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Functional Relevance of the IL-23–IL-17 Axis in Lungs In Vivo

Lung Pharmacology and Immunology Groups, Department of Internal Medicine/Respiratory Medicine and Allergology, Institute of Medicine, Sahlgrenska Academy at Göteborg University, Gothenburg, Sweden; Lung Disease Research Group, Cooperative Research Centre for Chronic Inflammatory Diseases, Departments of Pharmacology and Medicine, The University of Melbourne, Parkville, Victoria, Australia; and Division of Pulmonology, Department of Pediatrics, Children's Hospital of Pittsburgh and the University of Pittsburgh, Pittsburgh, Pennsylvania

Correspondence and requests for reprints should be addressed to Stefan Ivanov, M.D., Lung Pharmacology & Immunology Groups, Department of Internal Medicine/Respiratory Medicine and Allergology, Institute of Medicine, Sahlgrenska Academy at Göteborg University, Guldhedsgatan 10A, S-413 46 Gothenburg, Sweden. E-mail: Stefan.Ivanov{at}lungall.gu.se

	Abstract

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It is known that interleukin (IL)-23, an IL-12-family cytokine, can be released by certain antigen-presenting cells in response to bacterial pathogens. Recent in vitro studies indicate that this cytokine stimulates a unique subset of CD4 cells, the T helper cell (Th)17 subset, to produce and release the proinflammatory cytokine IL-17. However, it has not been known whether this is an action of IL-23 per se that has bearing for the early innate response in lungs in vivo and whether there is an IL-23–responsive population of IL-17–producing CD4 cells in the bronchoalveolar space. We now present evidence that IL-23 can be involved in the early innate response to both gram-negative and gram-positive bacterial products in the lungs: Recombinant IL-23 protein per se accumulates inflammatory cells in the bronchoalveolar space in part via endogenous production of IL-17, and this IL-17 production occurs locally in IL-23–responsive CD4 cells. This IL-17 response to IL-23 occurs without any pronounced impact on Th1/Th2 polarization. Moreover, recombinant IL-23 protein increases the local MMP-9 activity, which is generated by neutrophils mainly. CD4 cells in the lungs may thus respond to IL-23 from antigen-presenting cells exposed to gram-negative and gram-positive pathogens and thereby reinforce the early innate response. These findings support that IL-23 and IL-17 form a functionally relevant "immunological axis" in the lungs in vivo.

Key Words: interleukin-23 • interleukin-17 • innate response • lungs

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   We show that in lungs in vivo, the antigen-presenting cell cytokine IL-23 and the T cell cytokine IL-17 form a functionally relevant "axis" linking innate and adaptive responses in the early phase of host defense.

 
Antigen-presenting cells (APCs), such as macrophages and dendritic cells, play a central role in recognizing various pathogenic stimuli from the outer environment. After this type of pathogen detection, APCs generate signals necessary for the induction and maintenance of appropriate immune responses, thus bridging the arms of innate and adaptive immunity (1). The members of the IL-12 cytokine family are considered to be important molecular components of this link (2, 3).

IL-23, a recently described member of the IL-12 cytokine family (3, 4), is produced by activated myeloid APCs in response to gram-negative as well as gram-positive bacteria and their products (5). This cytokine is a heterodimer, consisting of a unique p19 subunit plus a p40 subunit, the latter being shared with IL-12 (4). Whereas IL-12 is an important factor for the differentiation of naïve T cells into IFN-–producing T helper type 1 (Th1) cells (6), IL-23 may act mainly on memory T cells of the CD4 subtype, as indicated by previous studies on mouse splenocytes and human peripheral blood mononuclear cells in vitro (4, 7, 8). IL-23, unlike IL-12, induces the production of the proinflammatory cytokine IL-17 (synonymous to IL-17A) in isolated CD4 cells from mouse spleen in vitro (7). It has also been shown that IL-23 causes IL-17 production in homogenized mouse lungs in vitro, but there is no corresponding evidence in vivo (9).

With reference to the pathogenic context of IL-23, there is evidence from mouse models of autoimmune disease in the central nervous system and in joints, respectively, that this APC cytokine drives the proliferation of a unique subset of T helper (CD4 or Th) cells producing IL-17 protein, named the Th17 subset (10–14). IL-23 does induce the secretion of the proinflammatory cytokines IL-17F, IL-6, TNF, and low amounts of the Th1 cytokine IFN- in this Th17 subset in vitro (11–13). Moreover, IL-23 may induce IL-17 production in the CD8 subset as well, as indicated in human cord blood cells and mouse splenocytes in vitro (8, 15). However, the existing evidence for IL-23 per se playing a role in host defense of the lungs in vivo is incomplete; this evidence is limited to gram-negative bacteria causing less increase in IL-17 protein in homogenized lungs from IL-23–deficient mice, compared with wild-type controls (16). In analogy, IL-23–deficient mice display reduced IL-17 production in draining lymph node cells from mouse models of autoimmune disease in the central nervous system and in joints ex vivo (11, 12). Specifically, it is not known whether IL-23 per se is important for the early innate response in lungs in vivo and whether there is an IL-23–responsive population of IL-17–producing CD4 or CD8 cells in the bronchoalveolar space that contributes to the innate response within this organ compartment (3, 17). Nor is it known whether stimulation with IL-23 alters Th1/Th2 polarization in IL-17–producing CD4 cells in the lungs.

