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Infection and Immunity, September 2005, p. 5350-5357, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5350-5357.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Involvement of Fractalkine/CX3CL1 Expression by Dendritic Cells in the Enhancement of Host Immunity against Legionella pneumophila

Toshiaki Kikuchi,1* Sita Andarini,1 Hong Xin,1 Kazunori Gomi,1 Yutaka Tokue,1 Yasuo Saijo,1 Tasuku Honjo,2 Akira Watanabe,1 and Toshihiro Nukiwa1

Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan,1 Department of Medical Chemistry, Graduate School of Medicine, Kyoto University, Kyoto, Japan2

Received 11 January 2005/ Returned for modification 24 February 2005/ Accepted 13 April 2005


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ABSTRACT
 
Legionnaires' disease is clinically manifested as severe pneumonia caused by Legionella pneumophila. However, the dendritic cell (DC)-centered immunological framework of the host defense against L. pneumophila has not been fully delineated. For this study, we focused on a potent chemoattractant for lymphocytes, fractalkine/CX3CL1, and observed that the fractalkine expression of DCs was somewhat up-regulated when they encountered L. pneumophila. We therefore hypothesized that fractalkine expressed by Legionella-capturing DCs is involved in the induction of T-cell-mediated immune responses against Legionella, which would be enhanced by a genetic modulation of DCs to overexpress fractalkine. In vivo immunization-challenge experiments demonstrated that DCs modified with a recombinant adenovirus vector to overexpress fractalkine (AdFKN) and pulsed with heat-killed Legionella protected immunized mice from a lethal Legionella infection and that the generation of in vivo protective immunity depended on the host lymphocyte subsets, including CD4+ T cells, CD8+ T cells, and B cells. Consistent with this, immunization with AdFKN/Legionella/DC induced significantly higher levels of serum anti-Legionella antibodies of several isotypes than those induced by control immunizations. Further analysis of spleen cells from the immunized mice indicated that the AdFKN/Legionella/DC immunization elicited Th1-dominated immune responses to L. pneumophila. These observations suggest that fractalkine may play an important role in the DC-mediated host defense against intracellular pathogens such as L. pneumophila.


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INTRODUCTION
 
Legionella pneumophila is an intracellular gram-negative bacillus that causes acute febrile pneumonia, or Legionnaires' disease (5, 31). Pulmonary infection with L. pneumophila usually occurs by direct inhalation of contaminated aerosols and aspiration (5, 31). Thus, mononuclear phagocytes in the respiratory tract (i.e., alveolar macrophages) are regarded as a critical component of the first-line host defense against Legionella infection (5, 7, 31). In addition to the innate immune response to the microorganism, the importance of adoptive cellular immunity, especially that mediated by CD4+ T cells, is underscored by the clinical observation that Legionnaires' disease is often contracted by individuals with depressed cell-mediated immunity, including transplant recipients, patients receiving corticosteroids, and patients suffering from AIDS (5, 7, 31). There is also substantial evidence that adoptive humoral immunity (i.e., a specific antibody response) plays a secondary role in the host defense against Legionella by improving phagocytosis and bacterial killing by phagocytes (5, 7, 31). However, the cellular and molecular mechanisms that induce an effective adaptive immune response to confer host protection against Legionella infection have yet to be clarified.

Dendritic cells (DCs) are professional antigen-presenting cells which possess an exquisite capacity to stimulate T cells and generate primary immune responses (2, 20). DCs are strategically situated in peripheral tissues to sense and capture invading pathogens, and after antigen uptake, they undergo maturation and move into secondary lymphoid organs to present the microbial fragments to T lymphocytes (2, 20, 25, 28). In many cases, DC maturation is also accompanied by the production of T-cell-attracting chemokines that assist DCs in attracting T cells for efficient antigen presentation (4, 19, 32). Fractalkine (CX3CL1) is an example of such T-cell attractants expressed by mature DCs and is a unique membrane-bound CX3C chemokine, with the chemokine domain perched atop a long mucin-like stalk at the cell surface (3, 11, 13, 26, 27). Fractalkine can be cleaved by ADAM10 (a disintegrin and metalloproteinase) or tumor necrosis factor alpha-converting enzyme (TACE or ADAM17) to produce a soluble 80-kDa glycoprotein (3, 9, 10, 26). The structure of fractalkine allows not only the shed soluble form to recruit T cells and monocytes expressing its specific receptor, CX3CR1, in local chemoattractant gradients but also the membrane-anchored form to act upon CX3CR1-positive cells directly and promote cell-cell adhesion (3, 11, 26).

