| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
* Molecular Microbiology Group, and
Immunobiology Group, Center of Molecular Biosciences, University of the Ryukyus, Okinawa, Japan;
Division of Host Defense and Vaccinology, Department of Microbiology, and
Division of Child Health and Welfare, Department of Investigative Medicine, Graduate School of Medicine, University of the Ryukyus, Okinawa, Japan;
¶ Division of Microbiology and Immunobiology, Department of Medical Technology, School of Health Sciences, Tohoku University and Infection Control Research Center, Tohoku University Hospital, Miyagi, Japan;
|| Division of Immunology and Molecular Biology, Cancer Research Institute, Kanazawa University, Ishikawa, Japan; and
# Center for Experimental Medicine, Institute of Medical Science, University of Tokyo, Tokyo, Japan
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that 
T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN- in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
10% of affected individuals show evidence of symptoms and develop the clinical disease. The immunological mechanism for the breakdown of host resistance in these individuals is unclear. Cell-mediated immunity is thought to be the major component of host defense against M. tuberculosis. The necessity of CD4+T cells in the control of M. tuberculosis in humans is illustrated by the strong association of CD4+T cell impairment and the reactivation of M. tuberculosis in patients with HIV infection (2). The reactivation of latent infection that stems from a failure of tissue granulomas to contain the organism (particularly in the lung) has also been reported in experimental models (3). Although neutrophils are not considered to be effective antimycobacterial effector cells, it has been reported that neutrophils participate in the immune response against mycobacterial infection (4, 5). Depletion of neutrophils at the early stage of mycobacterial infection resulted in an increase of bacterial burden in several reports (5, 6) and reduced pulmonary granuloma formation in another report (7). The depletion of neutrophils roughly 2 wk after mycobacterial infection has been reported to induce an increase in the bacterial count in the lung (8), although the bacterial burden did not increase under similar conditions in another report (5). In contrast, the induction of neutrophils by LPS or rG-CSF resulted in enhanced protection against infection. Because mycobacteria induce cytokine production by neutrophils (5), cytokines are likely candidates in the mechanism of neutrophil-mediated enhancement of protection and granuloma formation. These reports suggest that neutrophils participate in the immune response to mycobacterial infection.
IL-17 is a proinflammatory cytokine secreted by T lymphocytes (9, 10, 11, 12, 13) that enhances the generation, activation, and migration of neutrophils through the induction of CXC chemokines, IL-6, IL-8, G-CSF, and TNF (14, 15, 16). Studies have shown the importance of IL-17 in various physiological and pathophysiological processes, including the induction of granulopoiesis (17, 18), host defense against Klebsiella or Candida infections (19, 20), rheumatoid arthritis (21, 22), allograft rejection (23, 24), and asthma (25, 26). IL-17 also triggers neutrophil migration to the lung (15). It has recently been reported that IL-17 is required for the optimal induction of Th1-type and Th2-type immune response, although the mechanism has not yet been clarified (27).
IL-23 was recently identified as a cytokine which induces IL-17 expression (28). IL-23 is a disulfide-bonded heterodimeric cytokine composed of an IL-23-specific p19 subunit and a p40 subunit common to IL-12 and IL-23. It is produced by macrophages and dendritic cells (29). The IL-23R also consists of two components a unique IL-23R subunit (IL-23R) and a subunit shared by IL-12 and IL-23 receptors (IL-12R
1) (30). The biological functions of IL-12 and IL-23 are similar but not identical. IL-23 is not as potent as IL-12 in the induction of IFN- production (29). Instead, IL-23 (but not IL-12) induces the proliferation of memory CD4+ T cells and the IL-17 production of CD4+ T cells (29, 28, 31). IL-23-transgenic mice consistently develop multiorgan inflammation associated with neutrophilia (32). Furthermore, mice deficient in IL-23 but not IL-12 are highly resistant to experimental autoimmune encephalomyelitis, whereas mice deficient in IL-12 but not IL-23 display defective development of Th1-type immune responses (33). A recent study reported that IL-23 enhances protective immunity to Cryptococcus neoformans infection and a deficiency of IL-23 is linked to a decrease in IL-17 production (34). All of these results indicate that IL-23 is an important regulator of the inflammatory immune response mediated by IL-17.
The role of IL-23 in mycobacterial infection has also been investigated. Mice lacking both IL-12 and IL-23 (IL-12/23p40-deficient mice) had a higher bacterial burden in Mycobacterium bovis bacille Calmette-Guérin (BCG)- or Mycobacterium tuberculosis-infected organs than mice deficient in IL-12 alone (IL-12p35-deficient mice) (35, 36). These results suggest an involvement of IL-23 in host defenses against mycobacterial infection. In contrast, an analysis of IL-23p19-deficient mice showed that IL-23 is superfluous in the response to M. tuberculosis infection (37). It is therefore controversial whether the IL-23/IL-17 axis is important in protective immunity against mycobacterial infections.
In the present study, we hypothesized that IL-17 participates in the immune response against mycobacterial infection through neutrophil induction and Th1 enhancement. We analyzed IL-17 expression in wild-type mice as well as the immune responses of IL-17 gene-knockout (KO)3 mice after lung Mycobacterium bovis BCG infection. Our results demonstrated that IL-17 is expressed during the early stage of M. bovis BCG infection and that IL-23 is required for the induction of IL-17. Although IL-17 is widely considered to be a CD4+ T cell product, we identified TCR + T cells and non-T cells as the major IL-17-producing cells during the early stage of infection. Furthermore, the lack of IL-17 resulted in reduced IFN- production by mycobacteria-specific CD4+ T cells and impaired granuloma formation after M. bovis BCG infection. We will discuss the implications of these results later in this report.