We designed the current study to determine the role of IL-23 in the innate response of the lungs. For this purpose, we employed local stimulation with Escherichia coli–derived lipopolysaccharide as well as Staphylococcus aureus–derived peptidoglycan, representing products of gram-negative and gram-positive bacteria, respectively (18). We also determined whether short-term stimulation with species-homologous and extracellular IL-23 protein per se actually leads to significant neutrophil accumulation and whether this effect is mediated via endogenous IL-17 production in lungs in vivo. In addition, we addressed the corresponding impact of IL-23 protein on the local proteolytic burden, using the gelatinase MMP-9 as a model marker of neutrophil and/or macrophage activity (19). Finally, we evaluated whether there is an IL-23–responsive population of CD4 or CD8 cells in the bronchoalveolar space that can account for local production of IL-17 and, if so, whether stimulation with IL-23 alters Th1/Th2 polarization in the responsive lung cells.

	MATERIALS AND METHODS

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Mice
Pathogen-free mice (BALB/c, male, 6–8 wk old; Mollegard-Bommice A/S, Ry, Denmark) were kept at the animal facilities of University of Göteborg under conditions approved by the Animal Ethics Committee of the University of Göteborg (Diary numbers 298–01, 177–04, and 138–2005). The animals received standard laboratory food and water ad libitum.

Anesthesia and Intranasal Treatment
Animals were transiently anaesthetized using isofluorane (Apoteksbolaget, Gothenburg, Sweden) and were subsequently stimulated intranasally with IL-23 (cat. No. 1887-ML; R&D Systems, Abingdon, UK) (1–3µg) or vehicle_LPS (Phosphate-buffered saline [PBS] + 0,1% bovine serum albumin [BSA] + 0.1–0.3 ng lipopolysaccharide [LPS]) in corresponding concentration and volume for 1 or 3 d (i.e., one or three daily doses). LPS was added to the vehicle to compensate for the maximal possible endotoxin content of the recombinant IL-23 protein (to avoid a false positive outcome of the study). In separate experiments, mice were intranasally stimulated with either vehicle_PBS (PBS + 0.1% BSA), vehicle_LPS, or IL-23 (1 µg) for 3 consecutive days to evaluate the impact of the added LPS.

The animals recovered fully after each anesthesia and did not display any clinical signs of long-term side effects. Twenty-four hours after the last intranasal stimulation, the mice were injected intraperitoneally with a mixture of ketamine (670 mg/kg) and xylazin (130 mg/kg) (both from Apoteksbolaget). After reaching deep anesthesia, the mice were killed by bleeding of the right ventricle of the heart.

Harvest of BAL Fluid
Tracheotomy was performed and the airways were washed twice with PBS (2 x 250 µl) through a tracheal cannula, followed by gentle aspiration. The recovered BAL suspension was kept on ice until centrifugation (model 5403; Eppendorf-Netheler, Hamburg, Germany; at 1,000 rpm corresponding to 100 x g, 10 min, 4°C). The cells were re-suspended in PBS containing 0.03% BSA and the total cell number was determined. The cell-free BAL supernatant was frozen (–80°C) until further analyses.

Cell differential counts were performed on cytospin slides prepared from BAL fluid (Cytospin 3; Shandon Life Sciences, Astmor, UK) after May-Grünwald-Giemsa staining as previously described (20). Briefly, all slides were evaluated in a light microscope (Zeiss Axioplan 2; Carl Zeiss AG, Jena, Germany) at x1,000 magnification, and standard morphologic criteria were used. Six hundred cells per sample were counted.

Harvest of Lungs
Lungs were washed with PBS (4 x 400 µl) through the tracheal cannula, then perfused with warmed (37°C) PBS (10 ml) through the right ventricle of the heart. Afterward the lungs were carefully excised, immediately placed in Eppendorf tubes, snap-frozen in liquid nitrogen, and stored (–80°C) until further use (see DETERMINATION OF IL-23 PROTEIN LEVELS BY ONE-DIMENSIONAL WESTERN BLOT ANALYSIS and MESSENGER RNA FOR IL-17 below).

For flow cytometry analysis of lung cells, lungs were placed in a mixture of Hanks' balanced salt solution (HBSS; Sigma-Aldrich, Stockholm, Sweden) and Golgi stop (cat N 51–2092KZ [554,724]; BD, Mountain View, CA), to block IL-17 protein secretion until staining of the cells. Lung tissue was disrupted mechanically and tissue fragments were removed via filtration through a cell strainer with a 100-µm nylon mesh followed by a filtration through a 40-µm nylon mesh-strainer (BD Falcon, BD Biosciences, San Jose, CA). The generated lung cell suspension was washed twice, re-suspended in PBS containing 0.5% BSA, and kept on ice until further use.

