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IL-12 and IL-18 Are Increased and Stimulate IFN- Production in Sarcoid Lungs

Katsunori Shigehara^*,,, Noriharu Shijubo^1,, Mitsuhide Ohmichi, Ryuji Takahashi, Shin-ichiro Kon, Haruki Okamura^¶, Masashi Kurimoto^||, Yohmei Hiraga, Tachio Tatsuno^*, Shosaku Abe and Noriyuki Sato

^* Hokkaido Branch of Japan Anti-Tuberculosis Association, Sapporo, Japan; Third Department of Internal Medicine and First Department of Pathology, Sapporo Medical University School of Medicine, Sapporo, Japan; Department of Respiratory Disease, Sapporo Hospital, Hokkaido Railway Co. Sapporo, Japan; ^¶ Laboratory of Host Defense, Institute for Advanced Medical Sciences, Hyogo Medical College, Nishinomiya, Japan; and ^|| Fujisaki Institute, Hayashibara Biochemical Laboratories, Okayama, Japan

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Sarcoidosis is a systemic chronic granulomatous disease of unknown cause. Recent investigations revealed that the cytokine profile in inflamed lesions of sarcoidosis is Th1 dominant. To obtain better immunopathologic understanding of sarcoidosis, we examined the expression of IL-12 and IL-18 and their roles in IFN- production in pulmonary sarcoidosis. Sarcoid cases had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in bronchoalveolar lavage (BAL) fluids compared with healthy subjects. IL-12 p70 and IL-18 were immunohistochemically expressed in the epithelioid cells and giant cells of sarcoid granulomas. Significant induction of IFN-, IL-12 p70, and IL-18 was observed from sarcoid BAL fluid cells with LPS stimulation, whereas LPS tended to induce only IL-12 p70 in BAL fluid cells from healthy subjects. Sarcoid cases had significantly greater IFN- induction with LPS stimulation than healthy subjects did. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells as well as in LPS-stimulated sarcoid BAL fluid cells, but IFN- and IL-12 mRNA expression was observed only in LPS-stimulated BAL fluid cells. Treatment with anti-IL-12- and anti-IL-18-neutralizing Abs significantly inhibited IFN- production from LPS-stimulated BAL fluid cells of sarcoid cases. Coadministration of rIL-12 or rIL-18 induced greater IFN- production in sarcoid BAL fluid cells than in normal BAL fluid cells. We concluded that bioactive IL-12 and IL-18 were produced in sarcoid BAL fluid cells and synergistically induced IFN- production, indicating important cytokines in the Th1 response of sarcoidosis.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Interleukin-18 is a recently described member of the IL-1 cytokine family and was initially defined as an IFN--inducing factor (1). Subsequent studies have elucidated a broad array of effector functions beyond lymphocyte activation that implicates IL-18 as an important regulator of both innate and acquired immune responses (2, 3). Several recent reports have indicated that IL-18 is expressed at sites of chronic Th1-mediated inflammatory diseases such as Crohn’s disease (4, 5) and rheumatoid arthritis (6). IL-18 promotes type 1 cytokine production from NK cells and T cells in leprosy (7). Increased levels of circulating IL-18 correlate with increased levels of circulating IFN- in human tuberculosis (8).

In the type 1 immune response (Th1 responses), IL-12 and IL-18 are important cytokines that synergistically stimulate IFN- production (9, 10, 11) and enhance NK and T cell-mediated cytotoxicity (12, 13). Furthermore, IL-12 induces the differentiation of T cell precursors (Th0 cells) into Th1 cells (14). IL-12 and IL-18 play critical roles in resistance to infection with intracellular parasites and bacteria and in some kinds of autoimmune diseases and tumor toxicity (2, 3, 15).

Sarcoidosis is a systemic disease of unknown origin that is characterized by noncaseating epithelioid cell granuloma with dominant infiltration of CD4⁺ Th cells and macrophages (16). Many cytokines and chemokines play an important role during the processes of sarcoid granuloma (17). On the basis of recent Th1 and Th2 paradigms, the cytokine profile of sarcoidosis is Th1 dominant when disease activity is high (17, 18, 19). Recent studies have demonstrated enhanced expression of IL-12 (20, 21, 22) and IL-12R (23, 24) in sarcoid lungs. IL-18 expression was reported to be increased in the airway epithelial cells of sarcoid cases (25). However, the cooperative roles of IL-12 and IL-18 in sarcoidosis have not been fully resolved in Th1 response.

In this study we investigated local production of IL-12 and IL-18 in patients with sarcoidosis and their influences on the Th1 response of sarcoidosis concerned with IFN- production in pulmonary sarcoidosis.

	Materials and Methods

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Study population

The diagnosis of sarcoidosis was established in 58 individuals (23 men (mean age, 26.0 years) and 35 women (mean age, 47.2 years); 36 nonsmokers and 22 smokers). They had histologic evidence consistent with sarcoidosis in the lung (showing noncaseating epithelioid cell granuloma) without any evidence of mycobacterial, fungal, or parasitic infection. None had a history of exposure to organic or inorganic materials known to cause granulomatous lung diseases. No patient had received corticosteroid therapy at the time of the study. Forty-eight patients had abnormal chest x-ray findings; 36 patients demonstrated hilar lymphadenopathy alone, 11 patients had hilar lymphadenopathy and interstitial infiltrates of the lung field, and 1 patient had interstitial infiltrates of the lung field alone. Ten patients had no abnormal chest x-ray findings, but had positive biopsy findings of the lung and uveitis and thus were strongly suspected to have sarcoidosis. The assessment of disease activity included clinical features, chest x-ray, high resolution computed tomography, lung function tests, bronchoalveolar lavage (BAL)² analysis, ⁶⁷Ga scan, and routine blood studies. The examinations revealed 35 extrapulmonary sarcoid lesions (27 uveitis, and 6 skin, 1 bone, and 1 parotid gland sarcoid lesions) in 28 patients; five patients had two extrapulmonary lesions, and one patient had three extrapulmonary lesions.