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
IL-17 gene-KO mice were generated as described previously (27). IL-17 KO mice of (129/Sv x C57BL/6)F1 hybrid background were backcrossed to the C57BL/6 for more than eight generations. The genotyping of IL-17 KO mice was conducted using the following PCR primers: primer 1, 5'-ACT CTT CAT CCA CCT CAC ACG A-3'; primer 2, 5'-GTA CAC CAG CTA TCC TCC AGA TAG-3'; primer 3, 5'-GCC ATG ATA TAG ACG TTG TGG C-3'. Primers 1 and 2 were used to detect the wild-type allele and primers 1 and 3 were used to detect the mutant allele. IL-12/23p40-deficient mice of C57BL/6 background (38) were purchased from The Jackson Laboratory. TCR C-deficient mice of C57BL/6 background were reported previously (39, 40). C57BL/6 Cr mice were purchased from Japan SLC. All animals were used for experiments at 8–12 wk of age. These mice were kept under conventional conditions in an environmentally controlled clean room at the Center of Molecular Biosciences (University of the Ryukyus, Okinawa, Japan). The experiments were conducted according to the institution’s ethical guidelines for animal experiments and the safety guideline for gene manipulation experiments.
Microorganisms and bacterial infection
M. bovis BCG (Japan BCG Association) was grown in 7H9 medium (Difco) supplemented with albumin-dextrose-catalase enrichment (Difco). Small aliquots of M. bovis BCG suspended in 7H9 medium containing 10% glycerol were stored at –80°C until use. The viable bacterial numbers were determined by 7H10 (Difco) plate supplemented with oleic acid-albumin-dextrose-catalase enrichment (Difco). The concentration of bacteria was quantified by colony counting. The bacteria were resuspended in PBS before use. Mice were inoculated intratracheally (i.t.) with 5 x 106 CFU of M. bovis BCG in 50 µl of PBS. M. bovis BCG-infected mice were sacrificed on days 0, 1, 3, 5, 7, 10, 14, 21, and 28. For the in vitro mycobacterial infection of splenocytes, 5 x 106 splenocytes were infected with 5 x 105 CFU of M. bovis BCG in 0.5 ml of antibiotic-free RPMI 1640 medium (Sigma-Aldrich) containing 10% heat-inactivated FBS for 90 min and an equal volume of RPMI 1640 containing 10% FBS, 100 U/ml gentamicin was subsequently added. The splenocytes were incubated for either 24 or 48 h.
Delayed-type hypersensitivity (DTH) responses
M. bovis BCG-infected mice were tested for a DTH response to the purified protein derivative (PPD; Japan BCG Association) derived from M. tuberculosis by the injection of 10 µg of PPD into the right hind footpad 28 days after the infection. The footpad was measured 24 and 48 h later using a spring-loaded micrometer (Mitutoyo) and the swelling was determined by the following formula: (footpad thickness of the PPD-injected right footpad (millimeter)) – (footpad thickness of the uninjected left footpad (millimeter)).
rIL-17
The stable transfectant producing IL-17 was described previously (41). Briefly, FBL-3 erythroleukemia cells were transfected with pEF-BOS mammalian expression vector (42) carrying the cDNA for full-length mouse IL-17 and pBL-hygB carrying the hygromycin B-resistance gene. Hygromycin B-resistant clones producing the IL-17 were selected. The culture supernatant was collected and the IL-17 concentration was determined and used as rIL-17.
Cell preparation
The lung was perfused with PBS through the right ventricle before excision from the mice. The excised lung tissue, separated from all the associated lymph nodes (LNs), was minced and incubated for 1 h at 37°C in 5 ml of PBS containing 1.0% FBS, 125 U/ml collagenase I (Sigma-Aldrich), 60 U/ml DNase I (Sigma-Aldrich), and 60 U/ml hyaluronidase (Sigma-Aldrich). Single-cell suspensions (pulmonary infiltrated (PIF) cells) were prepared by passing through 30-mm stainless steel mesh. To enrich the pulmonary lymphocytes, PIF cells were resuspended in 8 ml of 45% Percoll solution (Amersham Biosciences), overlaid on 5 ml of 67.5% Percoll solution, and centrifuged at 2200 rpm for 20 min at 20°C. The cells at the interface were collected and used for in vitro culture or flow cytometric analysis. Single-cell suspensions from the mediastinal LNs and spleens were also prepared by passing through 30-mm stainless steel mesh.
Cells in the bronchoalveolar lavage fluid (BAL) were recovered at the indicated times after infection with M. bovis BCG. Briefly, the BAL was collected with 1 ml of RPMI 1640 medium containing 10% FBS and 68 mM EDTA. Cells in the BAL were collected and resuspended with 50% FBS-RPMI 1640 medium. Cytospin slides were prepared using a Cytospin model IV (Shandon). Fifty microliters of a 5 x 105 cells/ml cell suspension was placed into the chamber which was attached to cytospin slides, then centrifuged at 800 rpm for 3 min. The cells were morphologically examined after staining with May-Grünwald and Giemsa solutions (Wako Pure Chemical).
Cell culture and ELISA for cytokine production
Single-cell suspensions were prepared in complete RPMI 1640 medium supplemented with 10% FBS. The suspensions (5 x 105 cells in volume of 200 µl of complete RPMI 1640 medium) were added to each well in 96-well plates and incubated in triplicate with or without 5 µg/ml PPD. In some experiments, rIL-17, rIL-23 (R&D Systems), or rIL-12 (PeproTech) was added into the culture. Cells were also cultured in the presence of BCG with or without anti-IL-23p19 Ab (2.5 µg/ml; R&D Systems). The plates were incubated at 37°C in an atmosphere of 5% CO2 for 24 and 48 h, and the culture supernatants were collected and stored at –30°C until analysis. IFN- (R&D Systems), IL-13 (BD Biosciences), IL-23 (eBioscience), and IL-17 (R&D Systems) were assayed by ELISA kits.
