections of fms-like tyrosine kinase 3 ligand (Flt3L), which induces growth in hemopoietic cells, has also been used to expand DCs in all tissues, including the lung (2, 3, 4). Other techniques for DC isolation include culturing blood monocytes in the presence of IL-4 and GM-CSF, which induces the generation of primarily myeloid DCs (5, 6, 7, 8). Accordingly, the techniques used to isolate DCs could have profound changes on the phenotype and function of DCs. Furthermore, our prior report shows that DCs from different tissues may be phenotypically and functionally heterogenous (9). Therefore, studies examining the biology of a DC derived from blood or isolated from the spleen may not be applicable to DCs derived from other tissues, including the lung.

Chemokine receptor expression has been used to characterize immature DCs (iDCs) and mature DCs (mDCs). Studies of nonpulmonary tissues show that iDCs express the chemokine receptors CCR1, CCR2, CCR5, and CCR6 (10, 11), and that CCR6 expression has been used to differentiate iDCs from mDCs (reviewed in Refs.10 and 11). Although studies indicate that iDCs and mDCs have differential expression of CCRs, recent reports in nonpulmonary tissues show that receptor expression may also vary between CD11c+CD8{alpha}– and CD11c+CD8{alpha}+ DCs, two major groups of murine DCs: CD11c+CD8{alpha}– DCs express CCR6 while CD11c+CD8{alpha}+ DCs do not (12). In the lung, immature DCs are CD11c+CD8{alpha}– (myeloid DCs), and mature lung DCs are both CD11c+CD8{alpha}– and CD11c+CD8{alpha}+ (lymphoid DCs), and the migratory patterns of these lung DCs may vary (13). However, the ability to use CCR expression to differentiate iDCs or mDCs in the lung is unknown.

Our laboratory has reported that freshly isolated (nonadhered and immature) murine lung DCs are functionally distinct from liver or splenic DCs (9). However, there are no studies reporting the effects of adherence on the phenotype and function of lung DCs. In addition, the phenotype and function of lung DCs induced by Flt3L compared with freshly isolated DCs from untreated mice has been reported in only one study (14). Finally, the ability to propagate DCs from lung mononuclear cells is unknown, and chemokine receptor expression on lung DCs has not been reported. The goal of the current study was to characterize freshly isolated lung DCs that most closely resemble in situ lung tissue DCs in their immature state and examine the impact of isolation techniques on the phenotype and function of these cells.

The current study shows that DCs cannot be propagated from lung mononuclear cells using IL-4 and GM-CSF, and furthermore, that adherence and Flt3L induce DC maturation, thereby altering their phenotype. After adhering lung mononuclear cells overnight, we discovered the emergence of a CD8{alpha}+ (lymphoid) DC population, and that CD8{alpha}– and CD8{alpha}+ DCs differed in their ability to stimulate T cells. In addition, adherence altered the expression of CCRs on iDCs relative to mDCs. These data suggest that lung DCs isolated from untreated mice without overnight culture/adherence reflect most closely those cells in situ. Moreover, these data highlight the importance of studying freshly isolated DCs when determining the role of these cells in pulmonary immunity.


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

Female C57BL/6 (I-Ab, H-2b) and BALB/c (I-Ad, H-2d) mice (8- to 10-wk-old) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and The Jackson Laboratory (Bar Harbor, ME). CD8{alpha}-deficient mice (B6.129S2-Cd8a+m1Mak) 8–10 wk of age were from The Jackson Laboratory. Mice were euthanized by injection (i.m.) with a ketamine mixture (79.3% Ketaject, 17.5% atropine, and 3.2% acepromazine). All mice were housed in pathogen-free facilities in the Laboratory Animal Resource Center at Indiana University School of Medicine (Indianapolis, IN) in accordance with Institutional Animal Care and Use Committee guidelines.