For comparison, 23 normal individuals were studied (13 men and 10 women: mean age, 34.8 years). Thirteen individuals were nonsmokers, and 10 were smokers. No healthy individuals had a history of cardiopulmonary or other illness. There were no abnormal findings on physical examination or chest x-ray, and all results showed lung function tests within normal limits. Informed consent was obtained from the patients with sarcoidosis and normal volunteers.

BAL analysis

BAL analysis was performed on 58 sarcoid cases and 23 healthy subjects as previously described (26). Three 50-ml aliquots of 0.9% sterile saline were instilled into a bronchus in the right middle lobe through a fiberoptic bronchoscope in routine BAL analysis. To obtain more BAL fluid cells for further examinations, six 50-ml aliquots were instilled in some sarcoid cases and healthy subjects. BAL fluids were recovered by gentle suction immediately after the infusion of each aliquot. BAL fluids were filtered through a single layer of sterile gauze. After centrifugation (400 x g, 10 min), the cell pellets were washed twice in PBS. Cells were >95% viable as determined by trypan blue exclusion test. Cell differential counts were determined by Wright-Giemsa staining. Evaluation of BAL fluid cells for the proportions of CD3⁺, CD4⁺, and CD8⁺ T cells was performed using mAbs and flow cytometry. The BAL fluid supernatants of the first three 50-ml aliquots obtained from all sarcoid cases and healthy subjects were centrifuged again at 500 x g for 30 min. The supernatants were collected and cryopreserved at -30°C for cytokine measurements. The results of BAL analysis of the first three 50-ml aliquots were shown in Table I. There were no significant differences in cell differential counts of BAL analysis between the first three aliquots and aliquots four to six in sarcoid cases or healthy subjects.

To evaluate IFN-, IL-12, and IL-18 production from BAL fluid cells, BAL fluid cells were obtained from 33 sarcoid cases and 10 healthy nonsmokers. Due to limitation of cell number, BAL fluid cells from six sarcoid cases and three healthy subjects were unable to be studied. Because tobacco smoking decreases cytokine production from alveolar macrophages (27, 28), we excluded smoker populations for cell culture examinations. Cells were cultured at 1 x 10⁶ cells/ml in RPMI 1640 medium supplemented with 10% heat-inactivated (56°C, 30 min) FCS, 2 mM L-glutamine, and 0.1% gentamicin (complete medium) in 24-well plates (Nunc, Roskilde, Denmark) for 48 h at 37°C under 5% CO₂ in the presence or the absence of LPS (1 µg/ml; Escherichia coli, serotype 0111:B4, Sigma Aldrich, Steinheim, Germany).

To investigate the effects of IL-12 and IL-18 on IFN- production in BAL fluid cells, neutralization for IL-12 and IL-18 and stimulation with IL-12 and IL-18 were performed. For neutralization, goat anti-human IL-12-neutralizing polyclonal Ab (10 µg/ml; R&D Systems, Minneapolis, MN) and/or mouse anti-human IL-18-neutralizing mAb (125-2H, IgG1, 1 µg/ml; Hayashibara Biochemical, Fujisaki Institute, Okayama, Japan) were cultured under the conditions described above. The concentrations of goat anti-human IL-12 polyclonal Ab and mouse anti-human IL-18 mAb used completely neutralized 1.0 ng/ml human rIL-12 and 500 pg/ml human rIL-18, respectively. Normal goat IgG and mouse IgG1 were simultaneously used as negative controls.

For stimulation with IL-12 and IL-18, rIL-12 (R&D Systems) and/or rIL-18 (Hayashibara Biochemical, Fujisaki Institute) were added in combination with different concentrations. In this analysis 2 x 10⁵ BAL fluid cells were cultured in 0.2 ml of complete medium in 96-well microtiter plates for 48 h at 37°C under 5% CO₂, and then culture supernatants were collected.

Analysis of cytokine gene expression

For cytokine gene expression analysis in BAL fluid cells, nine sarcoid cases and six healthy nonsmokers were selected from among increased total BAL fluid cell count study subpopulations (sarcoid cases, >3 x 10⁷ cells; healthy subjects, >2 x 10⁷ cells of six 50-ml aliquots). Total RNA of 5 x 10⁶ cultured BAL fluid cells was isolated using Isogen (Nippon Gene, Toyama, Japan) and quantified by measurement of absorbance at 260 nm. To synthesize cDNA, 1 µg of total RNA was incubated in the presence of Moloney murine leukemia virus reverse transcriptase (Life Technologies, Gaithersburg, MD) and 5 ng/ml oligo(dT)_12–18 primers (Pharmacia Biotech, Uppsala, Sweden) for 30 min at 42°C using reaction conditions described by the manufacturer (Life Technologies), and samples were stored at -80°C.

