IL-18 and IL-12 synergy induces matrix degrading enzymes in the lung.

Interleukin (IL)-18 is a pro-inflammatory cytokine suggested to be involved in the development of pulmonary emphysema and inflammation. Studies involving immunology and cancer have revealed that IL-18 can have synergistic effects with IL-12. We have studied the presence of IL-18 and IL-12 receptors (IL-18R/IL-12R) in the lungs and whether IL-18 and IL-12, alone or in combination, have the ability to initiate the induction of mediators related to the development of emphysema and inflammation. The expression of the IL-18R was abundant in lungs compared to other organs (heart, liver, and spleen), and the IL-12R was also expressed in lung tissue. Mice treated with i.p. injection of recombinant murine IL-18 or IL-12 expressed significantly higher pulmonary mRNA levels of the matrix degrading enzymes metalloproteinase (MMP) 12 and cathepsin S, in addition to interferon-γ, tumor necrosis factor-α, and CXC chemokine ligand 9 (CXCL9) (all P < .05) than controls (received PBS). Treatment with IL-18 and IL-12 in combination showed an even more pronounced induction of these mediators, as well as a significant increase in MMP-9, IL-6, IL-1β, and transforming growth factor-β (P < .05). Furthermore, cellular apoptosis in lung tissue was induced. Immunohistochemical analysis revealed T-cell infiltration in pulmonary vessels following co-stimulation. In summary, IL-18 and IL-12 exert a synergistic effect on the lungs by inducing MMPs, cathepsins S, and pro-inflammatory cytokines, which may promote pulmonary emphysema and inflammation. The synergy between IL-18 and IL-12 involves infiltration of T-cells in the lungs, possibly induced by the T-cell chemoattractant CXCL9.


INTRODUCTION
Interleukin (IL)-18 is a pro-inflammatory cytokine in the IL-1 superfamily [1][2][3], and it is structurally related to IL-1β. The activation of both cytokines is associated with the inflammasome NLRP3 [4]. The inflammasome contains caspase-1 that cleaves the biologically inactive precursor pro-IL-18 to bi-disease (COPD), a disease entity of which emphysema is a major component, is supported by a study showing that patients with severe COPD have increased circulating levels of IL-18 [17], and also in studies showing increased expression of  in Tcells, alveolar macrophages, and in epithelial cells from the lungs of these patients [17][18][19][20]. The increased serum levels of IL-18 correlate negatively with lung function, suggesting a role for IL-18 in the pathogenesis of COPD. Interestingly, mice subjected to cigarette smoke, the most common cause of pulmonary emphysema, exhibit increased levels of IL-18 and activated caspase-1 in the lungs that may facilitate release of active IL-18 into the circulation and increase circulating levels of IL-18, indicating activation of the IL-18 pathway [19].
Experimental studies in immunology research have shown that inflammatory effects of IL-18 can be potentiated by the cytokine IL-12 [21,22]. Interestingly, COPD patients express increased levels of IL-12 in exhaled breath condensate [23], in addition to increased blood levels of IL-18 [17,24], and in experimental models both cytokines are induced by stimuli such as cigarette smoke [19,20] and alveolar hypoxia [25], which many COPD patients are repetitively being exposed to.
COPD is a composite term comprising patients with emphysema and chronic bronchitis. The development of emphysema in COPD has causally been related to inflammation, and involves metalloproteinases (MMPs) such as MMP-9 and MMP-12, cathepsins, infiltration of inflammatory cells, and various pro-inflammatory cytokines [26][27][28][29][30][31]. MMPs and cathepsins are proteolytic enzymes that degrade the lung matrix causing pulmonary emphysema. Cytokines, such as IL-1β and TNF-α, are able to induce or activate these proteinases [32,33]. The activity of MMPs is counteracted by tissue inhibitors of metalloproteinases (TIMPs) and the balance between MMPs and anti-MMPs is fundamental in the pathogenesis of emphysema [34].
Since there are indications of a link between IL-18, possibly in concert with IL-12, and the development of emphysema in both experimental and clinical studies, it would be of interest to study whether these cytokines in fact have the ability to induce the generation of mediators related to the emphysema development in the lungs. Thus, the aim of the present study was to investigate the early response of IL-18 with regard to induction of emphysema-related mediators, such as proteinases, and whether the upregulation of mediators is potentiated by IL-12. In addition, we wanted to study the presence of IL-18 and IL-12 receptors in the lungs, compared to other organs, in order to obtain information about the lung as a possible target organ for these cytokines. To examine these aspects, the expression of the IL-18 receptor (IL-18R) and IL-12 receptor (IL-12R) in  lung tissue from mice was determined. Subsequently,  animals were injected with murine IL-18, IL-12, or  IL-18 and IL-12 in combination, and induction of  relevant MMPs, cathepsins, cytokines, and growth factors were examined by RT-PCR. The upregulated proteinases were also examined by Western analysis. In addition, histological and immunohistochemical (IHC) examination of lung tissue was performed.

