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Different Roles for Human Lung Dendritic Cell Subsets in Pulmonary Immune Defense Mechanisms

Department of Respiratory Diseases, Ghent University Hospital, Ghent, Belgium

Correspondence and requests for reprints should be addressed to Ingel K. Demedts, Department of Respiratory Diseases, Ghent University Hospital 7K12IE, De Pintelaan 185, B-9000 Ghent, Belgium. E-mail: M.DemedtsIngelK{at}UGent.be

	Abstract

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Dendritic cells (DC) have a central role in the initiation of adequate immune responses. They recognize pathogens by means of Toll-like receptors (TLR) and link innate to adaptive immune responses by releasing proinflammatory cytokines and inducing T cell proliferation. We conducted this study to evaluate the expression and function of TLR on human lung DC subsets and to study their T cell stimulatory capacity. TLR gene expression by human pulmonary DC was evaluated by RT-PCR, while protein expression was analyzed by flow cytometry. We investigated cytokine release by DC in response to different TLR ligands. T cell stimulatory capacity was evaluated by mixed leukocyte reactions of purified lung DC with allogeneic T cells. Myeloid dendritic cells type 1 (mDC1) and myeloid dendritic cells type 2 (mDC2) express mRNA transcripts for TLR1, TLR2, TLR3, TLR4, TLR6, and TLR8. Flow cytometric analysis demonstrated high TLR2 protein expression for mDC1 and moderate TLR4 expression for mDC2. mDC1 and mDC2 release proinflammatory cytokines (TNF-, IL-1, IL-6, and IL-8) in response to TLR2 and TLR4 ligands. TLR3 ligands induce cytokine release in mDC1, but not in mDC2. Plasmacytoid DC (pDC) express TLR7 and TLR9 and release proinflammatory cytokines in response to imiquimod and IFN- in response to CpG oligonucleotides. mDC1 are strong inducers of T cell proliferation, while pDC hardly induce any T cell proliferation. mDC2 have an intermediate T cell–stimulatory capacity. Our results show divergent roles for the different human lung DC subsets, both in innate and adaptive immune responses.

Key Words: adaptive immunity • innate immunity • Toll like receptors

	Introduction

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Dendritic cells (DC) are antigen-presenting cells that play a central role in host immune defense, by linking innate to adaptive immune responses (1, 2). They recognize danger signals from invading pathogens and are able to prime naïve T cells and to initiate appropriate T cell immune responses against these microorganisms (3). Moreover, by the release of proinflammatory cytokines, dendritic cells influence the polarization of the adaptive T cell response into either a T helper (Th)1, Th2, or a T regulatory (Treg) direction (4).

DC use Toll-like receptors (TLR) (and other pattern recognition receptors) to detect the presence of infection (5). These TLR are a group of highly conserved pattern recognition receptors that were first described in Drosophila (6). They recognize "pathogen associated molecular patterns" (PAMP), which are molecular signatures that are unique to the microbial world and invariant among pathogens of a given class. TLR4, for example, recognizes lipopolysaccharide (found in all gram-negative bacteria) and thus enables the recognition of this large group of microorganisms by the immune system (7). With as few as 10 TLR, the human immune system can initiate adequate immune responses against almost any threatening microorganism. In addition to the detection of PAMPs, TLR also recognize endogenous ligands, such as heat shock proteins (8, 9), extracellular matrix breakdown products (10, 11), and intracellular contents from necrotic cells (12, 13). Recognition of PAMPs by TLR induces the release of inflammatory cytokines (14) and antimicrobial peptides (15), which allows a rapid reponse to invading pathogens.

In addition to this important role in innate immunity defense mechanisms, DC are involved in the initiation of adaptive immune responses (1, 16). They capture antigens from possibly harmful microorganisms at the epithelial surfaces, migrate to the draining lymph nodes (17) where they present antigens to T cells, and induce T cell proliferation and polarization.

Previously, several groups described the presence of DC in human lung (18–21), but a detailed description on their role in innate and adaptive immune responses in human lung is lacking. Recently, we demonstrated the presence of three different DC subsets in human lung (22): myeloid DC type 1 (mDC1, identified by the expression of BDCA1 and MHCII); myeloid DC type 2 (mDC2, identified by the expression of BDCA3 and MHCII); and plasmacytoid DC (pDC, identified by the expression of BDCA2 and CD123). These three DC subsets have different phenotypes regarding the expression of both lineage and maturation markers. The goal of this study was to evaluate the role of these DC subsets in pulmonary immune defense mechanisms. For this purpose, we analyzed the expression and function of TLR on human lung DC subsets. Indeed, while the expression of TLR on human blood DC has been described in detail (14, 23), no data are available on the expression of TLR by human pulmonary DC. Moreover, we investigated the capacity of the different human lung DC subsets to induce T cell proliferation.