Magnetic separation
To enrich T cells, spleen cell suspension was passed through a nylon wool column. The T cell subsets were further fractionated by high-gradient MACS (Miltenyi Biotec). The cells were incubated with FITC-conjugated anti-CD8a (BD Biosciences), PE-conjugated anti-CD4, and allophycocyanin-conjugated anti-CD3e mAbs (BD Biosciences) for 15 min at 4°C. After washing, cells were resuspended and incubated with anti-FITC microbeads (Miltenyi Biotec) for 15 min at 4°C. After another washing step, CD8+ cells were isolated using an AutoMACS (isolation mode: deplete). Negative fractions containing CD4+ and CD4–CD8– cells were incubated with anti-PE microbeads (Miltenyi Biotec) for 15 min at 4°C. After washing, CD4+ cells were isolated using an AutoMACS (isolation mode: deplete). Negative fractions containing CD4–CD8– cells were incubated with anti-allophycocyanin microbeads (Miltenyi Biotec) for 15 min at 4°C. CD3-positive or -negative CD4–CD8– cells were isolated using an AutoMACS (isolation mode: deplete). Aliquots of the unsorted (whole splenocytes) and sorted cell fractions (CD4+, CD8+, CD3+CD4–CD8–, and CD3–CD4–CD8– cells) were analyzed by flow cytometry (FCM) as described below. The CD4+, CD8+, CD3+CD4–CD8–, and CD3–CD4–CD8– cell populations were sorted to a purity of >98, >98, >95, or >93%, respectively.
Expression of cytokine/chemokine genes
Total RNA was extracted from various organs, such as the lung, mediastinal LNs, or the spleen, using TRIzol reagent (Invitrogen Life Technologies). First-strand cDNA was synthesized from 2 µg of RNA using reverse transcriptase (Superscript II; Invitrogen Life Technologies) and 20 pM of random primer in 20 µl of reaction buffer. The synthesized first-strand cDNA were amplified by quantitative real-time PCR using 20 pM of each primer pair with 2.5 U of the Taq polymerase (Takara Shuzo) in a total volume of 20 µl of the reaction buffer consisting of 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatin, 0.2 mM dNTP, and SYBR Green I (Cambrex Bio Science). Thermal cycling was initiated with a first denaturation step of 5 min at 95°C, followed by 40 cycles of 95°C for 10 s and 60°C for 30 s. The fluorescence emitted from amplified DNA was read at 60°C at the end of each cycle. The data of the real-time PCR amplification were analyzed using the iCycler iQ and the Real-Time PCR Optical System Software version 3.0 (Bio-Rad). The cycle number at which the various transcripts were detectable, referred to as the threshold cycle (Ct), was compared with that of -actin and referred to as 
Ct. The relative gene level was expressed as 2–(Ct), in which Ct equals Ct of the experimental sample minus Ct of the control sample. The specific primers were as follows: IL-17 sense (5'-AAG GCA GCA GCG ATC ATC C-3'), IL-17 antisense (5'-GGA ACG GTT GAG GTA GTC TGA G-3'); IL-23p19 sense (5'-CCT GCT TGA CTC TGA CAT CTT C-3'), IL-23p19 antisense (5'-TGG GCA TCT GTT GGG TCT C-3'); IL-12/23p40 sense (5'-ACA TCA AGA GCA GTA GCA GTT C-3'), IL-12/23p40 antisense (5'-AGT TGG GCA GGT GAC ATC C-3'); IL-12p35 sense (5'-CCA CCC TTG CCC TCC TAA AC-3'), IL-12p35 antisense (5'-GGC AGC TCC CTC TTG TTG TG-3'); IL-15 sense (5'-AAA CCC ATG TCA GCA GAT AA-3'), IL-15 antisense (5'-AAG TAG CAC GAG ATG GAT GT-3'); KC sense (5'-TCG CCA ATG AGC TGC GCT GTC-3'), KC antisense (5'-GCT TCA GGG TCA AGG CAA GCC-3'); MIP-2 sense (5'-GAG CTT GAG TGT GAC GCC CCC AGG-3'), MIP-2 antisense (5'-GTT AGC CTT GCC TTT GTT CAG TAT C-3'); G-CSF sense (5'-TCA TTC TCT CCA CTT CCG-3'), G-CSF antisense (5'-GTA TTT ACC CAT CTC CTT CC-3'); IL-6 sense (5'-TCC AGT TGC CTT CTT GGG AC-3'), IL-6 antisense (5'-GTG TAA TTA AGC CTC CGA CTT G-3'); TNF-
sense (5'-TTC TGT CTA CTG AAC TTC GGG GTG ATC GGT CC-3'), TNF- antisense (5'-GTA TGA GAT AGC AAA TCG GCT GAC GGT GTG GG-3'); -actin sense (5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3'), and -actin antisense (5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3').
In some experiments, expression of IL-17 and -actin was confirmed by agarose gel electrophoreses of PCR products and staining of the gel with ethidium bromide after adjustment of cDNA amount by the quantitative real-time PCR of -actin.
Flow cytometric analysis of intracellular cytokine assay
To analyze the IL-17 expression of the cells from the in vitro infection system, spleen cells were incubated with or without M. bovis BCG for 48 h at 37°C and 5% CO2, with GolgiPlug-containing brefeldin A (BD Biosciences) added for the last 8 h in 24-well flat-bottom plates (Nalge Nunc International) in 1 ml of RPMI 1640 medium containing 10% FBS. After 8 h of culture, the cells were harvested and washed once in PBS containing 1.0% newborn calf serum and 0.1% NaN3 (staining buffer).
To analyze the IL-17 expression of the cells of the in vivo infection system, PIF cells or fractionated pulmonary lymphocytes 3 days after M. bovis BCG infection were incubated with or without 1 µg/ml calcium ionophore A-23187 (Calbiochem) and 25 ng/ml PMA (Sigma-Aldrich) for 4 h at 37°C and 5% CO2 in the presence of GolgiPlug.