Abs and Reagents

The following fluorochrome-conjugated anti-mouse Abs were used for flow cytometry experiments: FITC CD11c, PE CD80, PE CD86, PE CD40, PE CD8{alpha}, and PE I-Ab (BD Pharmingen, San Diego, CA). Biotinylated anti-mouse OX40 ligand (OX40L) mAb (15), a generous gift of Dr. N. Ishii (Tohoku University School of Medicine, Sendai, Japan), was followed by a PE-conjugated anti-rat secondary Ab. The corresponding isotype control Abs and an Fc blocking Ab were all purchased from BD Pharmingen. Complete medium (cRPMI): RPMI 1640, 10% FBS, 400 mM L-glutamine, 100 U of penicillin/streptomycin, 1% 2-ME (5 x 10–4 M) (Invitrogen, Carlsbad, CA); DNase I (Sigma-Aldrich, St. Louis, MO), and collagenase D (Roche Diagnostics, Indianapolis, IN) were used in these studies. In some experiments, IL-4 and GM-CSF (R&D Systems, Minneapolis, MN) were both used at 20 ng/ml in cRPMI. The hemopoietic growth factor, Flt3L, was a kind gift from Immunex (Seattle, WA).

DC isolation

Different techniques were used to isolate lung DCs. First, lung mononuclear cells were isolated as previously described with minor modifications (9, 16). Briefly, lungs were digested in a DNase/collagenase solution. DCs were isolated from lung mononuclear cells (iDCs), obtained after a Percoll density gradient (Amersham Biosciences, Uppsala, Sweden), by magnetic microbead isolation using CD11c (N418) microbeads (Miltenyi Biotec, Auburn, CA).

A second isolation technique used was performed as reported by Lipscomb et al. (17, 18) with some modifications. First, culturing lung mononuclear cells for 1 h at 37°C in cRPMI on a tissue culture plate enriched the lung DC population. Nonadherent cells were removed by gentle pipetting, fresh medium was added, and the adherent cells were cultured for 18 h at 37°C. Previous studies have identified the loosely adherent cells after overnight culture as cells with a typical DC morphology and function (17, 18). Lung DCs were isolated from the loosely adherent cells by CD11c microbead selection (mDCs). In a third technique, either CD11c iDCs or mDCs were isolated from mice that received Flt3L injections (i.p.) for 9 days. Lastly, to expand the DC population, lung mononuclear cells were cultured in cRPMI containing IL-4 and GM-CSF (20 ng/ml) for 2, 4, 6, and 9 days followed by isolation of CD11c+ cells.

Liver and splenic DCs were isolated similar to lung mononuclear cells with minor modifications. Livers underwent a collagenase digestion, Percoll density gradient, and then DCs were positively selected by CD11c microbeads. After RBC lysis, splenic DCs were immunomagnetically sorted using CD11c microbeads.

In separate experiments examining the function of CD8{alpha} on DCs, CD11c+CD8{alpha}+ lung DCs were isolated by culturing CD11c+ lung mononuclear cells overnight, stained with PE-CD8{alpha} (BD Pharmingen), and then electronically sorted using a FACStarPlus (BD Biosciences, Mountain View, CA). All DC isolations consistently yielded a 75–90% pure population as assessed by flow cytometry staining of purified cells.

Flow cytometry

Cells were preincubated with an FcR (Fc{gamma} RIII/II)-blocking Ab (BD Pharmingen) to block a nonspecific FcR:Ab interaction (9). Cells were then incubated with the indicated fluorochrome-conjugated Abs followed by washing with 1% paraformaldehyde. Samples were analyzed on a FACSCalibur flow cytometer (BD Biosciences).

T cell isolation

BALB/c splenic T cells were isolated using CD90 magnetic microbead selection (Miltenyi Biotec), which yielded a ≥97% pure population by CD3-specific staining assessed by flow cytometry analysis.

Mixed leukocyte reaction

Gamma irradiated (2000 rad) C57BL/6 DCs from wild-type or CD8{alpha}-deficient mice were cocultured with BALB/c T cells in 96-well flat-bottom plates (BD Biosciences). Eighteen hours before completion of the 72-h incubation, thymidine was added and T cell proliferation was reported as mean cpm thymidine incorporation in triplicate wells (±SD).