With regard to the pathophysiology of L. pneumophila infection, we and others have recently observed that DCs are efficient stimulators of a protective immune response against this bacterium and several other microbes (15, 24). In the present study, we have advanced the understanding of the role of DCs in boosting anti-Legionella immunity and attempted to determine if fractalkine production by DCs is involved in the molecular mechanisms of the DC-mediated immune response to Legionella. To accomplish this, we first evaluated the endogenous expression of fractalkine in DCs pulsed with heat-killed L. pneumophila and subsequently examined in vivo the impact that the exogenous overexpression of fractalkine in DCs had on the induction of anti-Legionella immunity by using Legionella-pulsed DCs which had been genetically engineered with an E1– recombinant adenovirus vector expressing fractalkine (AdFKN). The data demonstrate that pulsing DCs with heat-killed Legionella enhances their fractalkine expression to some extent and that immunization with AdFKN-modified DCs pulsed with heat-killed L. pneumophila renders mice more resistant to a lethal respiratory challenge with L. pneumophila than control immunization without AdFKN modification.


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MATERIALS AND METHODS
 
Mice. Female C57BL/6 (H-2b) and A/J (H-2a) mice of 6 to 8 weeks of age were purchased from Japan Charles River (Atsugi, Japan) and Japan SLC (Hamamatsu, Japan), respectively. CD4+ T-cell-deficient (B6.129S2-Cd4tm1Mak), CD8+ T-cell-deficient (B6.129S2-Cd8atm1Mak), and B-cell-deficient (B6.129S2-Igh-6tm1Cgn) mice that had been backcrossed to the C57BL/6 background were obtained from Jackson Laboratory (Bar Harbor, Maine).

Adenovirus vectors. The E1– E3– adenovirus vectors based on human adenovirus type 5 used for this study included AdFKN, expressing mouse fractalkine cDNA under the control of the cytomegalovirus immediate-early promoter/enhancer, and AdNull, an identical vector with no transgene (13). As previously described, the recombinant adenovirus vectors were propagated, purified by CsCl gradient centrifugation, and titrated by a serial-dilution end-point assay. Both vectors were free of replication-competent adenovirus (1, 16).

Legionella and DC preparation. L. pneumophila (clinically isolated "Suzuki" strain provided by K. Yamaguchi, Toho University School of Medicine, Tokyo, Japan; serogroup 1) was grown, washed, and suspended in sterile phosphate-buffered saline, pH 7 (PBS), before use as previously described (15). DCs were generated from mouse bone marrow precursors in complete RPMI-1640 medium (10% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 µg/ml streptomycin, and 100 units/ml penicillin) with 10 ng/ml recombinant mouse granulocyte-macrophage colony-stimulating factor (R&D Systems Inc., Minneapolis, MN) and 2 ng/ml recombinant mouse interleukin-4 (IL-4; R&D Systems), as described previously (14, 17, 18). The DCs used for this study were prepared from A/J mice unless otherwise noted. In some experiments, CD11c+ DCs were purified with the MACS system (Miltenyi Biotech). The fractalkine expression of Legionella-pulsed DCs was examined by staining DCs with rat antifractalkine (clone 126315; R&D Systems) followed by detection with a fluorescein isothiocyanate (FITC)-conjugated anti-rat immunoglobulin G (IgG) antibody (BD Biosciences Pharmingen, San Jose, CA).

Fractalkine expression of genetically modified DCs. To demonstrate that AdFKN expressed fractalkine in DCs, DCs were transduced with AdFKN, AdNull, or PBS alone (naive) at a multiplicity of infection of 50 for 3 h, washed, and then cultured in complete RPMI-1640 medium for 48 h at 37°C. For semiquantitative reverse transcriptase PCR (RT-PCR), total cellular RNA was extracted from transduced DCs using ISOGEN (Nippon Gene, Tokyo, Japan) and subjected to RT-PCR using an RNA PCR kit (Takara Shuzo, Kyoto, Japan) and the following PCR primers, as described previously (1, 16): for exogenous fractalkine, 5'-TGCCAAGAGTGACGTGTCCA-3' (designed for specific amplification of exogenous fractalkine) and 5'-CACTGGCACCAGGACGTATG-3'; for exogenous and endogenous fractalkine, 5'-GCTTACGGCTAAGCCTCAGA-3' and 5'-CACTGGCACCAGGACGTATG-3'; for macrophage-derived chemokine (MDC), 5'-GTGGCTCTCGTCCTTCTTGC-3' and 5'-GGACAGTTTATGGAGTAGCT-3'; and for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 5'-ATGGTGAAGGTCGGTGTGAACGGA-3' and 5'-TTACTCCTTGGAGGCCATGTAGGC-3'. For immunocytochemical analysis, transduced DCs were stained using 10 µg/ml rat antifractalkine (R&D Systems) and 10 µg/ml mouse anti-I-Ak (major histocompatibility complex [MHC] class II, clone 11-5.2; BD Biosciences Pharmingen) antibodies for 30 min, followed by visualization with 10 µg/ml Cy3-conjugated anti-rat IgG (Chemicon, Temecula, CA) and 10 µg/ml FITC-conjugated anti-mouse IgG (Chemicon) antibodies for 30 min. The chemoattracting activity of AdFKN-modified DCs was assessed by a chemotaxis assay using mouse T-lymphocyte EL4 cells (American Type Culture Collection, Manassas, VA) and a 1:9 dilution of the supernatant of transduced DCs, as described previously (14). Where indicated, an antifractalkine neutralizing antibody (R&D Systems) or rat control IgG (BD Biosciences Pharmingen) was added to the supernatant in the lower chambers at 20 µg/ml.