The cells were pretreated with culture supernatant from 2.4G2 hybridoma producing mAb specific for FcR II/III (Fc blocker), and were then surface stained with allophycocyanin-conjugated anti-CD3, NK1.1, CD8, or CD11b (Mac-1), FITC-conjugated anti-TCR C or Gr-1, and biotin-conjugated anti-TCR C, CD4, or CD45R/B220 (BD Biosciences) mAbs plus TriColor-streptavidin. Surface-stained cells were subjected to intercellular IL-17 staining. For intracellular cytokine staining, we used PE-conjugated anti-IL-17 mAb after permeabilization of the cells using Cytofix/Cytoperm kits (BD Biosciences).
To examine the Ag-specific Th1 immune-response in the in vivo infection system, pulmonary lymphocytes at 7, 14, and 28 days after M. bovis BCG infection were incubated with or without 5 µg/ml PPD in the presence of mitomycin C-treated spleen cells (1 x 105 cells) from naive mice for 24 h at 37°C and 5% CO2, with the addition of GolgiPlug for the last 6 h. Cells were pretreated with Fc blocker, and subsequently surface stained with allophycocyanin-conjugated anti-CD3 mAb. To detect Th1 cells, we used PE-conjugated anti-IFN- mAb.
For both intracellular IL-17 and IFN- staining, cells were detected by a flow cytometer, FACSCalibur (BD Biosciences). The data were analyzed with CellQuest software (BD Biosciences).
Bacterial counts in organs
Seven, 14, and 28 days after M. bovis BCG infection, the mice were sacrificed and their lungs, spleens, and livers were removed. The organs were homogenized in saline containing 0.05% Tween 80. Ten-fold serial dilutions of the homogenates were placed onto Middlebrook 7H10 agar (Difco). The plates were incubated at 37°C for 3 wk. After incubation, the colonies were counted and the bacterial counts in organs were calculated as log10 CFU per organ.
Histopathology
The mice were sacrificed at 3, 7, 14, and 28 days after infection with M. bovis BCG. Approximately one-third of the lung and the spleen were fixed in buffered formalin and embedded in paraffin for histopathological examination. Thin sections with 4-mm thickness were prepared and stained with H&E.
To quantify the granuloma area, histological data were acquired using a charge-coupled device camera (Olympus). The digital data were analyzed using the Image J program distributed by the National Institutes of Health. The threshold was set to discriminate between granuloma tissue and normal tissue and the percentage granuloma area was calculated by the Analyze Particle command. Ten to 15 sections were analyzed and the mean and SD of the percentage granuloma area were calculated.
Statistical analysis
The statistical significance of the data was determined by Student’s t test. A p value of <0.05 was considered to indicate a significant difference.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
To assess the involvement of IL-17 in the immune response against mycobacteria, we initially analyzed the expression of IL-17 in the lungs of mycobacteria-infected mice. IL-17 mRNA in the lung of M. bovis BCG-infected C57BL/6 mice was detected on day 1 postinfection, peaked on day 5, and then returned to the baseline by day 7 (Fig. 1). We also investigated the factors which induced the IL-17 expression. Other studies have reported that IL-15 and IL-23 are potential inducers of IL-17 production (28, 43). We detected no correlation between IL-15 and IL-17 expression (Fig. 1). In contrast, IL-23p19 expression increased on day 1 and stayed at the same level for up to 3 days (Fig. 1). IL-23p19 expression increased again on day 7 and maintained high expression level up to day 21. IL-12p35 expression also increased on days 1–3, but returned to baseline by day 5. IL-23 is a heterodimeric cytokine consisting of a unique p19 subunit and a p40 subunit shared by IL-12 and IL-23, and IL-12 consists of unique p35 and common p40. The expression of p40 was not induced at the early stage of infection although its expression was enhanced at a later stage. This finding suggests that constitutively expressed IL-12/23p40 is used to form IL-12 or IL-23. We subsequently tested the involvement of both IL-23 and IL-12 in the induction of IL-17. rIL-23 but not rIL-12 induced IL-17 production in resident pulmonary lymphocytes in a dose-dependent manner, thus suggesting that IL-23 but not IL-12 plays an important role in IL-17 production (Fig. 2A). Furthermore, CD3+ T cells in the resident pulmonary lymphocytes were the major IL-17-producing cells significantly induced by rIL-23; however, they were only slightly induced by rIL-12 (Fig. 2B). We also found that IL-23 but not IL-12 induced IL-17 production in resident peritoneal exudate cells (44). To further confirm the importance of IL-23 in the induction of IL-17 after M. bovis BCG i.t. infection, we infected wild-type and IL-12/23p40 KO mice with M. bovis BCG. The expression of IL-17 in the lungs of the IL-12/23p40 KO mice was markedly diminished in comparison to that in the wild-type mice on days 3 and 28 after the M. bovis BCG infection (Fig. 2C). However, it is still possible that IL-12 contributes to IL-17 induction because IL-12/23p40 KO mice lack both IL-12 and IL-23. To confirm the role of IL-23 in the induction of IL-17 production in the resident pulmonary lymphocytes, the lymphocytes of wild-type mice were infected in vitro with M. bovis BCG in the presence or absence of neutralizing Ab to IL-23p19 or control Ig. As shown in Fig. 2D, IL-17 expression increased significantly after the mycobacterial infection, while the addition of neutralizing Ab to IL-23p19 to the culture suppressed the induction of IL-17 expression. Furthermore, we detected comparable levels of IL-23p19 mRNA (Fig. 2E) and IL-23 protein (data not shown) in the lungs of the wild-type and IL-17KO mice after M. bovis BCG infection, indicating that IL-23 production is independent of IL-17. These data indicate that IL-23 is required for IL-17 production by M. bovis BCG-infected mice.