	MATERIALS AND METHODS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Preparation of Single-Cell Suspensions from Lung
Lung tissue was obtained from patients who underwent lobectomy or pneumectomy for various reasons (mostly lung cancer). In total, resection specimens from 36 patients were processed. Written informed consent was obtained from all subjects according to protocols approved by the Medical Ethical Committee of the Ghent University Hospital. Tissue distant from the primary pathologic lung tissue was collected by a pathologist. No mediastinal or hilar lymph nodes were included in the analyzed tissue. Resection specimens were processed as previously described (22) to obtain a single-cell suspension of pulmonary mononuclear cells. Details are provided in the online supplement.

Labeling of Single-Cell Suspensions for Flow Cytometry
Single-cell suspensions of pulmonary mononuclear cells were labeled with monoclonal antibodies (see online supplement for a more detailed protocol and for a list of the antibodies used). Three different lung DC subsets were identified as previously described (22), and TLR expression was evaluated on the different subsets by flow cytometry. Details on the gating procedure can be found in the online supplement.

Purification of Human Lung DC
Human lung DC subsets were isolated from mononuclear cell suspensions by fluorescence-activated cell sorting to obtain highly purified DC subsets (purity > 95%). Details on the sorting strategy can be found in the online supplement.

RNA Extraction
RNA from sorted cells was extracted with the ChargeSwitch Total RNA Cell Kits (Invitrogen, Carlsbad, CA). All RNA extractions included an additional DNase step.

RT-PCR
Expression of TLR1, -2, -3, -4, -6, -7, -8, and -9 mRNA, relative to GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA, was analyzed with the Assays-on-Demand Gene Expression Products (Applied Biosystems, Foster City, CA). RT-PCR was performed on an ABI PRISM 7700 Sequence Detection System with MuLV RTase (Applied Biosystems). Reverse transcription was performed at 48°C for 30 min followed by 12 min incubation at 95°C for denaturation of RNA–DNA heteroduplexes, and 50 cycles of 95°C for 15 s and 60°C for 60 s. Monitoring of the RT-PCR occurred in real time using an FAM/TAMRA probe. All reactions were performed starting from 10 ng of total RNA.

Stimulation of DC with TLR Ligands
Purified lung DC subsets were cultured for 24 h in the presence or absence of different TLR ligands. TLR ligands used were 100 ng/ml lipopolysaccharide from Escherichia coli type 0111:B4 (LPS; Sigma-Aldrich, St. Louis, MO), 3 µg/ml CpG-oligodeoxynucleotide (ODN 2216; Invivogen, San Diego, CA), 1 µg/ml peptidoglycan from Staphylococcus aureus (PGN; Sigma-Aldrich), 10 µg/ml imiquimod-R837 (Invivogen, San Diego, CA), and 25 µg/ml poly(I:C) (Invivogen). Culture supernatant was collected and several cytokines were measured in culture supernatant by cytometric bead array (CBA, Human inflammation kit; BD Pharmingen, San Diego, CA). IFN- was measured with a commercially available ELISA (PBL Biomedical Laboratories, Piscataway, NJ). (See online supplement for details.)

Mixed Leukocyte Reaction
Purified pulmonary DC subsets were cocultured with allogeneic T cells for 5 d, and the degree of T cell proliferation was compared between the different subsets (see online supplement for details). T cells were isolated from human peripheral blood mononuclear cells by magnetic depletion of non–T cells using a commercially available Pan T Cell Isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany).

Statistical Analysis
Statistical analysis was performed with GraphPad Instat version 3.01 for Windows. Data are presented as mean (± SEM). Repeated measures analysis of variance with Tukey post testing was used to compare differences in TLR RNA expression. Paired t tests were used to compare cytokine levels with or without stimulation with TLR ligands. Statistical significance was defined as P < 0.05.

	RESULTS

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

 
Differential Expression of TLR mRNA in Human Lung DC Subsets
The expression of TLR transcripts was analyzed by quantitative RT-PCR. We compared the level of mRNA expression between the different lung DC subsets.