Animals and Reagents
The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1996)

Real-Time Polymerase Chain Reaction
A RT-PCR system (

Histology
For histological analysis, 4 mice from every group were used. The lungs were sectioned transversely, stained with hematoxylin and eosin, and examined in a blinded manner by an experienced pathologist.

Immunohistochemistry
Formalin-fixed, paraffin-embedded sections from 4 mice in each group were prepared as previously described [39]. The sections were subjected to staining by using DAKO autostainer (DAKO, Glostrup, Denmark)  To study apoptosis, the percentage of caspase-3 positive cells over total cells was determined, as previously described [40].

Statistical Analysis
Data are presented as means ± SE. Comparisons between groups were made by using unpaired student's t-test or the nonparametric Mann-Whitney rank sum test depending on the distribution of data (SigmaStat 3.1.1, Systat Software, Richmond, CA, USA). Differences were considered significant for P < .05.

Animal Model
No significant changes in body weight were found in the IL-18, IL-12, or IL-18 + IL-12 stimulated groups compared to respective controls. All organ weights were related to TL. There were no differences between the stimulated groups and the controls with regard to the left ventricle, right ventricle, and lung weight normalized to TL.

IL-18 and IL-12 Receptors
High mRNA and protein levels of the IL-18R were measured in the lungs compared to liver, spleen, and heart ( Figure 1A, C, and E). The most abundant level of IL-12R mRNA was observed in the spleen, followed by lung tissue, while the IL-12R protein level was highest in liver followed by spleen and lung ( Figure 1B, D, and F). Immunohistochemical analysis showed expression of IL-18R and IL-12R in bronchial epithelium and in alveolar macrophages and lymphocytes. (Figure 2A,

Antiproteases
Injection of IL-18 alone did not induce upregulation of antiproteases, while upregulation of TIMP-1 and TIMP-4 was observed following IL-12 stimulation ( Figure 4A and D). Co-stimulation with IL-18 and IL-12 resulted in increased expression of TIMP-1, TIMP-3, and PAI-1 ( Figure 4A, C, and E), whereas TIMP-2 was downregulated ( Figure 4B). FIGURE 1. mRNA levels of IL-18Rα (A) and IL-12Rβ1 (B) in liver, heart, spleen, and lungs. The mRNA expression in the liver was set to 100%. The mice did not receive any injections. Representative Western blots and bar graphs showing the relative abundance of IL-18Rα (C, E) and IL-12Rβ1 (D, F) protein in heart, spleen, and lungs compared to liver that was set to 100%. Values are presented as mean ± SE. * P < .05, * * P < .001.

CXCL9
The gene expression of the T-cell chemoattractant CXCL9 was strongly induced in animals treated with IL-18 and IL-12 alone, compared to controls ( Figure 6A). An even more pronounced increase was observed in animals co-stimulated with IL-18 and IL-12 ( Figure 6A).

Treatment with IL-18 and IL-12 in combination induced infiltration of lymphocytes aggregating in the wall of pulmonary blood vessels. IHC examination revealed that the majority of the lymphocytes were T-cells, positively stained with CD3 (Figure 6B). Increased infiltration of B-cells, macrophages, or regulatory T-cells was not observed in any of the groups.
Treatment with either IL-18 or IL-12 alone did not induce any histological changes compared to control animals.