Immunization and infection of mice. DCs were incubated with AdFKN, AdNull, or PBS at a multiplicity of infection of 50 for 3 h at 37°C in the presence of heat-killed (80°C, 10 min) L. pneumophila at a ratio of 10 bacteria to 1 DC. DCs were washed extensively with PBS and injected intravenously at 5 x 105 cells per mouse for immunization. Three weeks after immunization, a lethal respiratory infection with L. pneumophila was induced as described below. Briefly, anesthetized mice were placed in a supine position, and 50 µl containing 5 x 107 CFU of L. pneumophila was inoculated via the trachea into the lung. All animals were monitored daily for 14 days after inoculation. Obviously moribund mice were sacrificed, and this was recorded as the time of death. Antibodies against L. pneumophila in sera were assessed by an enzyme-linked immunosorbent assay (ELISA) in microtiter plates coated with 107 CFU of L. pneumophila per well using secondary and tertiary antibodies as previously described (all antibodies were from Pierce Biotechnology, Rockford, IL) (15).

Immune responses of immunized mice. To define the immunological features of mice immunized with AdFKN-modified DCs, mice were immunized with genetically modified DCs as described above. Splenocytes were isolated 14 days after immunization, and 6 x 106 splenocytes were cocultured for 4 days in complete RPMI-1640 medium with heat-killed L. pneumophila (106 CFU). After being cocultured, the splenocytes were stained with a FITC-conjugated monoclonal antibody against CD4 (clone RM4-5; BD Biosciences Pharmingen) or CD8a (clone 53-6.7; BD Biosciences Pharmingen) and were analyzed on an EPICS XL cytometer with EXPO32 ADC software (Beckman Coulter, Miami, FL). Dead cells and debris were excluded from the analysis by gating on the appropriate forward-scatter, side-scatter, and propidium-iodide-staining profile. Proliferation was measured by using Flow-Count fluorospheres (Beckman Coulter) to calibrate the count of the cells. The concentrations of gamma interferon (IFN-{gamma}) and IL-4 released into the medium were measured using ELISA kits for mouse IFN-{gamma} and IL-4 (BioSource International, Camarillo, CA), respectively. For immunofluorescent staining of intracytoplasmic IFN-{gamma} or IL-4 in CD4+ T cells, splenocytes stained with the FITC-conjugated anti-CD4 antibody were fixed and permeabilized by using a BD Cytofix/Cytoperm Plus kit (BD Biosciences Pharmingen) and were further stained with a phycoerythrin-conjugated monoclonal antibody against IFN-{gamma} (clone XMG1.2) or IL-4 (clone 11B11) or an appropriate isotype-matched control antibody (BD Biosciences Pharmingen). To determine the percentage of stained cells above the isotype control staining, 1% of false-positive events were accepted with the control antibody.

Statistical analysis. All data are reported as means ± standard errors, unless otherwise noted. Statistical comparisons were made using the two-tailed Student t test, and P values of <0.05 were accepted as indicating significance. Survival evaluation was carried out by using Kaplan-Meier analysis.


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RESULTS
 
Slightly upregulated expression of fractalkine on Legionella-pulsed DCs. L. pneumophila induced DCs to express endogenous fractalkine on their surfaces, but at only a slightly increased level compared to that on naive DCs (Fig. 1). Interactions of fluorescence-labeled L. pneumophila with DCs were studied by confocal laser scanning microscopy, and the phagocytosis of Legionella by DCs was confirmed by demonstrating that fluorescence appeared intracellularly in Legionella-pulsed DCs and not in control naive DCs (Fig. 1A). Next, DCs were pulsed with heat-killed L. pneumophila and then cultured for 2 days to analyze their surface expression of endogenous fractalkine by flow cytometric analysis (Fig. 1B). For greater accuracy in the analysis, dual staining with propidium iodide (PI) was performed to distinguish viable (PI–) cells from nonviable cells lest nonviable cells be read as false positive. Two-color flow cytometric analysis showed that the percentage of fractalkine-positive, PI– cells was somewhat higher in Legionella-pulsed DCs than in control naive DCs (6.3% of Legionella-pulsed DCs versus 2.4% of naive DCs; Fig. 1B). Consistent with this observation, the fractalkine upregulation of Legionella-pulsed DCs was also confirmed by semiquantitative RT-PCR analysis (Fig. 1C).