Determination of IL-23 Protein Levels by One-Dimensional Western Blot Analysis
Mice were stimulated intranasally with either PBS (50 µl), LPS (10µg in 50µl PBS, from E. coli, Serotype 026:B6; Sigma), or peptidoglycan (PepG: 50 µg in 50 µl PBS, from S. aureus, cat. no. 77140; Fluka, Sigma-Aldrich) (see ANESTHESIA AND INTRANASAL TREATMENT above), and after 1.5 h lung tissue and BAL fluid were harvested (see HARVEST OF BAL FLUID and HARVEST OF LUNGS above).

After thawing, the lung tissue was finely minced and placed in a mixture of anti-protease solution (Antiprotease Complete Mini; Roche Diagnostics, Mannheim, Germany) and modified RIPA buffer (50 mM Na₂HPO₄, 150 mM NaCl, 1% NP4, 0.5% Deoxycholate [NA-based], 0,1% SDS, 2 mM EDTA, and 1 mM pefablock). To homogenize the lung tissue, 0.2 g of acid-washed glass beads were added. After ultrasonification (Trofusonic, 80%, 10 min; Elma, Singen, Germany), cell debris was removed by centrifugation (10 min x 12,000 x g; Sigma, Labex Instrumental, Helsinborg, Sweden) and the protein concentration was determined using commercial protein assay kits (BSA protein assay kit, cat. no. 500-0113, and Bio-Rad protein assay kit, cat. no. 500-0114; Bio-Rad Laboratories, Hercules, CA). The cell lysate was boiled for 5 min in SDS sample buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% -mercaptoethanol, before loading onto a 10% SDS-polyacrylamide gel. After electrophoresis (Mini Protean 3 Cell; Bio-Rad), the gel was electroblotted onto a Hybond ECL nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden). The nonspecific binding sites on the membrane were blocked by exposure to 0.5% fat-free milk powder in Tris-buffered saline (TBS) with 0.5% Tween-20 for 4 h. A rat anti-mouse IL-23 p19 monoclonal antibody (0.5µg in 30 ml TBS, clone G23-8, lot No E017737; Nordic BioSite, Täby, Sweden) was added, followed by horseradish peroxidase–conjugated anti-rat IgG (0.5µg in 30 ml TBS9, cat. no. 6130-05; SouthernBiotech, Birmingham, AL). Bound antibodies were visualized with ECL Plus (Amersham Pharmacia Bioscience). The level of IL-23 protein was determined by exposure in a charge-coupled device camera (LAS-1000; Fuji Science Imaging, Fujifilm, Dusseldorf, Germany).

In addition to lung tissue, we processed cell-free BAL fluid in a similar manner, the procedures starting from the addition of the antiprotease solution and on, as described above.

Systemic Blockade of Endogenous IL-17
Mice were pretreated with a neutralizing anti-mouse IL-17 antibody (Monoclonal anti-mouse, clone 50104; R&D Systems) (21); (100 µg in 500 µl PBS) or its isotype control (Purified Rat IgG2, clone R35-95; BD Biosciences Pharmingen, Erembodegen, Belgium) (100 µg in 500 µl PBS) intraperitoneally, 12 h before each intranasal stimulation (IL-23 1 µg or vehicle_LPS for 3 d). BAL samples were harvested 24 h after the last intranasal stimulation and processed as described above.

Messenger RNA for IL-17
Total RNA was isolated from 10 mg of lung tissue ground to a fine powder under liquid nitrogen using a commercial kit (RNeasy; Qiagen, Doncaster, Victoria, Australia) according to the manufacturer's instructions. The purified total RNA was used to generate first-strand cDNA synthesis with Super Script III (Invitrogen, Mount Waverley, Victoria, Australia) as described previously (22). The reaction mix containing 1 µg of RNA, 250 ng of random hexamers (Promega, Melbourne, Australia), and 10 mM dNTP mix was diluted to 12 µl in sterile water, heated (65°C) for 5 min, and chilled on ice for 5 min. First-strand synthesis was then performed in a 20-µl total reaction volume by adding 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM DTT, 40 U RNaseout, and 200 U Superscript III reverse transcriptase enzyme at 25C for 10 min followed by 50°C for 50 min. The reaction was inactivated by heating (70°C) for 15 min. Complementary DNA was stored (–20°C) before amplification. Quantitative real-time PCR was performed as described previously (ABI PRISM 7900 HT Sequence Detection System; Applied Biosystems, Melbourne, Australia) (23) using pre-developed Taqman primers from Applied Biosystems (Assays on Demand, Foster City, CA). Briefly, gene expression was quantified by multiplexing single reactions, where our gene of interest (IL-17) was standardized to control (18 s rRNA). An individual sample from the control group was then arbitrarily assigned as a calibrator against which all other samples are expressed as a fold change.

Measurement of Cytokines
Cell-free BAL fluid was analyzed for free, soluble IL-17 and total pro–MMP-9 using commercially available enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems).