|
|
It has been reported that IL-17 induces the production of neutrophil CXC chemokines such as human IL-8, murine KC/CXCL1, and murine MIP-2/CXCL2 (45, 46, 47). IL-17 also induces the production of cytokines important in the induction, activation, or survival of neutrophils, such as G-CSF, IL-6, and TNF (15, 48). Therefore, we investigated whether IL-17 is responsible for the chemokine/cytokine expression induced by M. bovis BCG infection. As shown in Fig. 3, the expression of KC and MIP-2 were severely impaired on days 1–3 and on day 1, respectively, in the lungs of infected IL-17 KO mice in comparison to that in wild-type mice. The expressions of G-CSF, IL-6, and TNF were also diminished in the IL-17 KO mice in comparison to those in the wild-type mice. In findings consistent with the decrease of chemokine/cytokine expression in the IL-17 KO mice, the BAL cells of the IL-17 KO mice contained significantly lower number of neutrophils on days 1–5 after infection (Table I). These results indicate that IL-17 participates in the induction of acute neutrophil-mediated inflammation in the lungs of M. bovis BCG-infected mice. In addition, the numbers of monocytes and lymphocytes in the BAL cells of the IL-17 KO mice were also lower in comparison to the wild-type mice after M. bovis BCG infection (Table I).
|
|
We established an in vitro infection system to determine the phenotype of IL-17-producing cells at the early stage of mycobacterial infection. Splenocytes from naive wild-type mice were infected with M. bovis BCG in vitro and were separated into several fractions after 24 and 48 h of culturing. The expression of IL-17 was subsequently analyzed by RT-PCR. Unexpectedly, strong IL-17 mRNA expression was detected in the CD4–CD8– cells (Fig. 4A). Because spleen TCR + T cells are CD4–CD8–CD3+ T cells, we theorized that CD4–CD8–CD3+TCR + T cells produce IL-17 in the culture system. Therefore, CD4–CD8– cells were separated into CD3-positive or -negative populations and analyzed. The expression of IL-17 induced by M. bovis BCG infection was detected not only in CD4–CD8–CD3+ T cells but also in CD4–CD8–CD3– (non-T) cells (Fig. 4B, left panels), thus indicating that IL-17 is expressed by both TCR + T cells and non-T cells. To confirm the expression of IL-17 by TCR + T cells, an in vitro infection analysis was conducted using spleen cells from TCR C KO mice. IL-17 expression of CD4–CD8–CD3+ T cells was markedly reduced in the TCR C KO mice (Fig. 4B, right panels). Interestingly, IL-17 production by CD4–CD8–CD3– (non-T) cells was also decreased in the TCR C KO mice after infection.
|
+ and TCR C+ cells produced IL-17, although C+ T cells represented >80% of the IL-17-producing T cells (Fig. 4D). The ratio of IL-17-producing cells in TCR C+ cells (45%) was higher than that in TCR C+ cells (3%).
To confirm IL-17 production by TCR + T cells and non-T cells in vivo in the mycobacteria-infected lung, we i.t. infected the wild-type mice with M. bovis BCG, and analyzed the IL-17 expression by PIF cells and pulmonary lymphocytes. CD3+ T cells were the major IL-17-producing cells in the infected lungs, although CD3– non-T cells produced IL-17 as well (Fig. 5A, left panel). To determine which T cells produce IL-17 upon M. bovis BCG-infection, pulmonary lymphocytes were stained with mAb against CD4 or CD8 and IL-17, and analyzed by FCM. Approximately 20% of the IL-17+ CD3+ T cells were CD4+, but the remaining IL-17-producing T cells were the CD4–CD8– phenotype (Fig. 5A, right panels). Among the CD3+ T cells, both the TCR C+ and TCR C+ T cells produced IL-17 (Fig. 5B). The ratio of IL-17-producing cells in TCR C+ T cells (45%) was higher than that in TCR C+ T cells (3.5%). In addition, the mean fluorescence intensity of IL-17 staining of the TCR C+ T cells (2150.6) was higher than that of the TCR C+ T cells (425.1). These results indicate that TCR + T cells produce IL-17 at a higher frequency and intensity than TCR + T cells. The data indicate that TCR + T cells are the major IL-17-producing cells in vivo. We further investigated the markers of IL-17-producing non-T cells. However, we could not detect IL-17 production in NK1.1+, CD45R/B220+, CD11b+, or Gr-1+ cells (Fig. 5C).
|
Because the participation of neutrophils in granuloma formation was suggested, and IL-17 itself was reported to enhance the Th1 response, we hypothesized that a lack of IL-17 during mycobacterial infection influences the establishment of the acquired immune response and granuloma formation. A histological examination was conducted on the lungs of M. bovis BCG-infected IL-17 KO mice on day 28 of infection, when the acquired immune response and granulomas were established in the lungs of the wild-type mice (Fig. 6). The size and number of granulomas in the lungs of the IL-17 KO mice were reduced in comparison to the granulomas in the wild-type mice on day 28 of infection (Fig. 6, A and B). The granulomas in the lungs of the IL-17 KO mice were less densely packed with mononuclear cells in comparison to those in the wild-type mice (Fig. 6A, e and f). This result indicates that IL-17 is an important factor in the establishment of granulomas.
|
1:0.6) was nearly identical with that in the wild-type mice. These results suggest that the accumulation of both monocytes and granulocytes was reduced in the granulomas in the IL-17 KO mice.