DISCUSSION
A high expression of IL-18 receptor in mouse lung tissue was observed, indicating the lung as a target organ for IL-18. The IL-12 receptor was also expressed in lung tissue, being a prerequisite for the synergistical effect of IL-12 and IL-18 in the lungs, as observed in the present study. Stimulation with IL-18 and IL-12 promoted an upregulation of the proteinases MMP9, MMP12, and cathepsin S that are all capable of degrading extracellular matrix in the lungs leading to

. Pulmonary gene expression of TIMP-1 (A), TIMP-2 (B), TIMP-3 (C), TIMP-4 (D), and PAI-1 (E) in mice receiving injections with IL-18, IL-12, or IL-18 and IL-12 in combination (gray bars) compared with control animals receiving PBS (black bars). The mRNA levels in controls were set to 100%. Gene expression was analyzed by using quantitative RT-PCR.
Values are presented as mean ± SE. * P < .05, * * P < .001 versus control mice. pulmonary emphysema [31,41,42]. IL-18 and IL-12 also induced an upregulation of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α, and IFN-γ that have been shown to promote pulmonary inflammation and to be of importance in the pathogenesis of emphysema [32,33,[43][44][45]. Treatment with IL-18 and IL-12 increased the expression of the antiproteases TIMP-1, TIMP-3, and PAI-1, whereas the expression of TIMP-2 was decreased. Interestingly, signs of increased apoptosis in the lungs were observed as early as 24 hours following IL-18 and IL-12 injections, and increased apoptosis in the lungs is claimed to be of importance in the development of pulmonary emphysema [46].

Homing of T-cells to the lungs, possibly due to upregulation of the T-cell chemoattractant CXCL9, was a part of the pulmonary inflammation induced by IL-18 and IL-12.
In the present study, we found that IL-18 has the ability to induce gene expression of proteolytic enzymes known to participate in the pathogenesis of pulmonary emphysema. In addition, mRNA levels of several inflammatory cytokines, also linked to the emphysema development, were increased in the lungs following stimulation with IL-18. Co-stimulation [47]. It has also been shown that prolonged co-stimulation with IL-18 and IL-12 is lethal in mice, and that the lungs of these mice show infiltration of mononuclear leukocytes [21]. Generation of IL-18 in COPD has been located to bronchial epithelial cells, T-cells, and macrophages [17]. Macrophages have also been shown to produce IL-12 [48]. The mechanism for the increased levels of these cytokines has not been fully elucidated, but with regard to IL-18, it has been shown that cigarette smoke, known to promote emphysema, may activate the innate immune system leading to the conversion of pro-IL-18 to its FIGURE 5