Flow Cytometry Analysis of Intracellular IL-17, IFN-, and IL-4 Proteins in Lung Cells
Flow cytometry analysis was performed as described earlier (24) with slight modifications. Unspecific binding was blocked (2% mouse sera; Dako, Glostrup, Denmark) for 15 min. The cells were thereafter incubated with a PerCP-conjugated anti-CD4 antibody (clone RM4–5; BD Biosciences) or its isotype-matched control (30 min at 4°C). Surface-immunostained cells were fixed in paraformaldehyde (4%) at room temperature (10 min), followed by two washings with PBS containing 0.5% BSA. After resuspension in 2 ml of SAP Buffer (HBSS containing 0.1% saponin and 0.05 NaN₃), the cells were incubated (45 min) with a PE-conjugated rat anti-mouse IL-17 monoclonal antibody (clone TC11–18H10; BD Bioscience), fluorescein-conjugated rat anti-mouse IFN- (clone 37,895; R&D Systems), or Alexa fluor–conjugated anti-mouse IL-4 (clone 11B11; BD Biosciences) or their proper isotype controls, at room temperature, followed by two washes with SAP buffer. Finally, the cells were washed with PBS containing 1% fetal calf serum (FCS), resuspended in the same buffer, and analyzed using a FACScan flow cytometer (BD Biosciences). Ten thousand cells were computed in a list mode and analyzed using the CellQuest Software (BD Biosciences).

Gelatinase Expression and Activity in BAL Fluid
Zymography was used to identify protease expression in response to IL-23 stimulation as previously described (25). Briefly, SDS-PAGE mini-gels (10%) were prepared with the incorporation of gelatin (2 mg/ml; Labchem, Pittsburgh, PA) before casting. Cell-free BAL fluid (20 ml) was run in the gels at a constant voltage of 200 V under nonreducing conditions. When the dye front reached the bottom, the gels were removed and washed twice for 15 min in 2.5% Triton X-100 and incubated at 37°C overnight in zymography buffer (50 mM Tris-HCl [pH 7.5], 5 mM CaCl₂, 1 mM ZnCl₂, and 0.01% NaN₃). The gels were then stained for 45 min with Coomassie blue background.

Cell-free BAL fluid was also tested for net gelatinase activity using fluorescence-conjugated gelatin (Molecular Probes, Invitrogen, Stockholm, Sweden) (23). The gelatin substrate (10 mg) was diluted in 50 mM Tris (pH 7.5), 150 mM NaCl, 5 mM CaCl₂, and 0.01% NaN₃ and incubated at room temperature for 16 h with 100 ml neat BAL fluid. The digested substrate has absorption/emission maxima at 495 nm/515 nm and its fluorescence intensity was measured in a microplate reader Flexscan II Benchtop scanning fluorometer workstation (Molecular Devices, Sunnyvale, CA) to detect quantitative differences in activity.

Immunocytochemistry for MMP-9 in BAL Cells
BAL cells from IL-23– (1 and 3 µg; 3 d) and vehicle_LPS-stimulated animals were fixed immediately after lavage with formaldehyde (2%; 30 min; on ice). Cells were then washed twice before making cytospin preparations. Air-dried samples were kept frozen (–80°C) until further use.

After thawing, slides were treated with donkey serum (10%) to avoid unspecific binding. Endogenous biotin was blocked using Biotin Blocking System (Dako). The intracellular expression of MMP-9 was assessed using polyclonal goat anti-mouse MMP-9 antibody (0.5 µg/ml, cat. no. AF 909, 1 h; R&D Systems). As secondary antibody, a biotinylated F(a)₂ fragment donkey anti-goat IgG antibody (cat. no. 705-066-147; Jackson ImmunoResearch Europe, Soham, UK) was used followed by alkaline phosphatase–conjugated streptavidin (Dako). Bound antibodies were visualized with Liquid Permanent Red (Dako). Mayer's Hematoxylin (Sigma-Aldrich, Steinheim, Germany) was used for counterstaining. The slides were evaluated using light microscopy at a 1,000-fold magnification (Carl Zeiss).

Stimulation of Bronchoalveolar CD3 Cells with IL-23 In Vitro
In vivo preparation. Pathogen-free mice (BALB/c, male, 6–8 wk old; Mollegaard-Bommice) were primed in vivo with endotoxin (LPS 30 µg in 50 µl PBS, LPS from E. coli, Serotype 026:B6; Sigma) for 42 h (see ANESTHESIA AND INTRANASAL TREATMENT above). BAL fluid was harvested as described above, with the modification that the airways were washed four times with PBS (4 x 1 ml). The recovered BAL suspension was pooled together, kept on ice until further centrifugation (at 1,000 rpm corresponding to 100 x g, 10 min, 4°C, model 5403; Eppendorf-Netheler, Hamburg, Germany). The cells were then resuspended in PBS containing 0.03% BSA and the total cell number was determined.

Culture of CD3 Cells and Stimulation with Exogenous IL-23
Enrichment of CD3 cells was conducted using positive selection as previously described (21). Briefly, BAL cells were incubated with a monoclonal biotinylated anti-CD3e Ab (clone 145–2C11; BD Biosciences) followed by incubation with streptavidin-coupled micro-beads (Miltenyi Biotec, Bergisch-Gladbach, Germany). The CD3 cells were then isolated by passing the BAL cells through a column (MACS; Miltenyi Biotec) in magnetic field.