Impaired IFN- production by mycobacterial Ag-specific T cells and DTH in the IL-17 KO mice after infection with M. bovis BCG
We investigated whether the absence of IL-17 affected the Ag-specific Th1 immune response to mycobacterial Ags after i.t. infection with M. bovis BCG. On day 14 after infection, there was no statistically significant difference in IFN- production by pulmonary lymphocytes in the wild-type and IL-17 KO mice, although IL-17KO mice tended to show slightly lower levels of production (Fig. 7A). On day 28 after infection, the lung lymphocytes from the IL-17 KO mice showed a significantly lower level of IFN- production than those from the wild-type mice (p < 0.01). In contrast, IL-4 and IL-13 were not produced in the pulmonary lymphocytes of either the wild-type or the IL-17 KO mice at any stage of M. bovis BCG infection (data not shown), indicating that the diminished IFN- production in the IL-17KO mice was not due to a deviation to the Th2-type immune response. To determine the population of IFN--producing cells, we analyzed the CD3+ pulmonary lymphocytes by intracellular IFN- staining and FCM analysis (Fig. 7B). T cells from the IL-17 KO mice showed a slightly lower percentage of IL-17-producing cells (1.7% of CD3+ T cells) in comparison to that of the wild-type mice (2.8% of CD3+ T cells) (Fig. 7B, upper panels). The ratio of IFN--producing cells was significantly lower in the T cells from the IL-17 KO mice than that from the wild-type mice (5.1 ± 0.7% in the IL-17 KO mice vs 11.7 ± 1.4% in the wild-type mice) on day 28 after infection (p < 0.01). These results indicated that the generation of Th1 cells was impaired in IL-17 KO mice infected with M. bovis BCG at 4 wk.
|
production of T cells. To examine the possibilities, we stimulated the pulmonary lymphocytes on day 28 after infection with graded concentrations of rIL-17. rIL-17 did not affect the IFN- production of the pulmonary lymphocytes from either the wild-type or the IL-17 KO mice (Fig. 7C). These results suggest that IL-17 is not a direct inducer of IFN- production in T cells. To further investigate the cell-mediated immune response in the IL-17 KO mice, we evaluated the ability of the IL-17 KO mice to mount DTH responses. We sensitized the wild-type and IL-17 KO mice by M. bovis BCG infection, elicited DTH responses 4 wk later by injection of PPD into the right hind footpads, and measured specific footpad swelling 24 and 48 h after the challenge. We found that the DTH to mycobacterial Ag was inhibited in the IL-17 KO mice in comparison to that of wild-type mice (Fig. 7D). Therefore, IL-17 is indispensable to the optimal induction of DTH responses and a lack of IL-17 leads to an inefficient cell-mediated immune response.
Bacterial loads of various organs in IL-17 KO mice after infection with M. bovis BCG
To analyze the role of IL-17 in protective immunity against mycobacterial infection, we examined the bacterial growth in various organs of the wild-type and IL-17 KO mice after i.t. infection with M. bovis BCG. As shown in Fig. 8, the bacterial numbers in the lungs, livers, and spleens of the IL-17 KO mice were similar to those in the wild-type mice on days 7, 14, and 28 after infection. The data suggest that IL-17 is superfluous during the early protective immune response that suppresses bacterial expansion in the infected organs.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
In this study, we found that TCR + T cells and unidentified non-T cells were the major IL-17-producing cells in the mycobacteria-infected spleen and lung. It has been reported that IL-17 is produced by TCR+CD4–CD8– thymocytes (54) as well as by activated CD4+ and CD4+CD45RO+ memory T cells (9). Activated CD8+ and CD8+CD45RO+ memory T cells are also produce IL-17 in humans (55). However, we previously reported that both TCR + T cells and TCR + T cells produced IL-17 after stimulation with the Fas ligand (41). The ratio of IL-17-producing cells in the TCR + T cells was higher than that in the TCR + T cells. Among the T cell subset identified by CD4/CD8 expression, CD4–CD8– cells were the major producers of Fas ligand-induced IL-17, although some CD4+CD8– cells produced it as well. This distribution of IL-17-producing T cell subsets in M. bovis BCG-infected spleen cells is similar to that observed in Fas ligand-induced IL-17 production. We confirmed that both TCR + T cells and TCR + T cells produce IL-17 in splenocytes and pulmonary lymphocytes after M. bovis BCG infection (Figs. 4D and 5B). The percentage of IL-17-producing TCR + T cells among the IL-17-producing T cells (60–70%) was higher than that of IL-17-producing TCR + T cells. It is noteworthy that the proportion of IL-17-producing cells among the TCR + T cells (45%) was higher than that in the TCR + T cells (3.5%) after M. bovis BCG infection, and that the mean fluorescence intensity of IL-17 staining is higher in TCR + T cells than TCR + T cells. These observations suggest that TCR + T cells are the major IL-17-producing cells in our system. Furthermore, a portion of IL-17-producing cells were non-T cells without CD3 expression (Figs. 4C and 5A). Recently, it was reported that neutrophils produce IL-17 in a model of LPS-induced lung inflammation (56); however, no IL-17 production by neutrophils was detected in our system. The identification of IL-17-producing non-T cells is ongoing.
The role of neutrophils at the early stage of mycobacterial infection is controversial. Several in vitro studies suggested that human neutrophils are able to kill virulent M. tuberculosis (57, 58) but other reports failed to reproduce the result (59). A recent report demonstrated the increased bacterial burden in infected organs from the early stage of mycobacterial infection when neutrophils are depleted from mice before and/or during this stage (6). However, another report failed to detect any difference in the bacterial counts in the neutrophil-depleted mice (60). We demonstrated that the absence of IL-17 resulted in a significant reduction of neutrophil accumulation in the lung (Table I and Fig. 6C) without compromising the control of bacterial growth on days 7–28 after infection (Fig. 8). The data indicate that IL-17-induced neutrophils themselves are not effective effector cells in the elimination of mycobacteria at the early stage of infection before the establishment of acquired immunity. Although neutrophils may not act as direct effector cells against mycobacteria, they may serve as important immunoregulators. Cytokines and chemokines are produced by mycobacteria-activated neutrophils (7, 61). Furthermore, Ab-mediated neutrophil depletion resulted in the formation of disorganized granulomas in the mycobacteria-infected lung (7). This observation is similar to that seen in the IL-17 KO mice. We hypothesize that the IL-17-induced migration of neutrophils into the lung may have an important role in the formation of organized granulomas.