Aliquots of 2 µl of cDNA were amplified by PCR using oligonucleotide primers specific for IFN-, IL-12 p40 and p35, IL-18, and GAPDH. In the DNA thermocycler 480 (Perkin-Elmer/Cetus, Norwalk, CT) each cycle of denaturation was run at 94°C for 1 min, annealing was performed for 1 min at 60°C (IFN-, IL-12 p40 and p35, and GAPDH) and at 55°C (IL-18), and extension was performed at 72°C for 1 min. PCR product was subjected to electrophoresis on 2% agarose gels and visualized by staining with ethidium bromide. The primers of examined cytokines were as follows: IFN-, 5'-atgaaatatacaagttatatcttggcttt and 3'-gatgctcttcgacctcgaaacagcat; IL-12 p40, 5'-ccaagaacttgcagctgaag and 3'-tgggtctattccgttgtgtc; IL-12 p35, 5'-cctcagtttggcagaaacc and 3'-ggtctttctggaggccaggc; IL-18, 5'-gcttgaatctaaattatcagtc and 3'-gaagattcaaattgcatcttat; and GAPDH, 5'-gaaggtgaaggtcgga.

With preliminary PCR amplification we confirmed the optimal number of PCR cycles in exponential amplification phase for each cytokine and GAPDH. Cycle numbers corresponding to exponential phase were individually determined for each primer set. The cycles numbers were 45 for IFN-, IL-12 p40, and IL-12 p35 and 30 for IL-18 and GAPDH. PCR products were stained by ethidium bromide after electrophoresis, and the intensities of the bands on photographs of the agarose gels were quantified using a CCD image sensor (Molecular Dynamics, Tokyo, Japan) using optical software (ImageQuant, version 3.3, Molecular Dynamics).

Immunoassay of BAL fluids and cultured supernatants of BAL fluid cells

BAL fluids were concentrated 10-fold by membrane dialysis using Vivapore (Vivascience, Stonehouse, Glostrup, Denmark) for cytokine immunoassays. We measured IFN-, IL-12 p70, and IL-12 (p40 and p70) in 10-fold concentrated BAL fluids and cultured supernatants using each specific commercial ELISA kit (IFN-: Cytoscreen, BioSource International (Camarillo, CA); sensitivity, 4 pg/ml; IL-12 p70: Quantikine, R&D Systems (Minneapolis, MN); sensitivity, 0.5 pg/ml; IL-12 (p40 and p70), Endogen (Cambridge, MA); sensitivity, 5 pg/ml), according to the manufacturers’ recommendations.

The specific ELISA system for human IL-18 was established at Hayashibara Biochemical, Fujisaki Institute (29). Briefly, a Maxisorp plate was coated with mAb 125-2H (20 µg/µl in PBS) at room temperature for 3 h and blocked with PBS containing 1% BSA (Sigma) at 4°C overnight. After washing with PBS containing 0.05% Tween 20 (Sigma), 50 µl of the assay buffer (PBS containing 1% BSA, 5% FCS, and 1 M NaCl) was dispensed, 50 µl of samples and standard human IL-18 were added to the assay buffer, and the plate was incubated at room temperature for 2 h. After washing, peroxidase-conjugated 159-12B (rat IgG2a, 0.5 µg/ml PBS containing 1% BSA, 5% FCS, 0.1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Dojin Chemicals, Tokyo, Japan), and 0.3 M NaCl) was added, and the plate was incubated at room temperature for 2 h. After washing, the substrate solution (100 µl of 0.1 M sodium phosphate-citrate buffer containing 0.5 mg/ml o-phenylenediamine and 0.003% H₂O₂, pH 5.0) was added. The reaction was stopped with 100 µl of 1 M H₂SO₄, and the absorbance at 490 nm was measured. In general, the assay was performed in duplicate. The detectable range of this ELISA was between 10 and 1000 pg/ml. The ELISA system can detect total IL-18 (proIL-18 and mature IL-18).

Immunohistochemical techniques of IL-12 and IL-18

IL-12 p70 mAb (20C2, rat IgG1, gift from Dr. Gately, Hoffmann-La Roche, Nutley, NJ) and IL-18 mAb (2-10C, established in Fujisaki Institute, Hayashibara Biochemical) were used to detect the expression of IL-12 p70 and IL-18. Lung specimens (five sarcoid lesions and three control lung tissues) were fixed with 10% buffered formalin and embedded in paraffin. Sarcoid lung lesions were obtained by lung biopsy at video-assisted thoracic surgery, and control lung tissues were obtained at operation of lung carcinoma. Five-micron sections were stained using a previously described technique (5). Briefly, the sections were permeabilized for 15 min with saponin buffer (1% FCS, 0.1% sodium azide, and 0.1% saponin in PBS) and then reacted with appropriate dilutions of mAbs. Positive cells were detected by sequential reaction with alkaline phosphatase avidin-biotin complexes and Fast Red substrate (alkaline phosphatase-ABC kit; Dako, Carpenteria, CA). No positive cells were identified when primary mAbs were replaced by isotype-matched control mAbs.

Statistical analysis

Data are presented as the mean ± SEM. For comparison of unpaired group data, unpaired t test or Mann-Whitney U test was used to evaluate statistical significance. For comparison of paired data, paired t test or Wilcoxon signed rank test was performed. Spearman rank correlation coefficient analysis was also used. The level of critical significance was assigned at p < 0.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
IL-12 and IL-18 protein levels in BAL fluids

We measured protein levels of IL-12 p70, IL-12 (p40 and p70), and IL-18 in BAL fluids using a specific ELISA system. IL-12 p70 was not detected in any BAL fluids, nor was it observed in 10-fold-concentrated BAL fluids of the examined cases. When IL-12 (p40 and p70) levels were compared between nonsmokers and smokers, sarcoid and healthy smokers had significantly lower IL-12 (p40 and p70) levels in BAL fluids than sarcoid (p < 0.0001) and healthy nonsmokers (p = 0.0008), respectively. Sarcoid nonsmokers and smokers had significantly increased IL-12 (p40 and p70) levels in BAL fluids compared with healthy nonsmokers (p < 0.0001) and smokers (p = 0.0012), respectively (Fig. 1).