The enriched CD3 cell population displayed 52% purity (n = 3) and it was seeded (0.5 x 10⁶ cells/well) in a 96-well plate (model 3072; BD Biosciences) in RPMI 1640 supplemented with FCS (10%), penicillin-streptomycin (100 U/ml and 100 g/ml), sodium pyruvate (1%), and L-glutamine (2 mM) (all from Sigma), and cultured in presence of media alone (vehicle, i.e., negative control), recombinant mouse IL-23 (cat. no. 1887-ML; R&D Systems) (100 ng/ml) or phorbol myristate acetate (PMA) and calcium ionophore (end conc. 2 ng/ml and 1 µg/ml respectively, both from Sigma) (positive control). In total, the cells were cultured for 12 h (5% CO₂, 37°C), during the last 6 of which Brefeldin A solution was added in a final concentration of 10 µg/ml, to block protein secretion.

Flow Cytometry Analysis of Intracellular IL-17 Protein in Bronchoalveolar CD3 Cells
The CD3 cells were processed as described above (see FLOW CYTOMETRY ANALYSIS OF INTRACELLULAR IL-17, IFN-, AND IL-4 PROTEINS IN LUNG CELLS), but with the modification that they were stained with a PerCP-conjugated anti-CD4 antibody (clone RM4-5; BD Biosciences) or anti-CD8 (clone 53-6.7; BD Biosciences) or their isotype-matched controls. Thereafter, the cells were incubated (45 min) with a PE-conjugated rat anti-mouse IL-17 monoclonal antibody (clone TC11-18H10; BD Biosciences) or its isotype control—PE-conjugated rat IgG1 (clone R3-34; BD Biosciences). The analysis was performed using a FACScan flow cytometer (BD Biosciences). Ten thousand cells were computed in a list mode and analyzed using the CellQuest Software (BD). Data for the impact of stimulus of interest (IL-23 or PMA+CI) on CD4 and CD8 cells are expressed as percent of the negative control (i.e., respective cells cultured in medium alone).

Data Presentation and Statistical Analysis
All data are presented as mean (SEM) unless otherwise stated. Statistical analysis of differences between groups was conducted using Student's t test (unpaired or paired, one- or two-way, as appropriate) or ANOVA if three variables were compared. A P value less than 0.05 was considered statistically significant.

	RESULTS

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IL-23 Protein in Lung Tissue and BAL Fluid
To determine the pathogenic context in which IL-23 is released, we measured the protein levels of IL-23 in lung tissue and BAL fluid after intranasal stimulation with PBS, LPS, or PepG. As early as 1.5 h after stimulation with LPS or PepG, IL-23 protein was substantially increased in both lung tissue and BAL fluid (Figures 1A and 1B).

View larger version (114K): [in this window] [in a new window]   Figure 1. IL-23 protein levels determined 1.5 h after intranasal stimulation in vivo with either PBS, LPS (10 µg), or Peptidoglycan (PepG) (50 µg). Data are shown as mean ± SEM; n = 3–5 per group analysis done with ANOVA. *P < 0.05 (A) Lung tissue. (B) BAL fluid.

Intranasal stimulation with IL-23 increased the number of total cells in the BAL fluid after 3 d of stimulation (Figure 2A), and there was a similar trend after 1 d of stimulation, although not statistically significant (data not shown). IL-23 increased the number of BAL macrophages after 1 and 3 d of stimulation as well (Figure 2B). The number of BAL neutrophils and BAL lymphocytes was elevated after 3 d of stimulation (Figures 2C and 2D) and there was a similar trend for both cell types after 1 d of stimulation, although not statistically significant (data not shown).

View larger version (33K): [in this window] [in a new window]   Figure 2. Inflammatory cells in BAL suspension after intranasal stimulation with recombinant mouse IL-23 (1µg) or vehicleLPS. BAL was performed 24 h after the last intranasal stimulation. Data are shown as mean ± SEM; n = 7–13 per group. *P < 0.05. (A) Total BAL cells. (B) BAL macrophages. (C) BAL neutrophils. (D) BAL lymphocytes.

Separate experiments showed that there was no statistically significant difference in the cellular characteristic of BAL fluid between vehicle alone and vehicle plus added LPS (Table 1).

View larger version (52K): [in this window] [in a new window]   Figure 3. Effect of a neutralizing anti-mouse IL-17 antibody on inflammatory cells in BAL suspension after intranasal stimulation with IL-23 (1 µg) or vehicleLPS during 3 d. Twelve hours before each intranasal stimulation, the animals were pretreated with anti-mouse IL-17 antibody (100 µg in 500 µl PBS) or its isotype control antibody (100 µg in 500 µl PBS) intraperitoneally. Data are shown as mean ± SEM; n = 5–8 per group. *P < 0.05; analysis done with ANOVA. (A) Total BAL cells. (B) BAL macrophages. (C) BAL neutrophils.

View larger version (27K): [in this window] [in a new window]   Figure 4. Relationship between the concentration of IL-17 protein in cell-free BAL fluid and the number of inflammatory cells in BAL suspension. Mice were stimulated intranasally with vehiclePBS, vehicleLPS, or IL-23 (see MATERIALS AND METHODS) during 3 d, and BAL suspension was harvested 24 h after the last stimulation. Data are presented as mean ± SEM; n = 4–6. A represents the data for BAL macrophages and IL-17; B, BAL neutrophils and IL-17; and C, BAL lymphocytes and IL-17.