Multiplex PCR for chemokine receptors

Total RNA was extracted from DC populations using the RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Approximately 1 µg of total RNA was reverse transcribed into first-strand cDNA by using a cDNA Cycle Kit (Invitrogen Life Technologies, Carlsbad, CA). The cDNA was used for enzymatic amplification with the specific MPCR kits for mouse chemokine receptors, CCR set 1 and CCR set 2, per manufacturer’s instructions (Maxim Biotech, San Francisco, CA).

RT-PCR for IDO

PCR for the detection of IDO was performed using methods described by Mellor et al. (19). Total RNA was isolated and reverse transcription performed. IDO transcripts (740 bp) were identified using IDO-specific forward and reverse primers as reported (9). Reaction products were run on a 2% agarose gel in TAE. Images were analyzed using a ChemiImager 4400 low light imaging system (Alpha Innotech, San Leandro, CA).

Statistics

The Student’s t test was used for analysis of data. Values of p < 0.05 were determined to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Overnight adherence and in vivo Flt3L treatment alter lung DC phenotype and function

To determine the impact of adherence and Flt3L treatment on lung DCs, we first examined their phenotypic profile compared with freshly isolated lung DCs (iDCs). Fig. 1A shows that DCs isolated by adherence steps (mDCs) up-regulated expression of all the tested costimulatory molecules, the most significant increase being observed with CD40 and OX40L. The increase in CD80, CD86, and MHC class II (MHC II) expression was not homogeneous as shown by the biphasic peaks, which suggest there are two populations of DCs that develop as a result of adherence (Fig. 1A).



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  		FIGURE 1. Flow cytometric analysis of freshly isolated DCs (iDCs) compared with cells obtained following adherence and/or Flt3L treatment A, iDCs; B, mDCs, isolated by overnight adherence; C, Flt3L-iDCs, isolated from Flt3L-treated mice; and D, Flt3L-mDCs, mDCs isolated from Flt3L-treated mice. Cells were washed, fixed, and analyzed on a FACSCalibur (BD Biosciences). Data is representative of more than five independent experiments.

 
To assess the effect of Flt3L treatment on lung DC phenotype, mice were given i.p. Flt3L injections for 9 days followed by isolation of either iDCs or mDCs. As expected, treating mice with Flt3L did expand the DC population, yielding almost a 9-fold greater cell number (data not shown). However, with this expansion, CD86 and MHC II expression significantly increased as shown in the Flt3L iDCs (Fig. 1C). Adherence of these Flt3L-treated cells did not increase CD86 and MHC II expression compared with nonadhered Flt3L-treated cells (Flt3L iDCs), however, CD80 expression was almost 2-fold higher and CD40 expression almost seven times greater than iDCs from untreated mice (Fig. 1, A and D).

Data in Fig. 1 showed that adherence and Flt3L treatment have a significant effect on the phenotype of lung DC. Data in Fig. 2 show the affect of adherence on the ability the DCs to stimulate T cells. Compared with iDCs, mDCs were significantly more potent in their ability to stimulate proliferation in allogeneic T cells, and Flt3L enhanced this effect. Even though mDCs are potent APCs, parallel experiments showed that Flt3L further enhanced the ability of mDCs to stimulate T cell proliferation.



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  		FIGURE 2. Overnight adherence and Flt3L treatment enhance the APC function of lung DCs. Cells were isolated as described in Materials and Methods, irradiated, and placed in an MLR with allogeneic splenic T cells at the indicated ratios for 72 h. APC function was measured by the amount of [3H]thymidine incorporation indicated as cpm. Data is representative of three individual experiments. Error bars indicate ± SD; *, p < 0.002; {dagger}, p < 0.02.

 
Comparative phenotypic analysis of freshly isolated lung DCs to liver and splenic DCs

In our prior study, we determined that lung iDCs were unique in their ability to suppress T cell proliferation (9). In the current study, we determined if lung iDCs were phenotypically unique compared with similarly isolated cells from liver and spleen. iDCs from lung, liver, and spleen had variable expression of MHC class II and costimulatory molecules. Data showing that lung iDCs expressed lower levels of MHC class II, CD80, CD86, CD40, and OX40L indicated that lung iDCs were less mature than these cells isolated from liver and spleen (Table I).