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  		FIG. 1. Upregulated fractalkine expression of L. pneumophila-pulsed DCs. (A) Confocal microscopic analysis. DCs were pulsed with DiI-labeled L. pneumophila at a ratio of 50 bacteria to 1 DC (L. pneumophila-pulsed DCs) or with PBS alone (naive DCs) for 3 h and viewed by confocal laser scanning microscopy for red fluorescence-labeled L. pneumophila and MHC class II (green fluorescence). (B) Flow cytometry analysis. DCs were pulsed with heat-killed L. pneumophila as for panel A and analyzed 2 days later for endogenous fractalkine expression. Propidium iodide (PI) staining was used to discriminate between viable and nonviable cells. The percentage of fractalkine-positive, PI– cells is shown in each panel. (C) Semiquantitative RT-PCR analysis. DCs were pulsed with heat-killed L. pneumophila as for panel A, and 2 days later the total cellular RNA from DCs was reverse transcribed into cDNA. The generated cDNA was used as a template to amplify endogenous fractalkine and GAPDH cDNA fragments by PCR. Samples were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. Lp, L. pneumophila.

Genetic modification of DCs. To enhance the chemoattracting property of Legionella-pulsed DCs, by which the endogenous fractalkine expression is upregulated to some extent, we genetically modified DCs with an E1– recombinant adenovirus vector expressing mouse fractalkine (AdFKN) and examined the FKN expression of the modified DCs by semiquantitative RT-PCR and a chemotaxis assay (Fig. 2). When cDNAs were subjected to PCR amplification with primer sets specific for AdFKN-derived fractalkine mRNA, exogenous (i.e., AdFKN-derived) fractalkine mRNA expression was detected at a high level only in AdFKN-modified DCs (Fig. 2A). In contrast, with common primer sets specific to AdFKN-derived and endogenous fractalkine mRNAs, the fractalkine transcript was robustly or feebly amplified from cDNAs of AdFKN-modified DCs or AdNull-modified DCs and naive DCs, respectively. As a control, similar low levels of mRNA expression of another chemokine, MDC/CCL22, were found in DCs with or without genetic modification. Comparable levels of expression of the housekeeping gene GAPDH confirmed the intactness of the RNA samples. No PCR amplification of RNA samples without reverse transcription (RT–) demonstrated a lack of genomic or vector DNA amplification. The chemoattracting activity of the fractalkine produced by AdFKN-modified DCs was assayed by using mouse T-lymphocyte EL4 cells (Fig. 2B). A significant migratory response of EL4 cells was observed in the culture supernatant of AdFKN-modified DCs compared with that of AdNull-modified DCs and naive DCs (P < 0.0005). The relevance of the observed chemotaxis to adenovirus-mediated fractalkine expression was demonstrated by the observation that the chemotactic activities of the AdFKN-conditioned medium were significantly inhibited by the addition of a neutralizing anti-mouse fractalkine antibody to the medium (P < 0.00005) but were unaffected by an isotype-matched control antibody (P > 0.2).



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  		FIG. 2. DCs were genetically modified to overexpress fractalkine. (A) Semiquantitative RT-PCR analysis of exogenous and endogenous fractalkine mRNA expression in genetically modified DCs. The total cellular RNA from DCs transduced with AdFKN, AdNull, or PBS alone (naive DCs) was reverse transcribed into cDNA with or without reverse transcriptase (RT+ or RT–). The generated cDNA was used as a template to amplify exogenous fractalkine, exogenous and endogenous fractalkine, MDC (control chemokine), and GAPDH cDNA fragments by PCR. Samples were separated by 1% agarose gel electrophoresis and stained with ethidium bromide. (B) Chemotaxis of T lymphocytes to conditioned medium from genetically modified DCs. Mouse T-lymphocyte EL4 cells placed in the upper chamber of a transwell chamber were assayed for chemotaxis in response to the supernatant from DCs transduced with AdFKN, AdNull, or PBS alone (naive DCs) in the lower chamber. Where indicated, an antifractalkine neutralizing antibody or rat control IgG was added to the supernatant from AdFKN-transduced DCs at the initiation of the assay. The number of cells migrating to the lower chamber was counted by flow cytometry. Results represent means ± standard errors (n = 3 per data point).