View larger version (18K): [in this window] [in a new window]   Figure 5. Assessment of the impact of IL-23 on IL-17 production. (A) Relative expression of IL-17 mRNA in lung tissue cells after intranasal stimulation with IL-23 (1 µg) or vehicleLPS during 1 and 3 d, respectively. Lungs were harvested 24 h after the last intranasal stimulation. Data are shown as mean ± SEM; n = 7 per group. *P < 0.05. (B) Concentration of free, soluble mouse IL-17 protein in cell-free BAL fluid after intranasal stimulation with IL-23 (1 µg) or vehicleLPS for 1 or 3 d. BAL was performed 24 h after the last intranaal stimulation. Data are shown as mean ± SEM; n = 7–13 per group. *P < 0.05.

Intracellular Protein Expression after Stimulation with IL-23
To determine a cellular source of IL-17, we isolated endotoxin-primed BAL cells harvested from mice in vivo, and subsequently enriched them for CD3 cells through positive selection with MACS and then cultured these cells in vitro. After culture in three different conditions (plain medium, IL-23 [100 ng/ml], or positive control [PMA+CI]), a flow cytometry analysis was conducted. This analysis showed that in vivo–primed CD4 and CD8 cells express intracellular IL-17 protein without additional stimulation (i.e., cultured in plain medium only) in vitro (CD4⁺/IL-17⁺: 1.25 ± 0.4% of CD4; CD8⁺/IL-17⁺: 0.9 ± 0.6% of CD8). The flow cytometry analysis also revealed that additional stimulation with IL-23 in vitro increased the IL-17–expressing fraction of CD4-positive CD3 cells significantly more than the IL-17–expressing fraction of CD8-positive CD3 cells (Figures 6A and 6C). Additional stimulation with IL-23 in vitro also increased intensity of IL-17 expression within each CD4 cell (Figure 6B).

View larger version (39K): [in this window] [in a new window]   Figure 6. Impact of IL-23 stimulation on IL-17 expression in murine CD 3 cells. All data represent mean values ± SEM from three separate and independent experiments. (A) BAL CD3 cells harvested from endotoxin-primed mice (see MATERIALS AND METHODS), were in vitro stimulated with IL-23 (100 ng/ml) or PMA+CI for 12 h. The fractions of CD4-positive, IL-17–positive CD3 cells (CD4+/IL-17+; solid bars) and of CD8–positive, IL-17–positive CD3 cells (CD8+/IL-17+; open bars) are shown. Data are expressed as percent of the negative control (Neg ctrl), that is, cells cultured in plain medium only. *P < 0.05, paired t test. (B) Intensity of intracellular expression of IL-17 in CD4-positive CD3 cells (CD 4+/IL-17+) and of CD8–positive CD3 cells (CD 8+/IL-17+) after in vitro stimulation with IL-23 or PMA+CI, detected by flow cytometry. Data for the IL-23– and PMA+CI-stimulated cells are presented as percent of the mean fluorescence intensity (MFI) of the negative control (see panel A), normalized to background (isotype control staining). *P < 0.05, paired t test. (C) Representative flow cytometry scattergrams of intracellular staining for IL-17 with the IL-17–positive BAL fluid cells (IL-17+) among CD4+ or CD8+ subpopulations after in vitro stimulation with IL-23 encircled (see also legend for panel A above). (D) Fraction of CD4-positive, IL-17–positive lung cells harvested 24 h after 3-d intranasal stimulation with IL-23 or vehicleLPS (see MATERIALS AND METHODS). Data are expressed as percent (%) of CD4 cells. *P < 0.05, unpaired t test.

Gelatinases in BAL Fluid
To assess the local proteolytic burden, we ascertained the identity (pro–MMP-9 ELISA and zymography) and activity (gelatinase substrate assay) of gelatinases in BAL fluid. Three days of stimulation with IL-23 (3 µg) in vivo substantially elevated the concentration of soluble pro–MMP-9 protein in BAL fluid (Figure 7A). There was a similar trend after three days of stimulation with IL-23 (1 µg) in vivo that did not reach statistical significance (data not shown). Zymography analysis of each single BAL sample revealed a dominant 92-kD band accounting for most gelatinase activity in all samples after stimulation with IL-23 (1 µg) during 3 d in vivo (Figure 7B). Moreover, 3 d of stimulation with IL-23 (1 µg) in vivo clearly increased the gelatinase activity in BAL fluid (Figure 7C). No such increasing effect was observed after one day of stimulation with IL-23 (1 µg) (data not shown).