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  		Table I. Phenotype of DCsa

 
IL-4 and GM-CSF fails to propagate DCs from lung mononuclear cells

IL-4 and GM-CSF are commonly used for the propagation of immature DCs in vitro from monocytic precursors (20), and monocytes are present within lung mononuclear cells (21). Therefore, we hypothesized that DCs may be propagated from lung mononuclear cells cultured in the presence of IL-4 and GM-CSF. Because Flt3L induces expansion of monocyte precursors (21), then lung mononuclear cells from Flt3L-treated mice were also cultured in the presence of IL-4 and GM-CSF. The quantity of cells, phenotype, and APC function were monitored serially throughout a 9-day culture period. Fig. 3A shows that the total number of cells decreased significantly by the second day in culture and remained stable thereafter. The phenotype of these cells was very heterogeneous with a peak in costimulatory molecule and MHC II expression observed at day 2 of culture and then inconsistent increases and decreases in these markers at later time points (data not shown). Variability of APC function was also observed in these cells during the culture period. Increasing quantities of DCs isolated at days 0, 2, and 6 induced modest increases in T cells proliferation. In contrast, DCs isolated on day 9 induce proliferation at DC:T cell ratios of 0.25:1 to 0.3:1. However, higher DC:T cell ratios markedly suppressed T cell proliferation (Fig. 3B).



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  		FIGURE 3. IL-4 and GM-CSF culturing of lung mononuclear cells (LMNCs) fails to propagate lung DCs. LMNCs were isolated from mice that had received 9 days of Flt3L injections. LMNCs were cultured with IL-4 and GM-CSF (20 ng/ml each) for up to 9 days. A, At the indicated time points, cultured LMNCs were harvested and counted by the trypan blue exclusion assay. Data from three separate experiments were first normalized to the cell number at the initiation of culture and then averaged. A statistically significant drop (*, p < 0.003) in cell number occurred at day 2. B, LMNCs at days 0, 2, 6, and 9 of culture were placed in an MLR at the indicated DC:T cell ratios and APC function of DCs indicated as mean ± SD cpm of T cell proliferation in ([3H]thymidine incorporation) in a 72-h triplicate coculture.

 
Chemokine receptor expression on iDCs and mDCs

Expression of the {alpha}{alpha} homodimer of CD8 (CD8{alpha}) identifies lymphoid DCs whereas, myeloid DCs do not express CD8{alpha} (22, 23, 24). We next determined if lung iDCs and mDCs differed in expression of CD8{alpha}. Fig. 4 shows that iDCs are primarily myeloid as very few cells express CD8{alpha}. In contrast, 36% of cells made mature by overnight adherence (mDCs) express CD8{alpha} (Fig. 4). A recent report shows that CD8{alpha}+ and CD8{alpha}– splenic iDCs and mDCs may be further characterized by the differential expression of CCRs (25). Therefore, we next determined the CCR expression patterns in CD8{alpha}+ and CD8{alpha}– iDCs and mDCs. iDCs (CD8{alpha}–) had variable expression of mRNA for CCR1, CCR2, CCR5, CCR6, and CCR7, with the lowest expression for CCR6. In contrast, expression of all CCRs, except CCR1, increased on mDCs (see Fig. 6). Fractionating the mDCs into CD8{alpha}+ and CD8{alpha}– populations revealed that CCR1 expression was unchanged in these cells compared with iDCs. Expression of CCR2, CCR5, CCR6, and CCR7 tended to be lower in CD8{alpha} – compared with CD8{alpha}+ DCs (Fig. 5).



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  		FIGURE 4. Increased CD8{alpha} expression on lung DCs following overnight adherence. iDCs and mDCs were isolated as described in Materials and Methods. CD8{alpha} expression analyzed by flow cytometry. Values in the upper right corner of the histograms indicate percent-positive cells. Data are representative of five separate experiments.