The overexpression of fractalkine in AdFKN-modified DCs was further verified by immunocytochemical analysis (Fig. 3). The immunofluorescent images from confocal laser scanning microscopy demonstrated the coexpression of fractalkine and the characteristic DC surface marker, MHC class II, in most of the AdFKN-modified DCs. In contrast, fractalkine expression was scarcely observed in AdNull-modified DCs and naive DCs, both of which expressed the MHC class II molecule, as did AdFKN-modified DCs.



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  		FIG. 3. Immunocytochemical evaluation of genetically modified DCs for fractalkine. Each fluorescent image of DCs transduced with AdFKN, AdNull, or PBS alone (naive DCs) was viewed by confocal laser scanning microscopy for fractalkine (red fluorescence, left and right panels) and MHC class II (green fluorescence, middle and right panels). Bars, 50 µm.

Protective effects of AdFKN-modified DCs. AdFKN modification reinforced the in vivo protective effect of DCs pulsed with L. pneumophila against a lethal Legionella bronchopulmonary infection (Fig. 4A). Prior studies from our laboratory demonstrated that the immunization of mice with DCs pulsed with L. pneumophila led to modest protection against a subsequent respiratory tract infection with Legionella. Consistent with this observation, the immunization of A/J mice with AdNull-modified DCs pulsed with heat-killed L. pneumophila (i.e., DCs pulsed with Legionella alone) provided 50% survival against a subsequent lethal challenge with Legionella (P < 0.01 for immunization with AdNull-modified DCs pulsed with L. pneumophila compared with no immunization). In contrast, 90% of mice immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila were protected against the lethal infection of L. pneumophila (P < 0.05 for immunization with AdFKN-modified DCs pulsed with L. pneumophila compared with all other groups). A control immunization with AdFKN-modified DCs alone or no immunization resulted in no survival against the Legionella challenge. As a further control, subcutaneous immunization with 750 ng recombinant mouse fractalkine and 8 x 106 CFU heat-killed L. pneumophila led to only 10% survival of the infected mice (P > 0.05 for comparison with no immunization; not shown).



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  		FIG. 4. Immunization of mice with AdFKN-modified DCs pulsed with L. pneumophila against lethal L. pneumophila respiratory infection. (A) A/J mice were immunized intravenously with AdFKN-modified DCs pulsed with heat-killed L. pneumophila ({blacksquare}, DC/AdFKN + Lp), AdNull-modified DCs pulsed with heat-killed L. pneumophila ({circ}, DC/AdNull + Lp), or AdFKN-modified DCs alone ({triangleup}, DC/AdFKN). Controls included naive mice without any immunization ({square}, no immunization). (B) CD4+ T-cell-deficient ({triangleup}), CD8+ T-cell-deficient ({circ}), B-cell-deficient ({diamond}), or wild-type C57BL/6 mice ({blacksquare}) were immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila. Controls included naive wild-type mice without any immunization ({square}). For both panels, mice were challenged by intratracheal administration of L. pneumophila 3 weeks after immunization. Survival was recorded as the percentage of surviving animals (n = 10 mice per group). Lp, L. pneumophila.

Using relevant knockout mice, we evaluated the role of CD4+ T cells, CD8+ T cells, and B cells in the protective immunity induced by immunization with AdFKN-modified DCs pulsed with heat-killed L. pneumophila (Fig. 4B). For this experiment, wild-type, CD4+ T-cell-deficient, CD8+ T-cell-deficient, and B-cell-deficient C57BL/6 mice were immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila and subsequently challenged with Legionella. The survival of CD4+ T-cell-, CD8+ T-cell-, and B-cell-deficient mice was not improved by the immunization compared with that of nonimmunized wild-type mice (P > 0.05). In contrast, 50% of immunized wild-type mice survived until the end of the experiment on day 14 (P < 0.001 compared with all other groups).

Anti-Legionella antibody responses. We next assessed the in vivo antibody responses of mice immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila by determining the serum levels of Legionella-specific antibodies (Fig. 5). A/J mice immunized with DCs pulsed with heat-killed L. pneumophila, regardless of their modification with AdFKN or AdNull, produced larger amounts of all serum anti-Legionella antibody isotypes than mice immunized with AdFKN-modified DCs alone and mice without any immunization (for IgM, P < 0.005; for IgG1, P < 0.05; for IgG2a, P < 0.05; for IgG2b, P < 0.05; for IgG3, P < 0.005; and for IgA, P < 0.05). Furthermore, for the IgG2b, IgG3, and IgA isotypes, immunization with AdFKN-modified DCs pulsed with heat-killed L. pneumophila significantly increased the levels of anti-Legionella antibodies compared with those induced by AdNull-modified DCs pulsed with heat-killed L. pneumophila (for IgG2b, P < 0.05; for IgG3, P < 0.05; for IgA, P < 0.05). As a control for the specificity of the anti-Legionella antibodies detected, the antibody levels generated by AdFKN-modified DCs alone were comparable for all isotypes with those of mice without any immunization (for IgM, P > 0.9; for IgG1, P > 0.1; for IgG2a, P > 0.6; for IgG2b, P > 0.9; for IgG3, P > 0.1; and for IgA, P > 0.4).