Our data further demonstrate that IL-17 is an important cytokine not only in the early neutrophil recruitment but also in the induction of Th1-type acquired immunity after pulmonary mycobacterial infection. The IL-17 KO mice showed a decreased level of mycobacterial Ag-specific Th1 response on day 28 after M. bovis BCG infection. It has been reported that IL-17 is required to induce optimum Th1 and DTH responses against haptens (27), which is consistent with our observation that the Th1-type response is decreased in the absence of IL-17. However, IL-17 failed to directly enhance mycobacterial Ag-specific IFN- production because the level of IFN- production was not altered when the T cells from the M. bovis BCG-infected IL-17 KO mice were cultured with rIL-17 (Fig. 7C). This finding is consistent with a report showing that exogenously added IL-17 affected neither Th1/Th2 phenotype differentiation nor IL-17 production (62). Yao et al. (63) reported that IL-17 stimulates the activity of the transcriptional factor NF-
B, which is known to up-regulate gene products involved in cell activation and growth control. Therefore, we speculate that IL-17-mediated NF-B activation of APCs or other immunoregulatory cells may be important in the induction of an optimum level of Th1 response. The mechanism of IL-17-mediated enhancement of the Th1 response is now under investigation.
In summary, we investigated IL-17 production and IL-17-mediated immune regulation in mycobacterial infection. We found that IL-23-induced IL-17 production in M. bovis BCG-infected mice in vivo and infected spleen cells in vitro. The major IL-17 producers were TCR + T cells. IL-17 induced by M. bovis BCG infection affected not only early pulmonary neutrophil induction, but also the development of IFN--producing T cells and granuloma formation. Our data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection. Because IL-17 exerts beneficial effects on the development of protective cell-mediated immunity against mycobacteria, it is likely to be used as an immune adjuvant to enhance the efficacy of vaccination inducing the protective Th1 response.
|
Acknowledgments |
|---|
|
Disclosures |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
|
Footnotes |
|---|
1 This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, The Naito Foundation, and Takeda Science Foundation. 
2 Address correspondence and reprint requests to Dr. Masayuki Umemura, Molecular Microbiology Group, Center of Molecular Biosciences, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa 903-0213, Japan. E-mail address: umemura{at}comb.u-ryukyu.ac.jp
3 Abbreviations used in this paper: KO, knockout; i.t., intratracheally; DTH, delayed-type hypersensitivity; PPD, purified protein derivative; LN, lymph node; PIF, pulmonary infiltrated; BAL, bronchoalveolar lavage fluid; FCM, flow cytometry; Ct, cycle threshold.
Received for publication March 24, 2006. Accepted for publication January 5, 2007.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionDisclosuresReferences |
|---|
, IL-12, and IL-1 and have a putative role in early host response. Clin. Immunol. 91: 354-358. [Medline] and TNF-, by human macrophages. J. Immunol. 160: 3513-3521. , IL-11, IL-17, and type I and type III collagen expression. J. Allergy Clin. Immunol. 111: 1293-1298. [Medline]1 and a novel cytokine receptor subunit, IL-23R. J. Immunol. 168: 5699-5708. production and type 1 cytokine responses. Immunity 4: 471-481. [Medline] T cells in the lungs and their regulatory roles in Th1 response and host defense against pulmonary infection with Cryptococcus neoformans. J. Immunol. 172: 7629-7634. gene mutant mice: independent generation of T cells and programmed rearrangements of TCR genes. Cell 72: 337-348. [Medline] chemokine from mesothelial cells. J. Immunol. 165: 5814-5821. , LPS and IL-1. Science 243: 1467-1469. stimulates KC synthesis in rat mesangial cells: glucocorticoids inhibit KC induction by IL-1. Am. J. Physiol. 266: (5 Pt. 2):F713-F722. . Eur. Respir. J. 21: 387-393. stimulates killing of Mycobacterium tuberculosis by human neutrophils. Infect. Immun. 70: 4591-4599. TCR+CD4–CD8– T cells. J. Interferon Cytokine Res. 16: 611-617. [Medline]This article has been cited by other articles:
|
|
 |
|
  H. Bai, J. Cheng, X. Gao, A. G. Joyee, Y. Fan, S. Wang, L. Jiao, Z. Yao, and X. Yang IL-17/Th17 Promotes Type 1 T Cell Immunity against Pulmonary Intracellular Bacterial Infection through Modulating Dendritic Cell Function J. Immunol., November 1, 2009; 183(9): 5886 - 5895. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. D'Angelo, A. De Luca, T. Zelante, P. Bonifazi, S. Moretti, G. Giovannini, R. G. Iannitti, S. Zagarella, S. Bozza, S. Campo, et al. Exogenous Pentraxin 3 Restores Antifungal Resistance and Restrains Inflammation in Murine Chronic Granulomatous Disease J. Immunol., October 1, 2009; 183(7): 4609 - 4618. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. von Vietinghoff and K. Ley IL-17A Controls IL-17F Production and Maintains Blood Neutrophil Counts in Mice J. Immunol., July 15, 2009; 183(2): 865 - 873. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X. Zhang, L. Gao, L. Lei, Y. Zhong, P. Dube, M. T. Berton, B. Arulanandam, J. Zhang, and G. Zhong A MyD88-Dependent Early IL-17 Production Protects Mice against Airway Infection with the Obligate Intracellular Pathogen Chlamydia muridarum J. Immunol., July 15, 2009; 183(2): 1291 - 1300. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Siegemund, N. Schutze, S. Schulz, K. Wolk, K. Nasilowska, R. K. Straubinger, R. Sabat, and G. Alber Differential IL-23 requirement for IL-22 and IL-17A production during innate immunity against Salmonella enterica serovar Enteritidis Int. Immunol., May 1, 2009; 21(5): 555 - 565. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Bordon, C. A. H. Hansell, D. P. Sester, M. Clarke, A. McI. Mowat, and R. J. B. Nibbs The Atypical Chemokine Receptor D6 Contributes to the Development of Experimental Colitis J. Immunol., April 15, 2009; 182(8): 5032 - 5040. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. W. Kappel, G. L. Goldberg, C. G. King, D. Y. Suh, O. M. Smith, C. Ligh, A. M. Holland, J. Grubin, N. M. Mark, C. Liu, et al. IL-17 contributes to CD4-mediated graft-versus-host disease Blood, January 22, 2009; 113(4): 945 - 952. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Godinez, M. Raffatellu, H. Chu, T. A. Paixao, T. Haneda, R. L. Santos, C. L. Bevins, R. M. Tsolis, and A. J. Baumler Interleukin-23 Orchestrates Mucosal Responses to Salmonella enterica Serotype Typhimurium in the Intestine Infect. Immun., January 1, 2009; 77(1): 387 - 398. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Shibata, H. Yamada, R. Nakamura, X. Sun, M. Itsumi, and Y. Yoshikai Identification of CD25+ {gamma}{delta} T Cells As Fetal Thymus-Derived Naturally Occurring IL-17 Producers J. Immunol., November 1, 2008; 181(9): 5940 - 5947. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lee, E. K. Reinke, A. L. Zozulya, M. Sandor, and Z. Fabry Mycobacterium bovis Bacille Calmette-Guerin Infection in the CNS Suppresses Experimental Autoimmune Encephalomyelitis and Th17 Responses in an IFN-{gamma}-Independent Manner J. Immunol., November 1, 2008; 181(9): 6201 - 6212. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Hamada, M. Umemura, T. Shiono, K. Tanaka, A. Yahagi, M. D. Begum, K. Oshiro, Y. Okamoto, H. Watanabe, K. Kawakami, et al. IL-17A Produced by {gamma}{delta} T Cells Plays a Critical Role in Innate Immunity against Listeria monocytogenes Infection in the Liver J. Immunol., September 1, 2008; 181(5): 3456 - 3463. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Kisielow, M. Kopf, and K. Karjalainen SCART Scavenger Receptors Identify a Novel Subset of Adult {gamma}{delta} T Cells J. Immunol., August 1, 2008; 181(3): 1710 - 1716. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. de Beaucoudrey, A. Puel, O. Filipe-Santos, A. Cobat, P. Ghandil, M. Chrabieh, J. Feinberg, H. von Bernuth, A. Samarina, L. Janniere, et al. Mutations in STAT3 and IL12RB1 impair the development of human IL-17-producing T cells J. Exp. Med., July 7, 2008; 205(7): 1543 - 1550. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Gerosa, B. Baldani-Guerra, L. A. Lyakh, G. Batoni, S. Esin, R. T. Winkler-Pickett, M. R. Consolaro, M. De Marchi, D. Giachino, A. Robbiano, et al. Differential regulation of interleukin 12 and interleukin 23 production in human dendritic cells J. Exp. Med., June 9, 2008; 205(6): 1447 - 1461. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Lochner, L. Peduto, M. Cherrier, S. Sawa, F. Langa, R. Varona, D. Riethmacher, M. Si-Tahar, J. P. Di Santo, and G. Eberl In vivo equilibrium of proinflammatory IL-17+ and regulatory IL-10+ Foxp3+ ROR{gamma}t+ T cells J. Exp. Med., June 9, 2008; 205(6): 1381 - 1393. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. M. Coquet, S. Chakravarti, M. J. Smyth, and D. I. Godfrey Cutting Edge: IL-21 Is Not Essential for Th17 Differentiation or Experimental Autoimmune Encephalomyelitis J. Immunol., June 1, 2008; 180(11): 7097 - 7101. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K Dheda, J-S Chang, S Lala, J F Huggett, A Zumla, and G A W Rook Gene expression of IL17 and IL23 in the lungs of patients with active tuberculosis Thorax, June 1, 2008; 63(6): 566 - 568. [Full Text] [PDF]
|
|
|
|
|
|
I. Godinez, T. Haneda, M. Raffatellu, M. D. George, T. A. Paixao, H. G. Rolan, R. L. Santos, S. Dandekar, R. M. Tsolis, and A. J. Baumler T Cells Help To Amplify Inflammatory Responses Induced by Salmonella enterica Serotype Typhimurium in the Intestinal Mucosa Infect. Immun., May 1, 2008; 76(5): 2008 - 2017. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. J. Scriba, B. Kalsdorf, D.-A. Abrahams, F. Isaacs, J. Hofmeister, G. Black, H. Y. Hassan, R. J. Wilkinson, G. Walzl, S. J. Gelderbloem, et al. Distinct, Specific IL-17- and IL-22-Producing CD4+ T Cell Subsets Contribute to the Human Anti-Mycobacterial Immune Response J. Immunol., February 1, 2008; 180(3): 1962 - 1970. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Ishii, H. Hamamoto, M. Kamimura, and K. Sekimizu Activation of the Silkworm Cytokine by Bacterial and Fungal Cell Wall Components via a Reactive Oxygen Species-triggered Mechanism J. Biol. Chem., January 25, 2008; 283(4): 2185 - 2191. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. H. Niess, F. Leithauser, G. Adler, and J. Reimann Commensal Gut Flora Drives the Expansion of Proinflammatory CD4 T Cells in the Colonic Lamina Propria under Normal and Inflammatory Conditions J. Immunol., January 1, 2008; 180(1): 559 - 568. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Smith, A. Zarbock, M. A. Stark, T. L. Burcin, A. C. Bruce, P. Foley, and K. Ley IL-23 Is Required for Neutrophil Homeostasis in Normal and Neutrophilic Mice J. Immunol., December 15, 2007; 179(12): 8274 - 8279. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. L. Roark, J. D. French, M. A. Taylor, A. M. Bendele, W. K. Born, and R. L. O'Brien Exacerbation of Collagen-Induced Arthritis by Oligoclonal, IL-17-Producing {gamma}{delta} T Cells J. Immunol., October 15, 2007; 179(8): 5576 - 5583. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