View larger version (123K): [in this window] [in a new window]   Figure 7. Assessment of impact of IL-23 on the proteolytic load in BAL fluid harvested 24 h after stimulation. (A) Concentration of total pro–MMP-9 protein in cell-free BAL fluid harvested after 3 d of stimulation with IL-23 (3 µg) or vehicleLPS intranasally. Data are shown as mean ± SEM; n = 7–8 per group. *P < 0.05. (B) A representative example of gelatin zymography of cell-free BAL fluid harvested after 3 d of stimulation with IL-23 (1 µg) or vehicleLPS intranasally. The molecular size (kD) is indicated on the left side of the panel. (C) Net gelatinase activity in cell-free BAL fluid harvested after 3 d of stimulation with IL-23 (1 µg) or vehicleLPS intranasally. The gelatinase activity was determined using a gelatin conjugated fluorescence substrate assay (see MATERIALS AND METHODS). Data are shown as mean ± SEM; n = 11–13 per group. *P < 0.05. (D) A representative result of immunocytochemical detection of intracellular mouse MMP-9 protein in a macrophage and a neutrophil from BAL suspension harvested 24 h after 3 d of stimulation with IL-23 (1 µg) intranasally.

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Using a mouse in vivo model, we show that IL-23 protein in the lungs is increased by LPS and PepG, respectively, and that recombinant, species-homologous IL-23 given intranasally per se increases the number of macrophages and neutrophils in BAL fluid. Anti–IL-17 given systemically blocks the increase in inflammatory BAL cells. Intranasal IL-23 increases mRNA for IL-17 and IL-17 protein as well as gelatinase activity locally. After IL-23 stimulation, the dominant gelatinase in cell-free BAL fluid is MMP-9, and soluble pro–MMP-9 is increased as well. Among inflammatory BAL cells, neutrophils account for the strongest intracellular signal for MMP-9 after IL-23 stimulation. Finally, we show that IL-23 stimulation of BAL cells causes an increase in IL-17 among CD4 cells mainly, and in lung CD4 cells, this impact is exerted without any major change in co-expression of IL-4 and IFN-.

Recent studies have shown that APC's can produce IL-23 when exposed to different bacterial stimuli (5). More specifically, bone marrow-derived mouse DC's cultures pulsed with Klebsiella pneumoniae show increased expression of p19 mRNA (i.e., the specific subunit of IL-23); expression that is Toll-like receptor (TLR) 4–dependent (15). On the other hand, S. aureus–derived PepG, acting via TLR 2/TLR 6 heterodimer (18), also increases the expression of p19 mRNA in human dendritic cells (18, 26). The fact that LPS, a TLR 4 agonist (18), and PepG, a TLR 2/TLR 6 agonist, both increase IL-23 production in our in vivo model, suggests that IL-23 could be released as part of the innate response to invasion of gram-negative as well as gram-positive bacteria. Moreover, our new findings consolidate those of a recently published study on the role of endogenous IL-23 in host defense of the lungs (16). In that particular study, production of IL-17 was assessed in homogenized lungs after exposure to K. pneumonie in vivo, and this was done using genetically engineered mice lacking functional IL-23 since birth. Consequently, that previous design included a clear risk of confounding interference from secondary and compensatory immunologic events, due to a redundancy in the immune system after a lifelong lack of IL-23 protein. The design of that previous study also made it impossible to distinguish between intracellular and extracellular IL-17 protein levels and therefore the in vivo relevance of extracellular IL-23 protein was not conclusively proven. Thus, our current study now supplements and strengthens the evidence previously published. It does this by demonstrating the immunologic relevance of IL-23 protein, that can be produced in response to gram-positive and gram-negative bacteria, for endogenous IL-17 production, subsequent neutrophil accumulation, and an MMP-9–related net increase in proteolytic activity in lungs in vivo.

Interestingly, the fact that the neutrophil and macrophage response to IL-23 is attenuated by pretreatment with a neutralizing anti–IL-17 antibody in our study may have indirect implications for the role of other members of the IL-17 family of cytokines (3, 17). This is because we have ascertained the specificity of the anti–IL-17 antibody that we used. In separate experiments, we confirmed this specificity by showing that the anti–IL-17 antibody used does not block IL-17F's activity in vitro, as measured by the induction of IL-6 in NIH3T3 cells by IL-17F, whereas it does block IL-17A induction of IL-6 in the same assay (S. Ivanov and coworkers, unpublished observations). Thus, our current study indirectly forward the possibility that IL-17 (synonymous with IL-17A), and not the structurally and functionally similar T cell cytokine IL-17F or other IL-17 cytokines, is functionally most important in mediating the direct immune response to IL-23 in lungs in vivo.

Using another mouse model, a model of allergic airway inflammation, we recently showed that endogenous IL-17 contributes to allergen-induced accumulation of monocyte-lineage cells in lungs in vivo (27). Specifically, we presented evidence that allergen-induced release of endogenous IL-17 exerts a direct action on the recruitment of monocyte-lineage cells and their subsequent survival in the bronchoalveolar space in vivo (27). It therefore seems likely that recombinant IL-23 protein in part exerts its effect on bronchoalveolar macrophages via endogenous IL-17 acting as a recruitment and survival-promoting factor. It is worth noting that our finding that systemic blockade of endogenous IL-17 with an antibody did attenuate IL-23's macrophage-accumulating effect in the bronchoalveolar space is fully compatible with recent findings in the central nervous system of mice (14). Using an in vivo model of autoimmune disease in the central nervous system, it was shown that the corresponding type of IL-17 blockade causes a significant reduction in IL-23–induced accumulation of macrophages (14).