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  		FIG. 5. In vivo Legionella-specific antibody responses of mice immunized with AdFKN-modified DCs pulsed with L. pneumophila. A/J mice were immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila (DC/AdFKN + Lp), AdNull-modified DCs pulsed with heat-killed L. pneumophila (DC/AdNull + Lp), or AdFKN-modified DCs alone (DC/AdFKN). Controls included naive mice without any immunization (no immunization). Two weeks after immunization, each isotype of anti-Legionella antibody was assessed in serum using a standard ELISA protocol. Each titer is the inverse of the dilution giving an optical density at 405 nm of less double equals0.1. Values represent means ± standard errors (n = 3 mice per data point). Lp, Legionella pneumophila.

Splenic immune responses. To analyze the cellular immune responses induced by AdFKN-modified DCs pulsed with heat-killed L. pneumophila, we evaluated the splenocytes of immunized mice for proliferating and cytokine-secreting reactivities against Legionella (Fig. 6A and B, respectively). For these studies, spleen cells were isolated 2 weeks after the immunization of mice with AdFKN-modified DCs pulsed with heat-killed L. pneumophila and were cocultured in vitro for 4 days with heat-killed L. pneumophila. Controls included spleen cells from mice immunized with AdNull-modified DCs pulsed with heat-killed L. pneumophila and with AdFKN-modified DCs alone and from mice without any immunization.



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  		FIG. 6. Responses of splenocytes from mice immunized with AdFKN-modified DCs pulsed with L. pneumophila. (A) T-cell proliferation against Legionella. A/J mice were immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila (DC/AdFKN + Lp), AdNull-modified DCs pulsed with heat-killed L. pneumophila (DC/AdNull + Lp), or AdFKN-modified DCs alone (DC/AdFKN). Controls included naive mice without any immunization (no immunization). Two weeks after immunization, splenocytes were isolated and cocultured for 4 days with heat-killed L. pneumophila. The number of viable CD4+ or CD8+ T cells was determined by flow cytometry. Results represent percentages of increase or decrease relative to the baseline at the start of the coculture. (B) Cytokine profile. Splenocytes from immunized A/J mice were cocultured with heat-killed L. pneumophila as for panel A. The culture medium was collected, and the levels of mouse IFN-{gamma} and IL-4 were assayed by ELISA. For both panels, results are shown as means ± standard errors (n = 3 per data point). Lp, Legionella pneumophila.

Both CD4+ and CD8+ T cells of mice immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila responded significantly to Legionella and proliferated compared to those of all other control mice (P < 0.0005 and P < 0.05, respectively), but the AdFKN/Legionella/DC-mediated elevation in CD8+ T-cell proliferation was not as striking as that in CD4+ T-cell proliferation (for CD4+ T cells, there was a 1.9- to 4.2-fold increase compared with control immunizations; for CD8+ T cells, there was a 1.1- to 1.3-fold increase compared with control immunizations) (Fig. 6A). Further analysis of their cytokine profiles demonstrated that spleen cells from mice immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila exhibited a significant enhancement of IFN-{gamma} production compared to those from all other control mice (P < 0.005) but that the increased IFN-{gamma} level induced by the AdFKN/Legionella/DC immunization did not markedly differ from that induced by the AdNull/Legionella/DC immunization (for AdFKN/Legionella/DC immunization, 27,680 pg/ml; for AdNull/Legionella/DC immunization, 24,741 pg/ml) (Fig. 6B). On the other hand, a coculture with heat-killed L. pneumophila induced comparable levels of IL-4 release from spleen cells of all immunized and nonimmunized mice (P > 0.05), which were approximately 100-fold less than the IFN-{gamma} production levels (Fig. 6B).

IFN-{gamma} and IL-4 production of CD4+ T cells from immunized mice. Based on these results, CD4+ T cells from mice immunized as described above were assayed for IFN-{gamma} and IL-4 production by immunofluorescent intracellular staining (Fig. 7). Immunization with AdFKN-modified DCs pulsed with heat-killed L. pneumophila significantly increased the percentage of IFN-{gamma}+ CD4+ T cells among spleen cells that had been isolated from immunized mice and then had been stimulated in vitro with heat-killed Legionella (P < 0.005 compare with all other control immunizations). The AdFKN/Legionella/DC immunization also resulted in an increased percentage of IL-4+ CD4+ T cells compared with immunization with AdFKN-modified DCs alone or no immunization (P < 0.05), but the increased level was comparable to that induced by immunization with AdNull-modified DCs pulsed with heat-killed L. pneumophila (P > 0.6). Taken together with the splenocyte analyses (Fig. 6 and 7), these data suggest that AdFKN/Legionella/DC immunization, and to a lesser extent AdNull/Legionella/DC immunization, promotes Th1-polarized immune responses against L. pneumophila.