 


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  		FIGURE 6. CD8{alpha}– lung DCs have greater APC function compared with CD8{alpha}+ DCs. CD11c+ DCs were isolated by magnetic microbead selection and adhered overnight. The DCs were then stained with a CD8{alpha}-specific mAb and sorted by FACS into CD8{alpha}+ and CD8{alpha}– DC populations. A, The APC function of both DC groups was examined in a MLR with splenic T cells for 72 h and T cell proliferation indicated as mean ± SD cpm of [3H]thymidine incorporation in triplicate wells. Data are representative of four individual experiments. B, Experiments were repeated in parallel to examine the function of CD8{alpha}+ and CD8{alpha}– lung DCs from wild-type mice compared with lung DCs isolated from CD8{alpha}-deficient mice (CD8KO). Proliferations measured as described in prior figures. Data are representative of four separate experiments.

 


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  		FIGURE 5. Chemokine receptor gene expression in lung DCs. mRNA was isolated from unfractionated iDCs and mDCs, as well as purified CD8{alpha}+ and CD8{alpha}– mDCS. CCR expression was determined by multiplex PCR as reported in Materials and Methods. The table shows densities of CCRs relative to a housekeeping gene, GAPDH. Data shown is from a representative experiment.

 
Differential function of CD8{alpha}+ and CD8{alpha}– DCs

Using cellular and genetic approaches, we next determined if APC function varied in CD8{alpha}+ compared with CD8{alpha}– DCs. Fig. 6A shows that CD8{alpha}– DCs were more potent stimulators of T cell proliferation than similarly isolated CD8{alpha}+ DCs. This same trend was seen in lung DCs genetically deficient in CD8{alpha} expression. For example, Fig. 6B shows that lung DCs isolated from CD8{alpha}-deficient mice were potent stimulators of allogeneic T cells compared with CD8{alpha}– DCs isolated from wild-type mice. Studies in nonpulmonary DCs have suggested that limited ability of CD8{alpha}+ compared with CD8{alpha}– DCs to stimulate T cells may be due to differential tryptophan metabolism resulting from expression of IDO (26). However, Fig. 7 shows that both subsets of these DCs express IDO. In addition, IDO expressed in both cell types were comparable in their ability to metabolize tryptophan to its metabolite, kynurenine (data not shown).



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  		FIGURE 7. IDO is expressed in CD8{alpha}+ and CD8{alpha}– DCs. The images show IDO (upper) and {beta}-actin (lower) gene expression in CD8{alpha}+ and CD8{alpha}– DCs as determined by RT-PCR. The graph shows the ratio of the intensities of IDO bands relative to {beta}-actin, a housekeeping gene.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Studies have established the central role of DCs in stimulating pulmonary immune responses. However, DCs exist in very small quantities in the lung thus limiting the ability to study these cells. Several isolation techniques have been used by many investigators to isolate and enrich these rare cells. However, the effects of these isolation techniques, reported for DCs isolated from nonpulmonary tissues, have not been described previously in lung DCs. The current study shows that lung DCs become phenotypically mature and are more potent stimulators of T cell proliferation after overnight adherence in vitro, or in response to treatment with Flt3L in vivo. Interestingly, freshly isolated DCs from Flt3L-treated mice (Flt3L-iDCS) or mature DCs from these mice (Flt3L-mDCs) were more potent APCs than iDCS or mDCs isolated from untreated mice. These data indicate the Flt3L treatment alone, results in lung DCs that are phenotypically and functionally distinct from DCs present in untreated mice (iDCs). Although previous studies have shown the maturational effect of overnight adherence on DCs (27), this is the first direct comparison of these cells to DCs from Flt3L-treated mice. The data also show how lung DCs in situ are myeloid (CD8{alpha}–) whereas mDCs are comprised of lymphoid-CD8{alpha}+ and myeloid-CD8{alpha}– populations, and that these cells differ in expression of mRNA transcripts for CCRs, but not IDO. Moreover, these studies show that CD8{alpha}+ and CD8{alpha}– DCs differ in their ability to stimulate allogeneic T cells.