View larger version (20K): [in this window] [in a new window]   FIGURE 1. The concentrations of IL-12 (p40 and p70) and IL-18 in BAL fluids of patients with sarcoidosis and in healthy volunteers. BAL fluids were concentrated 10-fold. IL-12 (p40 and p70) and IL-18 were measured by specific ELISA. Each circle represents a single individual. , Nonsmokers; •, smokers. The dashed lines indicate the levels of sensitivity of the IL-12 (p40 and p70) assay (5 pg/ml) and the IL-18 assay (10 pg/ml). Tobacco smoking affected BAL fluid IL-12 (p40 and p70) levels, but it did not affect BAL fluid IL-18 levels. Sarcoid patients had significantly elevated levels of IL-12 (p40 and p70) and IL-18 in BALF compared with healthy subjects. All comparisons were made using the Mann-Whitney U test.

Immunohistochemistry of IL-12 and IL-18 in sarcoid lungs

In normal lungs, very little positive staining of IL-12 and IL-18 was found. Only some alveolar macrophages showed weak staining for IL-12 and IL-18 (data not shown). In contrast, positive staining of IL-12 p70 and IL-18 was found in epithelioid cells of granulomas and macrophages in alveolar lumens and in the interstitium of the sarcoid lungs, especially in the cytoplasm (Fig. 2, A and C). Giant cells were also positive for IL-12 p70 and IL-18. Lymphocytes and fibroblasts around granulomas were negative.

View larger version (160K): [in this window] [in a new window]   FIGURE 2. Immunohistochemical localization of IL-12 p70 and IL-18 in the sarcoid lung. Anti-human IL-12 p70 mAb 20C2 (A) and IL-18 mAb 2-10C (C) were applied to paraffin-embedded sections using the alkaline phosphatase/avidin-biotin-peroxidase complex technique. IL-12 p70 and IL-18 are expressed in epithelioid cells and giant cells of sarcoid granulomas and in macrophages of the sarcoid lung. Control immunohistochemical reactions used isotype matched rat (B) or mouse (D) IgG. Original magnification of A–D, x100.

IFN-, IL-12, and IL-18 mRNA expression and their production in cultured BAL fluid cells

Freshly isolated sarcoid BAL fluid cells expressed IL-18 mRNA, but not IFN-, IL-12 p40, or IL-12 p35 mRNA. IFN- and IL-12 p40 mRNA expression was observed at 4 and 48 h after initial culture with LPS stimulation, and IL-12 p35 mRNA expression was observed at 4 h after LPS stimulation. IL-18 mRNA expression was also observed at 4 and 48 h after initial culture with LPS stimulation (Fig. 3A). IL-18 mRNA expression was observed in BAL fluid cells freshly isolated from healthy subjects, and it was observed at 4 and 48 h after initial culture with LPS stimulation (data not shown).

View larger version (33K): [in this window] [in a new window]   FIGURE 3. Expression of IFN-, IL-12, and IL-18 mRNA and their production in BAL fluid cells. A, IFN-, IL-12, and IL-18 mRNA in sarcoid BAL fluid cells. Sarcoid BAL fluid cells were collected and cultured with LPS (1 µg/ml). Stimulated BAL fluid cells were recovered at 4 and 48 h. Total RNA of the BAL fluid cells was isolated, and cDNA was synthesized using 5 ng/µl oligo(dT)12–18 and reverse transcriptase. Specific primers for IFN-, IL-12 p40 and p35, and IL-18 for PCR reaction underwent 45, 45, 45, and 30 cycles, respectively. IL-18 mRNA expression was observed in freshly isolated sarcoid BAL fluid cells, but IFN-, IL-12 p40, and IL-12 p35 mRNA were not. IL-12 p35 mRNA expression was observed only at 4 h, and IFN- and IL-12 p40 mRNA were observed at 4 and 48 h after initial culture with LPS stimulation. IL-18 mRNA was observed at 4 and 48 h after LPS stimulation as well. The GAPDH gene was used as an internal control. B, IFN-, IL-12 p70, and IL-18 production in LPS-stimulated and unstimulated BAL fluid cells of 27 sarcoid cases and 10 healthy subjects. BAL fluid cells were incubated in RPMI 1640 containing 10% FCS with or without LPS for 48 h. The concentrations of IFN-, IL-12 p70, and IL-18 in culture supernatants were measured using specific ELISAs. LPS significantly induced IFN-, IL-12 p70, and IL-18 production in sarcoid cases, whereas only IL-12 p70 was increased with LPS stimulation in normal BAL fluid cells (p = 0.06). Sarcoid cases had significantly greater IFN- induction with LPS stimulation than did healthy subjects. C, The relationships between IFN- and IL-18 levels in unstimulated sarcoid BAL fluid cells and between the IFN- and IL-12 p70 levels in LPS-stimulated sarcoid BAL fluid cells. There were significant positive correlations.

By culture with LPS stimulation, IFN-, IL-12 p70, and IL-18 production was significantly induced from sarcoid BAL fluid cells, whereas culture with LPS tended to induce only IL-12 p70 production (p = 0.06; Fig. 4) in BAL fluid cells from healthy subjects, but not IFN- or IL-18 production. When compared cytokine induction by LPS stimulation between sarcoid cases and healthy subjects, sarcoid cases had significantly higher IFN- production than healthy subjects (p < 0.0001). There were no significant differences in IL-12 p70 or IL-18 induction with LPS stimulation from BAL fluid cells between sarcoid cases and healthy subjects. There was a significant positive correlation between IFN- and IL-12 p70 production from LPS-stimulated BAL fluid cells in sarcoid cases (Fig. 3C), but not in healthy subjects.