There was a striking difference between the myeloid DC subsets (mDC1 and mDC2) compared with plasmacytoid DC (Figure 1). While both mDC1 and mDC2 expressed transcripts of TLR1, -2, -3, -4, -6, and -8, very few or no transcripts of TLR7 and TLR9 could be detected in these DC subsets compared with plasmacytoid DC (pDC). Expression of TLR1, -2, and -3 was significantly higher in mDC1 and mDC2 compared with pDC, and expression of TLR4 and -8 was significantly higher in mDC2 compared with pDC. mDC2 also had a significantly higher expression of TLR4 than mDC1. The differences in TLR6 expression did not reach statistical significance. Plasmacytoid DC expressed huge amounts of TLR7 and TLR9 transcripts, as well as some TLR1 and TLR6, while no transcripts of TLR2, -3, -4, or -8 could be detected. Expression of TLR7 and TLR9 was significantly higher in pDC compared with both mDC1 and mDC2.

View larger version (18K): [in this window] [in a new window]   Figure 1. Quantitative analysis of mRNA expression of TLR1, TLR2, TLR3, TLR4, TLR6, TLR7, TLR8, and TLR9 in pulmonary mDC1, mDC2, and pDC. mRNA expression is shown as the ratio of the number of transcripts for a given TLR to the number of transcripts for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data shown are representative of three experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

mDC1 expressed TLR2 at high levels, while they have low to moderate amounts of TLR1 and TLR4 expression at the cell surface (Figure 2). There was no expression of TLR3, -8, or -9 on mDC1.

View larger version (23K): [in this window] [in a new window]   Figure 2. Analysis of TLR protein expression by mDC1, mDC2, and pDC in human lung. Shaded histograms indicate expression of the specific TLR; open histograms indicate isotype control staining. Data shown are representative of seven experiments.

In plasmacytoid DC (pDC), no expression could be detected for any TLR at the protein level, except for a low expression of the intracellular TLR9 receptor.

Cytokine Release in Response to Different TLR Ligands
TLR recognize pathogen-associated molecular patterns and initiate an adequate immune response. To investigate the function of different TLR on human lung dendritic cells, we stimulated the purified lung DC subsets with different TLR ligands and measured the release of cytokines by these DC in response to TLR ligation.

Stimulation of mDC1 and mDC2 with PGN (a TLR2 ligand) induced an increase in the release of TNF-, IL-1, IL-6, and IL-8 (Figure 3), while none of these cytokines could be detected in supernatant of plasmacytoid DC cultures. mDC1 stimulated with PGN released significantly more TNF- (571.1 [± 261.8] pg/cell nr x 10⁶), IL-1 (143.8 [± 33.5]), IL-6 (283.1 [± 131.1]) and IL-8 (13,709.4 (± [,438.7]) compared with unstimulated mDC1 (TNF- 77.9 [± 30.5] pg/cell nr x 10⁶, IL-1 37.0 [± 21.2], IL-6 94.5 [± 34.9], IL-8 6,177.6 [± 3,016.5]) (P < 0.05, P < 0.01, P < 0.05, and P < 0.05 respectively). mDC2 stimulated with PGN released significantly more TNF- (500.6 [± 222.6] pg/cell nr x 10⁶), IL-1 (587.0 [± 228.2]) and IL-6 (3,325.0 [± 1,600.4]) compared with unstimulated mDC2 (TNF- 246.0 [± 109.8] pg/cell nr x 10⁶, IL-1 218.1 [± 80.4] and IL-6 2,127.1 [± 1,032.1]) (P < 0.05 for each of these three cytokines).

View larger version (29K): [in this window] [in a new window]   Figure 3. Analysis of cytokine release by pulmonary mDC1, mDC2, and pDC in response to stimulation with peptidoglycan (PGN, a TLR2 ligand). Levels of TNF- (A), IL-1 (B), IL-6 (C), and IL-8 (D) were measured in culture supernatant by cytometric bead array. Data shown are representative of three to five experiments. *P < 0.05, **P < 0.01.

View larger version (29K): [in this window] [in a new window]   Figure 4. Analysis of cytokine release by pulmonary mDC1, mDC2, and pDC in response to stimulation with LPS (a TLR4 ligand). Levels of TNF- (A), IL-1 (B), IL-6 (C), and IL-8 (D) were measured in culture supernatant by cytometric bead array. Data shown are representative of three to five experiments. *P < 0.05, **P < 0.01.