Data showing that freshly isolated lung murine lung DCs have low levels of MHC class II (I-a) expression and weak APC function are consistent with prior reports (28, 29). In those studies, Holt and colleagues demonstrated that rat lung APCs were immature as shown by low level expression of MHC class II (28), and that maturation by overnight adherence enhanced APC function (29). However, direct comparison of these reports to the current study may not be possible. The purity of DCs in the earlier reports was <40%, whereas, DCs in the current study were up to 90% pure. The contaminating cells in the prior studies were likely macrophages, which can have profound effects on DC function (18, 28, 29). Accordingly, functional data examining DCs in those reports could have been affected by the presence of macrophages. Investigators have reported that DCs isolated after overnight adherence have a mature phenotype and high levels of APC function (18, 30, 31). Although these studies examining the effect of adherence on DC function are consistent with the current study, only one study was conducted in mice (18), but it did not examine the effect of the isolation technique on DC function. Similar to the only report comparing iDCs and mDCs in humans (30), the present study is the first to directly compare the phenotype and function of a relatively pure population of iDCs and mDCs in murine lung.

The hemopoietic growth factor Flt3L, like IL-4 and GM-CSF, has also been used to increase the numbers of DCs both in vitro and in vivo. Administration of Flt3L in vivo results in an alteration of hematopoiesis in murine bone marrow, which results in significant increases in the quantity of functional DCs, both CD8{alpha}+ and CD8{alpha}–, in lymphoid organs (2). In addition, these cells were mature as shown by up-regulated expression of MHC II and CD86, and were potent APCs (4, 32). These results from murine studies are similar to a study from Pabst et al. (33), who reported that intratracheal instillation of Flt3L induces expansion of different DC subsets and up-regulated immune responses in the lungs of rats.

Similar to the effects of overnight adherence, Flt3L treatment in vivo induced maturation in lung DCs. These DCs appeared to be almost fully mature because adhering them overnight (Flt3L-mDC) only resulted in a slight increase in CD80, CD40, and OX40L expression, whereas CD86 and MHC II expression was actually somewhat less than with Flt3L treatment alone (Flt3L-iDCs). Also, adherence conferred no functional advantage to DCs isolated from Flt3L-treated mice. This suggests that Flt3L treatment in mice generates phenotypically and functionally mature DCs, which is in agreement with reports describing nonlung DCs, and a recent study from Masten et al. (14) examining lung DCs. The receptor for Flt3L is expressed on cells other than DCs. Indeed, Flt3L treatment induces the expansion of all myeloid-derived cells in vivo (34, 35). DC phenotype and function in situ may be related to interactions of DCs with cells in the local environment (18, 36). Accordingly, the phenotype and function of lung DCs isolated from mice treated with Flt3L may not be the result of Flt3L alone, but may also be affected by expansion of other immune cells in the lung. Collectively, these data suggest that analysis of DCs isolated from Flt3-treated mice is unlikely to represent the phenotype and function of resident lung DCs from untreated mice.

The ability of lung DCs to stimulate immune responses is dependent on their ability to migrate from the lung to draining lymph nodes where they activate T cells. Migration is dependent on expression of CCRs that allow DCs to respond to chemokines produced in the local environment. During maturation DCs are reported to down-regulate CCR6 and begin to express CCR7, a CCR critical for DC migration to and within lymphoid organs (10, 11, 37). Although studies indicate that iDCs and mDCs have differential expression of CCRs, recent reports show that receptor expression may also vary between CD11c+CD8{alpha}– and CD11c+CD8{alpha}+ DCs in nonpulmonary tissues: CD11c+CD8{alpha}– DCs express CCR6 while CD11c+CD8{alpha}+ DCs do not (12). In the lung, iDCs are CD11c+CD8{alpha}–, and mDCs are both CD11c+CD8{alpha}– and CD11c+CD8{alpha}+, and the migratory patterns of these lung DCs may vary (13). Contrary to these reports in nonpulmonary tissues, the current study shows that transcripts for CCR6 appear to increase in mDCs compared with iDCs. In addition, CCR7 transcripts are present in iDCs and mDCs but that expression may vary in CD8{alpha}+ compared with CD8{alpha}– mDCs. A limitation of the current study is the lack of phenotypic data showing CCR expression. However, a recent report from Colvin and colleagues (25) examining splenic DCs showed that reagents currently available to examine surface expression of murine CCRs yielded inconsistent results. Moreover, whereas iDCs did not migrate, only mDCS migrated in response to CC21 and CC19, but chemotactic activity did not correlate with CCR expression patterns in that report (25). Although it is intriguing to speculate about CCR expression relative to maturation status of DCs, novel reagents will be needed to determine definitively the CCR phenotype of murine DCs including those in the lung.