The critical role of IL-23 for the production of IL-17 has previously been indicated by several observations in vitro. Thus, it is known that deficiency of IL-12 p40 (inability to produce endogenous IL-12 and IL-23), but not deficiency of IL-12 p35 (inability to produce endogenous IL-12 alone), presumably in macrophages, results in abrogation of IL-17 production by T cells in mouse models of gram-negative and helmithic infections (15, 28). Furthermore, there is reduced production of IL-17 in vitro by T cells from IL-23–deficient mice that have been activated in vivo (12) or by T cells primed in vitro with IL-23–deficient APCs (29). Previous studies also indicate that CD4 as well as CD8 cells are capable of producing IL-17 in various types of inflammation (30–33). Importantly, the one and only previous demonstration of T cells residing within the bronchoalveolar space actually producing IL-17 in vivo is a study demonstrating intracellular IL-17 protein in "lymphoid" BAL cells from human volunteers exposed to organic dust (34). That particular study includes evidence of increased mRNA for IL-17 in the collective population of BAL cells but no information specifically proving a CD4 or CD8 origin. It is therefore of particular interest that we now show that CD4- and CD8-positive CD3 cells from the bronchoalveolar space of mice, primed and harvested in vivo, do express IL-17 protein without further stimulation in vitro. Moreover, we present evidence that among these CD3 cells, it is the CD4 subset that responds directly to IL-23 protein per se in vitro, by displaying a significantly increased fraction of cells and an increased individual intensity of each cell positive for intracellular IL-17 protein. Thus we provide evidence that there are IL-23–responsive CD4 cells in the bronchoalveolar space of mice. Importantly, we confirm these results in vivo as well, by demonstrating that short-term IL-23 stimulation increases the IL-17–positive CD4 lung cell fraction. This latter impact of IL-23 is exerted without any pronounced impact on Th1/Th2 polarization in the responsive CD4 cells, as assessed by co-expression of IL-4 and IFN-. Whereas it remains to be proven that these cells constitute a truly unique Th17 subset in the lungs (10), our novel data illustrate that IL-23 per se may act as a direct amplification stimulus of IL-17 production in CD4 cells of the lungs.

Our current demonstration of IL-23 per se exerting a clear-cut increasing effect on MMP-9–related gelatinase activity within the bronchoalveolar space in vivo suggests an impact on the local proteolytic burden; this may have bearing for several inflammatory lung diseases involving mobilization of MMP-9 (19). Even though our current study does not specifically prove the involvement of the secondary mediator IL-17 in this particular effect on MMP-9–related activity, endogenous IL-17 is very likely to be involved. This is because it is known that IL-17 per se can control the proteolytic burden in the bronchoalveolar space of mice in vivo, through its impact on neutrophil accumulation (23). The fact that bronchoalveolar neutrophils mainly did express MMP-9 protein in our current study is fully in line with what we have previously observed after local stimulation with recombinant and species-homologous IL-17 in the same organ compartment of mice (23). Indeed, in our current study on IL-23, a limited number of bronchoalveolar macrophages also displayed intracellular MMP-9 protein, similar to what has previously been shown in IL-17–stimulated macrophages derived from human blood monocytes (35, 36). However, in our current study on bronchoalveolar cells, this signal for intracellular MMP-9 protein in macrophages was weaker and less reproducible than that observed in the neutrophils. We also found that the concentration of total, pro–MMP-9 protein in the bronchoalveolar fluid was substantially increased after stimulation with the high dose of IL-23 alone, and this can be explained in at least two ways. Either this effect is dose dependent or this effect requires the involvement of additional IL-23–induced factors, apart from IL-17.

In conclusion, our current study forwards evidence that in lungs in vivo, the APC cytokine IL-23 and the T cell cytokine IL-17 form a functionally relevant "axis" linking innate and adaptive responses, in the early phase of host defense. Importantly, this axis is probably activated via TLR 2/6 and TLR 4 stimulation, respectively, and may involve IL-17 release mainly from local CD4 cells. In this context, IL-23 does not seem to markedly alter Th1/Th2 polarization. Of special functional importance is the finding that IL-23 per se does increase the proteolytic burden, a burden likely to be generated by the accumulation of MMP-9–producing neutrophils locally. These findings are all compatible with "Th17" cells acting in the lungs in vivo, even though more evidence is needed to prove that these cells are truly unique in relation to Th1 and Th2 cells (10). If similar mechanisms exist in humans, then IL-23 constitutes a potential target for pharmacotherapy in lung diseases characterized by an excess local accumulation of either APCs or of neutrophils, in an aberrant host defense (37, 38).

	Acknowledgments

 
The authors express their gratitude to Professor Jan Lötvall, the Department of Internal Medicine/Respiratory Medicine & Allergology, at Göteborg University, for his generous support and facilitation of the work presented in this manuscript.

	Footnotes

 
Funding: Financial support was obtained from the Swedish Heart-Lung Foundation and the Swedish Medical Research Council (K2005–74X-09048–16A), the NHMRC and CRC-CID Australia. No support was obtained from the tobacco industry.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0020OC on December 1, 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 January 18, 2006

Accepted in final form October 31, 2006

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