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  		FIG. 7. Characterization of CD4+ T cells from mice immunized with AdFKN-modified DCs pulsed with L. pneumophila. A/J mice were immunized with AdFKN-modified DCs pulsed with heat-killed L. pneumophila (DC/AdFKN + Lp), AdNull-modified DCs pulsed with heat-killed L. pneumophila (DC/AdNull + Lp), or AdFKN-modified DCs alone (DC/AdFKN). Controls included naive mice without any immunization (no immunization). Two weeks after immunization, splenocytes were isolated and cocultured for 4 days with heat-killed L. pneumophila. The percentage of CD4+ T cells that stained intracellularly with an antibody against mouse IFN-{gamma} or IL-4 was determined by flow cytometry (mean ± standard error; n = 3 per data point). Lp, Legionella pneumophila.


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DISCUSSION
 
Although L. pneumophila infection accounts for 2% to 15% of community-acquired pneumonia cases, the pathophysiology of Legionella infection has not been fully elucidated (5, 7, 8, 31). The aim of the present study was to provide insights into the cellular and molecular mechanisms that underlie the induction of protective immune responses against this bacterium, focusing on the endogenous and exogenous expression of a T-cell-attracting CX3C chemokine, fractalkine, by DCs. In this context, the endogenous fractalkine expression of DCs was found to be subtly increased after contact with heat-killed L. pneumophila in vitro. The in vitro data were relevant to the in vivo data because the exogenous overexpression of fractalkine in the Legionella-capturing DCs resulted in a significant enhancement of T-cell-mediated protective immunity to lethal Legionella infection. Thus, the fractalkine expression of DCs may be associated with the host defense against intracellular pathogens, including L. pneumophila.

DCs are professional antigen-presenting cells with a unique ability to induce primary T-cell-mediated immune responses compared to other antigen-presenting cells such as macrophages or B cells (2, 20). DCs of the immature phenotype are located at the interfaces of potential pathogen entry sites (2, 20, 25, 28). Once they have encountered a microbe, DCs migrate from the peripheral tissues to lymphoid organs and undergo maturation en route (2, 20, 25, 28). During maturation, they process the microbial products to present their fragments as complexes with MHC proteins to T cells, together with costimulatory and adhesion molecules (2, 20). In addition, the DC maturation process is accompanied by phenotypic and functional changes, including the upregulation of chemokines that bring T cells into close proximity to DCs as well as several other coordinated events such as changes in morphology, a loss of endocytic/phagocytic receptors, and an upregulation of costimulatory and adhesion molecules (2, 4, 19, 20, 32).

DC-derived chemokines known to be upregulated upon maturation include fractalkine (CX3CL1), interferon-inducible protein 10 (IP-10; CXCL10), MDC (CCL22), and TARC (thymus- and activation-regulated cytokine; CCL17) (4, 19, 32). Earlier findings that these chemokines differ in the efficacy with which they attract cells of the Th1 or Th2 cytokine-secreting phenotype (e.g., fractalkine and IP-10 or MDC and TARC are associated with the Th1 or Th2 phenotype, respectively) have suggested that the chemokine expression of mature DCs is involved in the selection of appropriate immune responses to be mounted for antimicrobial immunity (4, 6, 12, 19, 32). In particular, fractalkine has received much attention since the membrane-bound CX3C chemokine is a versatile molecule capable of inducing firm adhesion of receptor-bearing T cells by its membrane-bound form as well as T-cell chemotaxis by the local chemoattractant gradients of its shed form (3, 26).

Regarding the host immune response to L. pneumophila infection, we and others have recently observed that DCs help to initiate adaptive immunity, as in other bacterial infections, and that the MHC class II-restricted presentation of Legionella antigens by DCs facilitates priming of the protective CD4+ T-cell-mediated responses to Legionella infection (15, 24). In addition, previous clinical and experimental studies have demonstrated that the cellular immune response to Legionella infection appears to be dominated by CD4+ T cells that produce cytokines consistent with a Th1 phenotype, as follows: (i) significant increases in IFN-{gamma} and IL-12 levels often occur during the acute phase of Legionella infection, but increases in Th2 cytokine levels (i.e., IL-4 and IL-10) occur in very few patients; (ii) human blood lymphocytes activated with L. pneumophila express mRNA from the IFN-{gamma} gene but not from the IL-4 gene; and (iii) intratracheal administration of Legionella to A/J mice results in the development of pneumonia with increased serum levels of IFN-{gamma}, and the pneumonia is deteriorated by treatment with an anti-IFN-{gamma} antibody (5, 7).