Murine DCs may be described as myeloid (CD8{alpha}+) and lymphoid (CD8{alpha}–) (38). Data in the current study showing that freshly isolated lung DCs (iDCs) are CD8{alpha}– and that mDCs are both CD8{alpha}+ and CD8{alpha}– is consistent with prior reports from Masten and colleagues (17, 18). Investigators have reported that CD8{alpha}+ and CD8{alpha}– lung DCs may have differential functions in vivo relative to anatomic location and trafficking. However, the difference in the ability to stimulate T cells was not directly examined in that report (13). The current study shows that CD8{alpha} – lung DCs are more potent stimulators of T cell proliferation than CD8{alpha}+ DCs. These data are consistent with reports from other investigators showing that CD8{alpha}+ splenic DCs were primarily tolerogenic (24, 39) or comparable to liver CD8{alpha}– DCs in their ability to stimulate T cells (40). Collectively, these data highlight further the concept that DCs may have organ-specific functions. An unanswered question is related to the actual quantity of plasmacytoid DCs in the murine lung. Based on reports from nonpulmonary tissues (1), these DCs may constitute a portion of the CD8{alpha}+ pool reported in the current study, or possibly represent a phenotypically unique group of cells. It is interesting to speculate that plasmacytoid DCs are present in both iDC and mDC subsets. As such, since virtually all iDCs were CD8{alpha}– in the current study, then these data may suggest that plasmacytoid lung DCs may have a different phenotype relative to their maturation status. These questions are currently being evaluated.

CD8{alpha}+ DCs were originally thought to be derived from a lymphoid-committed progenitor and CD8{alpha}– DCs from a myeloid progenitor (24, 41, 42). However, other studies have suggested that both DC subsets may arise from the same myeloid progenitor (43, 44, 45, 46). In contrast to most T cells on which CD8 is expressed as an {alpha}{beta} heterodimer, CD8 is expressed as an {alpha}{alpha}-homodimer on DCs (43). In vitro studies show that both the homodimer and heterodimer of CD8 on T cells have affinity for MHC class I (47, 48), though the {beta}-chain of the {alpha}{beta} heterodimer may increase avidity for MHC class I (49). CD8-signaling in T cells is in part mediated by binding by p56lck to the cytoplasmic tail of CD8{alpha} (50). MHC class I is expressed on all nucleated cells including T cells, and the cytoplasmic tail of CD8{alpha} has a role in signaling. Therefore, it is interesting to speculate that CD8{alpha} expression on DCs may be involved in T cell responses as well as identifying DC subsets. For example, CD8{alpha} on DCs may contribute to DC-T cell interactions, as the CD8 heterodimer does on T cells, by helping to keep the DC and T cell in close proximity or perhaps contributing to T cell costimulation. These questions are currently being investigated.

Collectively, these data show that the isolation techniques used to isolate lung DCs have profound effects on the phenotype and function of these cells. Freshly isolated DCs (iDCs) are those that resemble most closely lung DCs in situ. In addition, the biologic activity reported for nonpulmonary DCs may not be extrapolated to lung DCs.

These data and our prior report (9) indicate that compared with DCs from other tissues, lung DCs are phenotypically and functionally unique.


   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 the National Institutes of Health Grants HL60797 and HL/AI67177 (to D.S.W). Back

2 Address correspondence and reprint requests to Dr. David S. Wilkes, Division of Pulmonary and Critical Care Medicine, Indiana University School of Medicine, Van Nuys Medical Sciences Building, 635 Barnhill Drive, Indianapolis, IN 46202. E-mail address: dwilkes{at}iupui.edu Back

3 Abbreviations used in this paper: DC, dendritic cell; iDC, immature DC; mDC, mature DC; OX40L, OX40 ligand; Flt3L, fms-like-tyrosine kinase 3 ligand; MHC II, MHC class II. Back

Received for publication April 26, 2004. Accepted for publication August 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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