View larger version (31K): [in this window] [in a new window]   FIGURE 4. Effects of anti-IL-12 and IL-18 neutralization on IFN- production of BAL fluid cells. A, BAL fluid cells from 13 sarcoid cases and five healthy subjects were incubated in the presence of anti-IL-12-neutralizing polyclonal Ab (10 µg/ml) and/or anti-IL-18-neutralizing mAb (125-2H, 1 µg/ml) with LPS stimulation for 48 h. Normal goat IgG and/or isotype-matched mouse IgG was used as a negative control. Culture supernatants were collected, and the IFN- concentration was measured using a specific ELISA. Data are expressed as the percent inhibition. *, p < 0.05, IFN- levels in cells treated with neutralizing Ab(s) vs IFN- levels in cells treated with negative control IgG. Treatment with anti-IL-12 and anti-IL-18 Abs markedly inhibited IFN- production by LPS-stimulated BAL fluid cells (sarcoid cases, 58.5 ± 5.6%; healthy subjects, 78.0 ± 4.7%). B, mRNA expression of IFN- in sarcoid BAL fluid cells with concomitant use of anti-IL-12- and anti-IL-18-neutralizing Abs. IFN- mRNA expression was obviously down-regulated with concomitant use of anti-IL-12- and anti-IL-18-neutralizing Abs.

Effects of IL-12- and IL-18-neutralizing Abs on IFN- production in BAL fluid cells

When using isotype-matched mouse IgG and/or nonimmune goat IgG as a negative control, LPS-stimulated sarcoid BAL fluid cells produced significantly higher IFN- (578 ± 219 pg/ml) than healthy subjects (67.3 ± 30.1 pg/ml). Anti-IL-12-neutralizing Ab or anti-IL-18-neutralizing mAb inhibited IFN- production from LPS-stimulated BAL fluid cells in 13 sarcoid cases (percent inhibition: anti-IL-12 Ab, 30.0 ± 5.1%; anti-IL-18 mAb, 39.8 ± 6.4%) and five healthy subjects (percent inhibition: anti-IL-12 Ab, 37.2 ± 7.8%; anti-IL-18 mAb, 44.4 ± 11.2%). With coadministration of anti-IL-12-neutralizing Ab and anti-IL-18-neutralizing mAb, IFN- production from BAL fluid cells was extremely decreased in sarcoid cases (percent inhibition, 58.5 ± 5.6%) and healthy subjects (percent inhibition, 78.0 ± 4.7%). Inhibition by treatment with anti-IL-12- and/or anti-IL-18-neutralizing Ab was significant in sarcoid cases (all p < 0.05). In addition, IFN- mRNA expression was obviously decreased in LPS-stimulated sarcoid BAL fluid cells when concomitantly treated with anti-IL-12- and anti-IL-18-neutralizing Abs. These results indicated that IL-12 and IL-18 produced from sarcoid BAL fluid cells were bioactive and were synergistic potent stimulators of IFN- in sarcoid lungs.

Effects of rIL-12 and/or rIL-18 on IFN- production in BAL fluid cells

We investigated IFN- production with stimulation of rIL-12 and/or rIL-18 in three sarcoid cases and three healthy subjects (Fig. 5). Under stimulation with rIL-12 alone, inducible IFN- levels were very low in both sarcoid cases and healthy subjects, whereas under stimulation with rIL-18 alone, IFN- was inducible in a dose-dependent manner in two sarcoid cases (cases 2 and 3) and one healthy subject (case 2). With the concurrent application of both recombinants, IFN- was inducible in a dose-dependent manner. With stimulation of rIL-12 and rIL-18, sarcoid BAL fluid cells exhibited greater IFN- production compared with normal BAL fluid cells. However, high dose stimulation reached a plateau of IFN- production in all sarcoid cases. Recombinant IL-12 and IL-18 synergistically stimulated IFN- production in BAL fluid cells of sarcoid cases and healthy subjects.

View larger version (28K): [in this window] [in a new window]   FIGURE 5. Effects of human rIL-12 and/or rIL-18 on IFN- production by BAL fluid cells. BAL fluid cells (three sarcoid cases and three healthy subjects) were incubated with rIL-12 and/or rIL-18 for 48 h. Culture supernatants were collected, and IFN- concentrations were measured by specific ELISA. Remarkable synergistic enhancement of IFN- production was observed with coadministration of rIL-12 and rIL-18. Enhancement of IFN- production was dose dependent; at high concentrations of rIL-12 and rIL-18, IFN- production reached a plateau. Sarcoid cases had greater IFN- induction with coadministration of rIL-12 and rIL-18 than did healthy subjects.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

IL-18 mRNA expression was observed in BAL fluid cells freshly isolated from sarcoid cases as well as healthy subjects, although IFN- and IL-12 mRNA expression was observed only after LPS stimulation in sarcoid BAL fluid cells. Constitutive IL-18 mRNA has been found in various organs and macrophage lineage cells and cell lines (2, 13, 30). The lungs are constantly stimulated through various exogenous stimuli. IL-18 may constantly produce and demonstrate an innate immunity even if virulent pathogens do not attack in the respiratory tract. This study demonstrated that IL-18 was significantly induced with LPS stimulation from sarcoid BAL fluid cells, while very weak induction was found in BAL fluid cells from healthy subjects. LPS activates capsase-1 in peripheral blood monocytes and the monocytic cell line THP-1 (31). Caspase-1 in alveolar macrophages may be activated by LPS stimulation, resulting in weakly enhanced secretion of IL-18 in healthy subjects. Tsutsui et al. (32) reported that IL-18 was markedly enhanced with LPS stimulation in a murine Propionibacterium acnes-conditioned liver injury model. Alveolar macrophages of sarcoidosis are thought be activated by unknown stimuli. Activation of alveolar macrophages may be a clue to elucidate marked induction of IL-18 with LPS stimulation from BAL fluids of patients with sarcoidosis.