Based on these considerations, we hypothesized that the DC-mediated immune response to L. pneumophila can be attributed at least in part to the DC-derived expression of the membrane-bound Th1 attractant fractalkine, which may promote both the chemotaxis of T cells toward Legionella-capturing DCs and the adhesion between them, leading to clonal expansion and a Th1-polarized differentiation of T cells recognizing Legionella antigens. Several pieces of evidence in the present study substantiate the validity of this hypothesis. In vitro pulsing of DCs with heat-killed Legionella subtly drove the endogenous DC expression of fractalkine, as demonstrated by the flow cytometric analysis. Consistent with the in vitro results, genetic modification of Legionella-pulsed DCs to overexpress exogenous fractalkine enabled DCs to provide the immunized mice with increased protection against the subsequent Legionella challenge in vivo, which could be viewed as a consequence of Legionella-specific immunoglobulin isotype responses induced by the immunization. In addition, these in vivo effects were well correlated with the finding that CD4+ T cells from the AdFKN/Legionella/DC-immunized mice proliferated and produced IFN-{gamma} in response to L. pneumophila. Further evidence comes from the observation that the survival of CD4+ T-cell-deficient and B-cell-deficient mice challenged with Legionella infection was not improved by the AdFKN/Legionella/DC immunization. Although the induction of Legionella-specific antibody responses by the AdFKN/Legionella/DC immunization may suggest the participation of IL-4 in it, little evidence to support this idea was provided by IL-4 analyses of immunized mice: the AdFKN/Legionella/DC and AdNull/Legionella/DC immunizations increased the IL-4-producing CD4+ T-cell frequencies in splenocytes of immunized mice, while the coculture with heat-killed L. pneumophila induced comparable levels of IL-4 release from spleen cells of all immunized and nonimmunized mice. The complete elucidation of the cellular mechanisms awaits further studies.

In terms of the chemokine involvement in the host immune defense against Legionella infection, three studies have been carried out so far. First, Yamamoto et al. showed that L. pneumophila infection of cultured mouse peritoneal macrophages increased the levels of cellular mRNAs for the neutrophil-attracting CXC chemokines, such as keratinocyte-derived chemokine and macrophage inflammatory protein 2 (both are mouse counterparts of human GRO/CXCL1-3), suggesting that these chemokines produced by macrophages contribute to the migration of neutrophils to sites of infection with this microorganism (22, 30). Second, Nakachi et al. characterized the Legionella-modulated gene expression profile of mouse alveolar macrophage MH-S cells by using a cDNA expression array technique and demonstrated that infection with virulent L. pneumophila significantly induced the gene expression of monocyte chemotactic protein 3 (CCL7), a CC chemokine that has a very broad range of target cells, including most leukocytic cell types (21, 23). Finally, Tateda et al. observed a neutrophil accumulation in Legionella-infected mouse lungs which was mediated by CXC chemokines such as keratinocyte-derived chemokine, macrophage inflammatory protein 2, and lipopolysaccharide-induced CXC chemokine (CXCL6) (22, 29). In contrast to these previous studies that emphasized the immunological roles of neutrophil chemoattractants and/or macrophages in Legionella infection, the present study points to DCs and their expression of a T-cell-attracting CX3C chemokine, fractalkine. These results may add to the growing evidence that fractalkine production by DCs has an effect on the development of Th1-cell-mediated immunity to intracellular bacteria such as L. pneumophila.


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ACKNOWLEDGMENTS
 
We thank K. Yamaguchi (Toho University School of Medicine, Tokyo, Japan) for the gift of the Legionella strain and B. Bell for reading the manuscript.

This study was supported in part by the Smoking Research Foundation (Tokyo, Japan), the Japan Research Foundation for Clinical Pharmacology (Tokyo, Japan), and the Ministry of Education, Culture, Sports, Science and Technology (Tokyo, Japan).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Respiratory Oncology and Molecular Medicine, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryomachi, Aobaku, Sendai 980-8575, Japan. Phone: (81)22-717-8539. Fax: (81)22-717-8549. E-mail: kikuchi{at}idac.tohoku.ac.jp. Back

Editor: J. N. Weiser


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Infection and Immunity, September 2005, p. 5350-5357, Vol. 73, No. 9
0019-9567/05/$08.00+0     doi:10.1128/IAI.73.9.5350-5357.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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