. Pulmonary gene expression of interferon (INF)-γ (A), interleukin (IL)-1β (B), IL-6 (C), tumor necrosis factor (TNF)-α (D), IL-10 (E), and TGF-β (F) in mice receiving injections with IL-18, IL-12, or IL-18 and IL-12 in combination (gray bars) compared with control animals receiving PBS (black bars)
. The mRNA levels in controls were set to 100%. Gene expression was analyzed by using quantitative RT-PCR. Values are presented as mean ± SE. * P < .05, * * P < .001 versus control mice. biologically active form [20]. Macrophages, which express both IL-18 and IL-12, also have the ability to produce several of the other mediators described in the present study, that is, IFN-γ , TNF-α and MMP12, MMP9, and cathepsin S [17,48,49]. Interestingly, these cells also express the receptors of IL-18 and IL-12, as shown in the current study. If the synergy between IL-18 and IL-12 participates in the pathogenesis of emphysema, macrophages may play a central role by both being target cells of these cytokines and a source of emphysema promoting proteases and cytokines.
It has, to the authors' knowledge, not previously been shown that IL-18 and IL-12 in synergy might induce pulmonary generation of proteases and inflammatory mediators strongly linked to the development of emphysema. MMP-9 is related to the development of emphysema in humans through increased plasma levels of MMP-9 observed both in α-1-antitrypsin deficiency-associated emphysema and in emphysema related to smoking [50,51]. Also, smoking leads to increased MMP-9 protein levels and activity in induced sputum [52]. Hence, increased levels of both IL-18 and IL-12 [19,53] in lungs exposed to cigarette smoke may induce pulmonary MMP-9 production that could participate in the development of emphysema. Smokers with severe COPD have increased MMP-9 mRNA in pulmonary tissue, and MMP-9 correlates negatively with FEV 1 , diffusion capacity of carbon monoxide, and the partial pressure of oxygen, indicating that MMP-9 is related to the severity of COPD [54]. The link between MMP-9 and emphysema was strengthened by a study showing that mice overexpressing human MMP-9 develop emphysema [42]. To our knowledge, it has not previously been shown that IL-18 and IL-12 synergy induces increased expression of MMP-9 in the lungs, suggesting a link between these cytokines and MMP-9, which might be of importance in the process of extracellular matrix degradation in the lungs.
MMP-12 has a broad and potent matrix degrading capacity, which can be induced by cigarette smoke [55]. In experimental studies, MMP-12 seems to play a central role in the development of emphysema [56,57]. In mice, knockout of MMP-12 protects against smoke-induced emphysema [41], and guinea pigs treated with a MMP-9/MMP-12 inhibitor did not develop emphysema after exposure to cigarette smoke [26]. With regard to human emphysema, however, data on MMP-12 are not that easy to interpret [58]. Some studies report increased levels of MMP-12 in sputum and alveolar macrophages from smokers and COPD patients [59,60], while other studies have not been able to confirm these findings [61,62]. In the present study, we found that stimulation with IL-18 and IL-12, both separately and in combination, resulted in increased MMP-12 expression, which, at least in mice, may contribute to the development of emphysema.
In addition to MMPs, the cystein proteinases cathepsins have the potential of degrading extracellular matrix, leading to pulmonary emphysema [63,64]. Cathepsin S in particular, seems to play a role in the development of emphysema by promoting matrix degradation and cell apoptosis [31,65]. In our study, cathepsin S was induced both by IL-18 and IL-12, and in combination the increase was pronounced, underlining the synergy between IL-18 and IL-12. It is possible that cathepsin S is a link between these cytokines and the observed signs of apoptosis in the present study [31]. Taken together, we have shown that IL-18 and IL-12 stimulation initiates production of the important matrix degrading enzymes MMP-9, MMP-12, and cathepsin S in the lungs, all having the potential to induce pulmonary emphysema.
The observed up-and downregulation of TIMPs and PAI-1 may influence the proteolytic effect on the lungs. Recent studies, however, show that prolonged stimulation with IL-18 in mice overexpressing this cytokine in the lungs leads to the development of severe emphysema, indicating a shift in the balance between proteases and antiproteases in the direction of increased proteolytic activity [19,66]. The importance of IL-18 in the development of emphysema has recently been highlighted, but the mechanism by which IL-18 leads to emphysema is not completely understood [66,67]. By showing generation of the matrix degrading enzymes MMP-9, MMP-12, and cathepsin S, our result may possibly contribute to the understanding of mechanisms participating in this complex process. The role of IL-12 in the development of emphysema is not clear, but in addition to having a synergistic effect with IL-18 on the generation of matrix degrading enzymes and emphysema promoting cytokines, as shown in the present investigation, increased levels of IL-12 in COPD has been documented [23,68]. In experimental models, it has been shown that IL-18 and IL-12 given both intraperitoneally and locally in the airways induce inflammatory changes in the lungs, and both elevated circulating and pulmonary levels of IL-18 and IL-12 have been observed in COPD patients [17,21,23,[69][70][71][72]. Thus, both systemic and local increase in the levels of IL-18 and IL-12 may promote pulmonary inflammation.