Sarcoid cases exhibited greater inducibility of IFN- and IL-12 p70 from BAL fluid cells with LPS stimulation than did healthy subjects. With regard to IL-12 production in APCs, not only LPS stimulation but also the interaction between CD40 and CD40 ligand are important (33). Significantly enhanced expression of CD40 was observed in alveolar macrophages of sarcoid cases compared with that in normal alveolar macrophages (34). The interaction of CD40 and CD40 ligand may be a clue to the greater induction of IL-12 p70 in sarcoid BAL fluid cells than in normal BAL fluid cells. BAL fluid T cells in sarcoid cases showed enhanced expression of IL-12₂R (22) and most BAL fluid T cells differentiated into Th1 (or T cytotoxic 1) cells (24). The expression of IL-18R was also up-regulated in Th1 cells (35). With coadministration of rIL-12 and rIL-18, sarcoid BAL fluid cells produced markedly greater IFN- production than did normal BAL fluid cells. By enhanced expression of IL-12₂R and IL-18R on sarcoid BAL fluid T cells, greater inducibility of IFN- may be elucidated.

Treatment of anti-IL-12- and anti-IL-18-neutralizing Abs markedly inhibited IFN- production by LPS-stimulated BAL fluid cells obtained from sarcoid cases (58.5%) and healthy subjects (78.0%). The results showed that with coadministration of anti-IL-12- and anti-IL-18-neutralizing Abs, IFN- production was not completely inhibited in sarcoid cases. Yang et al. (36) reported two regulatory mechanisms of IFN- production: the mechanism through the TCR and the costimulatory mechanism of IL-12 and IL-18 stimulation. In sarcoid lungs, accumulated T cells might be activated by unknown stimuli and various environmental factors. IFN- production by activated T cells occurs spontaneously even if the actions of IL-12 and IL-18 from macrophages are absent. TNF- and IL-1 produced by LPS-stimulated alveolar macrophages also function as weak inducers of IFN-. These cytokines were reported be significantly induced in sarcoid BAL fluid cells with LPS stimulation compared with that in normal BAL fluid cells (27, 37).

In the stimulation of BAL cells with rIL-12 and rIL-18, IFN- production was synergistically elevated in a dose-dependent manner until it plateaued, although the concentrations of rIL-12 and rIL-18 differed among cases. Similar results were observed in induced Th1 cells from PBMC and an IL-12-responsive T cell clone (9, 38). Plateau phenomenon in IFN- production may also be comprehensive, as the saturation of IL-122R and IL-18Rs with IL-12 and/or IL-18 occurs in high dosages. The molecular mechanisms of plateau phenomenon and synergism in IFN- production of BAL fluid cells are of great interest and must be evaluated.

We document that IL-12 and IL-18 are increased in patients with sarcoidosis. LPS-stimulated sarcoid BAL fluid cells had greater inducibility of IFN-, IL-12, and IL-18 than LPS-stimulated normal BAL fluid cells. IL-12 and IL-18 induced greater IFN- in sarcoid BAL fluid cells than in normal BAL fluid cells. IL-12 and IL-18 are considered key cytokines in the Th1-dominant response of pulmonary sarcoidosis.

	Acknowledgments

 
We thank Dr. M. Gately (Hoffmann-La Roche, NJ) for providing us anti-IL-12 mAb (20C2); M. Ikeda, H. Yamauchi, and T. Taniguchi (Hayashibara Biochemical Laboratories, Inc.) for their technical assistance and useful discussion; and Dr. H. Harada, H. Sasaki-Date, Y. Ueda-Morikawa, and Mayumi Sasaki, Sapporo Hospital, Hokkaido Railway Co., for providing us with important clinical and pathological information. We also thank K. Teraoka and J. Sasaki, Sapporo Hospital, Hokkaido Railway Co., for helping with the BAL fluid cell preparation.

	Footnotes

 
¹ Address correspondence and reprint requests to Dr. Noriharu Shijubo, Third Department of Internal Medicine, Sapporo Medical University School of Medicine, South-1, West-16, Chuo-ku, Sapporo 060-8543, Japan.

² Abbreviation used in this paper: BAL, bronchoalveolar lavage.