View larger version (28K): [in this window] [in a new window]   Figure 5. Analysis of cytokine release by pulmonary mDC1, mDC2, and pDC in response to stimulation with poly(I:C) (a TLR3 ligand). Levels of TNF- (A), IL-1 (B), IL-6 (C), and IL-8 (D) were measured in culture supernatant by cytometric bead array. Data shown are representative of five experiments. *P < 0.05.

View larger version (12K): [in this window] [in a new window]   Figure 6. Analysis of cytokine release by pulmonary pDC in response to stimulation with imiquimod (a TLR7 ligand). Levels of TNF- (A), IL-1 (B), IL-6 (C), and IL-8 (D) were measured in culture supernatant by cytometric bead array. Data shown are representative of five experiments. *P < 0.05.

View larger version (11K): [in this window] [in a new window]   Figure 7. Release of IFN- by pulmonary mDC1, mDC2, and pDC in response to stimulation with CpG (unmethylated DNA sequences, a TLR9 ligand). Cytokines were measured in culture supernatant by ELISA. Data shown are representative of three experiments. *P < 0.05.

Myeloid DC type 1 appeared to be strong inducers of T cell proliferation, while plasmacytoid DC hardly stimulated T cells to proliferate (Figure 8). Myeloid DC type 2 had an intermediate capacity to initiate T cell proliferation.

View larger version (10K): [in this window] [in a new window]   Figure 8. T cell proliferation induced by pulmonary mDC1 (squares), mDC2 (triangles), and pDC (circles) in a mixed leukocyte reaction. Allogeneic T cells were cocultured with the different human lung DC subsets for 5 d and T cell proliferation was measured by the amount of 3H thymidine incorporation. Data shown are representative of three experiments.

	DISCUSSION

Top Abstract Introduction MATERIALS AND METHODS RESULTS DISCUSSION References

Our results suggest that the different lung DC subsets in human lung have different tasks and functions in human immune defense against respiratory pathogens. Myeloid DC (both type 1 and type 2) are able to detect the presence of gram-positive and gram-negative bacteria by the expression of TLR2 and TLR4, which allow them to recognize peptidoglycan from the cell wall of gram-positive bacteria and lipopolysaccharide from the outer membrane of gram-negative bacteria, respectively. While both mDC1 and mDC2 expressed mRNA transcripts for TLR3 (a receptor for double-stranded viral RNA), we were unable to detect TLR at the protein level on these DC subsets.

A subset of mDC2 expressed TLR2 at high levels, while a limited number of mDC2 did not express TLR2 at all. This confirms our previous findings (22) that mDC2 are in fact a heterogeneous group of DC in human lung that probably can further be divided into two different subgroups with different expression patterns of CD11c, CD14, and TLR2. These BDCA3 expressing myeloid DC have also been described in human peripheral blood (24–26) and in human tonsils (26). In the latter study, Lindstedt and coworkers used transcriptional profiling to compare BDCA1+, BDCA2+, and BDCA3+ DC subsets in blood and tonsil. They demonstrated a significant overlap in gene expression between BDCA1+ and BDCA3+ DC (most pronounced in tonsillar DC). However, they found selective transcription of several genes specific for BDCA1+ and BDCA3+, respectively. These authors suggest that the BDCA1+ and BDCA3+ DC may have a common origin and that they could represent two different stages of a similar subset.

In contrast to mDC1 and mDC2, plasmacytoid DC in human lung did not express TLR2 or TLR4 but strongly expressed TLR7, which allows the recognition of single-stranded viral RNA (16) and TLR9, the intracellular receptor for unmethylated DNA sequences of viruses and bacteria.

Previously, other groups described the presence of TLR on human bronchial (27) and alveolar epithelial cells (28), airway smooth muscle (29), alveolar macrophages (30), and in lung granulomas from patients with active tuberculosis (31). Our data provide the first description on the expression of TLR on DC in human lung. Given the central role of DC in immune responses, this is an important finding. However, to confirm the data on gene and protein expression for TLR on human lung DC, we wanted to explore the functional consequences of TLR ligation on DC subsets in human lung.

In line with the distinct expression patterns for TLR, we found that the lung DC subsets also respond in different ways to recognition of several pathogen-associated molecular patterns. Stimulation of the DC subsets with LPS and PGN (as an in vitro approach to study the in vivo outcome of the encounter of the DC subsets with gram-negative and gram-positive microorganisms) confirmed the earlier findings on TLR expression: mDC1 and mDC2 released increased amounts of various proinflammatory cytokines in response to LPS and PGN, whereas pDC did not. Although the absolute levels of cytokines released were largest in mDC2, the relative increase in cytokine release was markedly higher in mDC1, suggesting that this is somehow a more dynamic population, with a swifter response to invading pathogens than mDC2.