The inflammation occurring in pulmonary emphysema involves invasion of inflammatory cells and production of pro-inflammatory cytokines in the lungs. We found that IL-18 and IL-12 induced infiltration of T-cells in the lungs, possibly via the T-cell chemoattractant CXCL9, which was substantially increased by stimulation with these cytokines. CXCL9 can be involved in homing of T-cells to the lungs in COPD [73]. Interestingly, patients with pulmonary emphysema have increased numbers of T-cells that produce the pro-inflammatory cytokine IFN-γ [30], which was markedly increased by the synergy between IL-18 and IL-12 in the present study. IFN-γ may promote pulmonary emphysema by inducing cathepsin S-dependent apoptosis, and also through MMP-12 in an apoptosis-independent pathway [31,44]. In our study, both pathways could be induced by IL-18 and IL-12. The powerful pro-inflammatory cytokines IL-6, IL-1β, and TNF-α, which were all induced by IL-18 and IL-12 synergy, have also been linked to the development of inflammation and emphysema in the lungs [32,33,43,45]. IL-6, IL-1β, and TNF-α have the potential to upregulate MMPs, and for instance, when IL-1β production is ectopically expressed in the lung epithelium, it causes increased expression of MMP-9 and MMP-12 and lung pathology that resembles emphysema [32]. MMP-9 is capable of activating IL-1β from its biologically inactive form, and instillation of MMP-12 into mouse airways induced an acute inflammatory response with leukocyte invasion, accompanied by increased pro-inflammatory cytokines such as IL-6 and TNF-α, and also MMP-9 [74]. The pro-inflammatory cytokine IL-13 has previously been linked to the development of pulmonary inflammation and emphysema in transgenic mice overexpressing IL-18 [16]. The increase in IL-13 following stimulation with IL-18 and IL-12 in the present study, however, did not reach statistical significance. According to previous studies, the interaction between pro-inflammatory cytokines, cathepsins, and MMPs suggests a complex network of mediators participating in the development of pulmonary emphysema, which in humans takes years to develop [16,[31][32][33][36][37][38][39][40]. The results of the present study suggest that IL-18 and IL-12 may take part in this process, but it should be noted that the duration of the study was too short to discover possible histological signs of emphysema. The 24 hours model only elucidates the acute responses induced by these two cytokines, and further studies using established mouse models of COPD and examinations of human tissues and cells are necessary to clarify the relevance of IL-18 and IL-12-induced pathways in the development of emphysema.
IL-18 and IL-12 stimulation induced the antiinflammatory cytokine IL-10, which has the ability to suppress pro-inflammatory cytokines such as TNF-α, IL-6, and IFN-γ [75,76]. IL-10 may also decrease the expression of MMPs [28,77]. In the present study, the anti-inflammatory effects of IL-10 were not able to inhibit a robust induction of proinflammatory cytokines, MMPs, and cathepsin S. Patients with COPD have reduced concentrations of IL-10 in sputum, which might be a mechanism for increasing lung inflammation [78]. Thus, the process of emphysema might both be initiated by an inflammatory response that causes destruction of pulmonary tissue or by reducing the defenses, such as a reduction in IL-10, which could protect lung tissue during inflammation [28,77]. An additional component in the development of emphysema is claimed to be an altered repair mechanism [79,80]. The growth factor TGF-β, which was modestly increased by the synergy between IL-18 and IL-12, is claimed to be involved in repair of lung tissue through stimulation of collagen production [81]. In our study, however, collagen type I and III was not upregulated by IL-18 and IL-12. Although fibroblasts from patients suffering from COPD can produce increased amounts of TGF-β, they seem to be less responsive to effects of TGFβ, thereby exhibiting a diminished repair response, which could contribute to the development of pulmonary emphysema [79].
In summary, IL-18 and IL-12, which have been found in increased levels in patients with COPD, may promote the development of pulmonary emphysema by induction of matrix degrading enzymes MMP9, MMP12, and cathepsin S and by inducing cellular apoptosis. The inflammatory response observed, involved influx of T-cells, possibly mediated through the chemokine CXCL9, and induction of pro-inflammatory cytokines in mouse lungs. These effects seem to be mediated through the IL-18 receptor, which is abundantly expressed in lungs compared to other organs, and the IL-12 receptor, which is also present in lung tissue.

Declaration of interest:
The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.