Received for publication December 27, 1999. Accepted for publication September 27, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Okamura, H., H. Tsutsui, T. Komatsu, M. Yutsudo, A. Hakura, T. Tanimoto, K. Torigoe, T. Okura, Y. Nukada, K. Hattori, et al 1995. Cloning of a new cytokine that induces IFN- production by T cells. Nature 378:88.[Medline]
2. McInnes, I. B., J. A. Gracie, B. P. Leung, X.-Q. Wei, F. Y. Liew. 2000. Interleukin-18: a pleiotropic participant in chronic inflammation. Immunol. Today 21:312.[Medline]
3. Dinarello, C. A.. 1999. IL-18: a TH1-inducing, proinflammatory cytokine and new member of the IL-1 family. J. Allergy Clin. Immunol. 103:11.[Medline]
4. Monteleone, G., F. Trapasso, T. Parrello, L. Biancone, A. Stella, R. Iuliano, F. Luzza, A. Fusco, F. Pallone. 1999. Bioactive IL-18 expression is up-regulated in Crohn’s disease. J. Immunol. 163:143.[Abstract/Free Full Text]
5. Pizarro, T. T., M. H. Michie, M. Bentz, J. Woraratanadharm, Jr M. F. Smith, E. Foley, C. A. Moskaluk, S. J. Bickston, F. Cominelli. 1999. IL-18, a novel immunoregulatory cytokine, is up-regulated in Crohn’s disease: expression and localization in intestinal mucosal cells. J. Immunol. 162:6829.[Abstract/Free Full Text]
6. Gracie, J. A., R. J. Forsey, W. L. Chan, A. Gilmour, B. P. Leung, M. R. Greer, K. Kennedy, R. Carter, X. Q. Wei, D. Xu, et al 1999. A proinflammatory role for IL-18 in rheumatoid arthritis. J. Clin. Invest. 104:1393.[Medline]
7. Garcia, V. E., K. Uyemura, P. A. Sieling, M. T. Ochoa, C. T. Morita, H. Okamura, M. Kurimoto, T. H. Rea, R. L. Modlin. 1999. IL-18 promotes type 1 cytokine production from NK cells and T cells in human intracellular infection. J. Immunol. 162:6114.[Abstract/Free Full Text]
8. Yamada, G., N. Shijubo, K. Shigehara, H. Okamura, M. Kurimoto, S. Abe. 2000. Increased levels of circulating interleukin-18 in patients with advanced tuberculosis. Am. J. Respir. Crit. Care Med. 161:1786.[Abstract/Free Full Text]
9. Yoshimoto, T., K. Takeda, T. Tanaka, K. Ohkusu, S. Kashiwamura, H. Okamura, S. Akira, K. Nakanishi. 1998. IL-12 up-regulates IL-18 receptor expression on T cells, Th1 cells, and B cells: synergism with IL-18 for IFN- production. J. Immunol. 161:3400.[Abstract/Free Full Text]
10. Bacon, C. M., E. F. Petricoin, J. R. Ortaldo, R. C. Rees, A. C. Larner, J. A. Johnston, J. J. O’Shea. 1995. Interleukin 12 induces tyrosine phosphorylation and activation of STAT4 in human lymphocytes. Proc. Natl. Acad. Sci. USA 92:7307.[Abstract/Free Full Text]
11. Akita, K., T. Ohtsuki, Y. Nukada, T. Tanimoto, M. Namba, T. Okura, R. Takakura-Yamamoto, K. Torigoe, Y. Gu, M. S.-S. Su, et al 1997. Involvement of caspase-1 and caspase-3 in the production and processing of mature human interleukin 18 in monocytic THP.1 cells. J. Biol. Chem. 272:26595.[Abstract/Free Full Text]
12. Kobayashi, M., L. Fitz, M. Ryan, R. M. Hewick, S. C. Clark, S. Chan, R. Loudon, F. Sherman, B. Perussia, G. Trinchieri. 1989. Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J. Exp. Med. 170:827.[Abstract/Free Full Text]
13. Ushio, S., M. Namba, T. Okura, K. Hattori, Y. Nukada, K. Akita, F. Tanabe, K. Konishi, M. Micallef, M. Fujii, et al 1996. Cloning of the cDNA for human IFN- inducing factor, expression in Escherichia coli, and studies on the biologic activities of the protein. J. Immunol. 156:4274.[Abstract]
14. Trinecheri, G.. 1993. Interleukin-12 and its role in the generation of TH1 cells. Immunol. Today 14:335.[Medline]
15. Trinecheri, G.. 1994. Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood 84:4008.[Free Full Text]
16. American Thoracic Society. 1999. Statement on sarcoidosis. Joint Statement of the American Thoracic Society (ATS), the European Respiratory Society (ERS) and the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG) adopted by the ATS Board of Directors and by the ERS Executive Committee. Am. J. Respir. Crit. Care Med. 160:736.
17. Müller-Quernheim, J.. 1998. Sarcoidosis: immunopathogenetic concepts and their clinical application. Eur. Respir. J. 12:716.[Abstract]
18. Robinson, B. W. S., T. L. McLemore, R. G. Crystal. 1985. interferon is spontaneously released by alveolar macrophages and lung T lymphocytes in patients with pulmonary sarcoidosis. J. Clin. Invest. 75:1488.
19. Bergeron, A., M. Bonay, M. Kambouchner, D. Lecossier, M. Riquet, P. Soler, A. Hance, A. Tazi. 1997. Cytokine patterns in tuberculosis and sarcoid granulomas correlation with histological features of the granulomatous response. J. Immunol. 159:3034.[Abstract]