Cytokines released by myeloid DC in response to LPS include TNF-, IL-1, IL-6, and IL-8. TNF- mediates endothelial activation and is a potent activator of neutrophils by mediating adherence, chemotaxis, degranulation, and respiratory burst (32, 33). IL-1 stimulates the synthesis of acute phase proteins, promotes endothelial cell adherence of leukocytes, and is able to activate T-lymphocytes by enhancing the production of IL-2 (33). IL-6 is the most important inducer of acute phase protein synthesis and mediates T cell activation, growth, and differentiation. IL-8 is an important chemokine that mediates the attraction of neutrophils to inflamed tissue. Altogether, in response to the recognition of signs of bacterial infection, myeloid DC subsets in human lung (both mDC1 and mDC2) release several proinflammatory cytokines and thus mediate the initiation of an adequate immune response against bacterial infections.

Stimulation of myeloid DC with a TLR3 ligand (poly[I:C], a synthetic analog of double-stranded viral RNA), induced the release of proinflammatory cytokines by mDC1, but not by mDC2. This suggests an additional role for mDC1 in the detection of viruses in the respiratory tract.

In contrast to myeloid DC, plasmacytoid DC did not release any of these cytokines in response to LPS, PGN, or poly(I:C). However, when stimulated with TLR7 or TLR9 ligands (intracellular TLR specialized in viral detection), pDC released high amounts of TNF- and IL-8 (in response to imiquimod) and IFN- (in response to CpG). IFN is a member of the type I interferon family and has strong antiviral activities (34, 35). The release of IFN- by blood pDC in reponse to viral infections has been described previously (14), and our data extend these findings to pulmonary plasmacytoid DC. In conclusion, human lung pDC seem to be specialized in the detection of respiratory viral infections and in the initiation of an antiviral immune response.

In addition to their role in innate immune responses, DC also interact with T cells and initiate adaptive immune responses. We found that mDC1 are much stronger inducers of T cell proliferation compared with mDC2 and pDC. While pDC hardly induced any T cell proliferation, mDC2 had a moderate capacity to initiate the proliferation of T cells. This suggests that mDC1 (and to a lesser extent also mDC2) are probably capable of inducing an active adaptive immune response, while pDC fail to do so and thus might be involved in the induction of tolerance. Data from a mouse model of allergic asthma, demonstrating that pDC suppress T cell effector generation in the lung, support this hypothesis (36). A possible explanation for the low capacity of pDC to induce T cell proliferation is the lack of co-stimulatory molecules on human lung pDC. Indeed, we have previously shown that pDC in human lung have significantly lower expression levels of co-stimulatory signals such as CD40, CD80, and CD86, when compared with myeloid DC (22). Second, the lack of release of proinflammatory cytokines (as demonstrated in this study) might account for the low T cell stimulatory capacity of pDC. However, it is important to remember that our results were obtained from mixed leukocyte reactions of purified lung DC cocultured with allogeneic T cells. While this is a frequently used method to evaluate T cell–stimulatory capacity of human DC, it does not reflect an antigen specific T cell response as it occurs in vivo.

In conclusion, we demonstrated that there are important differences in the way human lung DC subsets recognize microorganisms and initiate innate and adaptive immune responses. These findings provide important insights into immune responses in the human lung and contribute to the increasing knowledge (2, 37, 38) on the function of pulmonary DC.

	Acknowledgments

 
The authors thank Greet Barbier, Indra De Borle, Philippe Degryze, Kathleen Desaedeleer, Ann Neesen, Professor Marleen Praet, Professor Frank Vermassen, and Dr. Lieve Van Walleghem for their technical contribution to this work.

	Footnotes

 
This work was supported by the Fund for Scientific Research in Flanders (FWO Vlaanderen, Research Projects G. 0011.03 and G.0343.01N) and by Project grant 01251504 from the Concerted Research Initiative of the Ghent University. I.D. is a doctoral research fellow of the Fund for Scientific Research in Flanders (FWO Vlaanderen).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1165/rcmb.2005-0382OC on April 20, 2006

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 11, 2005

Accepted in final form April 3, 2006

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