20. Moller, D. R., J. D. Forman, M. C. Liu, P. W. Nobel, B. M. Greenlee, P. Vyas, D. A. Holden, J. M. Forrester, A. Lazarus, M. Wysocka, et al 1996. Enhanced expression of IL-12 associated with Th1 cytokine profile in active pulmonary sarcoidosis. J. Immunol. 156:4952.[Abstract]
21. Minshall, E. M., A. Tsicopoulos, Z. Yasruel, B. Wallaert, H. Akoum, H. Vorng, A.-B. Tonnel, Q. Hamid. 1997. Cytokine mRNA gene expression in active and nonactive pulmonary sarcoidosis. Eur. Respir. J. 10:2034.[Abstract]
22. Shigehara, K., N. Shijubo, M. Ohmichi, S. Kon, Y. Shibuya, R. Takahashi, S. Morita-Ichimura, T. Tatsuno, Y. Hiraga, S. Abe, et al 2000. Enhanced mRNA expression of Th1 cytokines and IL-12 in active pulmonary sarcoidosis. Sarcoidosis Vasc. Diffuse Lung Dis. 17:151.[Medline]
23. Rogge, L., A. Papi, D. H. Presky, M. Biffi, L. J. Minetti, D. Miotto, C. Agostini, G. Semenzato, L. M. Fabbri, F. Sinigaglia. 1999. Antibodies to the IL-12 receptor 2 chain mark human Th1 but not Th2 cells in vitro and in vivo. J. Immunol. 162:3926.[Abstract/Free Full Text]
24. Taha, R. A., E. M. Minshall, R. Olivenstein, D. Ihaku, B. Wallaert, A. Tsicopoulos, A. B. Tonnel, R. Damia, D. Menzies, Q. A. Hamid. 1999. Increased expression of IL-12 receptor mRNA in active pulmonary tuberculosis and sarcoidosis. Am. J. Respir. Crit. Care Med. 160:1119.[Abstract/Free Full Text]
25. Cameron, L. A., R. A. Taha, A. Tsicopoulos, M. Kurimoto, R. Olivenstein, B. Wallaert, E. M. Minshall, Q. A. Hamid. 1999. Airway epithelium expresses interleukin-18. Eur. Respir. J. 14:553.[Abstract]
26. Shijubo, N., Y. Itoh, K. Shigehara, T. Yamaguchi, K. Itoh, Y. Shibuya, R. Takahashi, T. Ohchi, M. Ohmichi, Y. Hiraga, et al 2000. Association of Clara cell 10-kDa protein, spontaneous regression and sarcoidosis. Eur. Respir. J. 16:414.[Abstract]
27. McCrea, K. A., J. E. Ensor, K. Nall, E. R. Bleecker, J. D. Hasday. 1994. Altered cytokine regulation in the lungs of cigarette smokers. Am. J. Respir. Crit. Care 150:696.[Abstract]
28. Yamaguchi, E., A. Itoh, K. Furuya, H. Miyamoto, S. Abe, Y. Kawakami. 1993. Release of tumor necrosis factor- from human alveolar macrophages is decreased in smokers. Chest 103:479.[Abstract/Free Full Text]
29. Taniguchi, M., K. Nagaoka, T. Kunikata, T. Kayano, H. Yamauchi, S. Nakamura, M. Ikeda, K. Orita, M. Kurimoto. 1997. Characterization of anti-human interleukin-18 (IL-18)/interferon--inducing factor (IGIF) monoclonal antibodies and their application in the measurement of human IL-18 by ELISA. J. Immunol. Methods 206:107.[Medline]
30. Tone, M., S. A. J. Thompson, Y. Tone, P. J. Fairchild, H. Waldmann. 1997. Regulation of IL-18 (IFN--inducing factor) gene expression. J. Immunol. 159:6156.[Abstract]
31. Schumann, R. R., C. Belka, D. Reuter, N. Lamping, C. J. Kirschning, J. R. Weber, D. Pfeil. 1998. Lipopolysaccharide activates caspase-1 (interleukin-1-converting enzyme) in cultured monocytic and endothelial cells. Blood 91:577.[Abstract/Free Full Text]
32. Tsutsui, H., K. Matsui, N. Kawada, Y. Hyodo, N. Hayashi, H. Okamura, K. Higashino, K. Nakanishi. 1997. IL-18 accounts for both TNF- and Fas ligand-mediated hepatotoxic pathways in endotoxin-induced liver injury in mice. J. Immunol. 159:3961.[Abstract]
33. Cella, M., D. Scheidegger, K. Palmer-Lehmann, P. Lane, A. Lanzavecchia, G. Alber. 1996. Ligation of CD40 on dendritic cells triggers production of high levels of interleukin-12 and enhances T cell stimulatory capacity: T-T help via APC activation. J. Exp. Med. 184:747.[Abstract/Free Full Text]
34. Nicod, L. P., P. Isler. 1997. Alveolar macrophages in sarcoidosis coexpress high levels of CD86 (B7.2), CD40, and CD30L. Am. J. Respir. Cell Mol. Biol. 17:91.[Abstract/Free Full Text]
35. Kunikata, T., K. Torigoe, S. Ushio, T. Okura, C. Usio, H. Yamauchi, M. Ikeda, H. Ikegami, M. Kurimoto. 1998. Constitutive and induced IL-18 receptor expression by various peripheral blood cell subsets as determined by anti-hIL-18R monoclonal antibody. Cell. Immunol. 189:135.[Medline]
36. Yang, J., T. L. Murphy, W. Ouyang, K. M. Murphy. 1999. Induction of interferon- production in Th1 CD4⁺ T cells: evidence for two distinct pathways for promoter activation. Eur. J. Immunol. 29:548.[Medline]
37. Yamaguchi, E., N. Okazaki, Y. Tsuneta, S. Abe, T. Terai, Y. Kawakami. 1988. Interleukins in pulmonary sarcoidosis: dissociative correlations of lung interleukins 1 and 2 with the intensity of alveolitis. Am. Rev. Respir. Dis. 138:645.[Medline]
38. Ahn, H.-J., S. Murao, M. Tomura, J. Mu, T. Hamaoka, K. Nakanishi, S. Clark, M. Kurimoto, H. Okamura, H. Fujiwara. 1997. A mechanism underlying synergy between IL-12 and IFN--inducing factor in enhanced production of IFN-. J. Immunol. 159:2125.[Abstract/Free Full Text]

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