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Keywords:

  • angiogenic chemokines;
  • bronchoalveolar macrophages;
  • cytokines;
  • IL-12;
  • IL-18;
  • innate immunity;
  • monocytes

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

During inflammation, interleukin (IL)-12 and IL-18 are produced by macrophages and other cell types such as neutrophils (IL-12), keratinocytes and damaged endothelial cells (IL-18). To explore the role of IL-12 and IL-18 in inflammatory innate immune responses we investigated their impact on human peripheral blood monocytes and mature bronchoalveolar lavage (BAL) macrophages. IL-12 and IL-18 together, but not alone, prevented spontaneous apoptosis of cultured monocytes, promoted monocyte clustering and subsequent differentiation into macrophages. These morphological changes were accompanied by increased secretion of CXC chemokine ligands (CXCL)9, CXCL10 (up to 100-fold, P < 0·001) and CXCL8 (up to 10-fold, P < 0·001) but not CCL3, CCL4 or CCL5. Mature macrophages (from BALs) expressed high basal levels of CXCL8, that were no modified upon stimulation with IL-12 and IL-18. In contrast, the basal production of CXCL9 and CXCL10 by BALs was increased by 10-fold (P < 0·001) in the presence of either IL-12 or IL-18 alone and by 50-fold in the presence of both cytokines. In conclusion, our results indicate a relevant role for IL-12 and IL-18 in the activation and resolution of inflammatory immune responses, by increasing the survival of monocytes and by inducing the production of chemokines. In particular, those that may regulate angiogenesis and promote the recruitment of monocytes, activated T cells (CXCL9 and CXCL10) and granulocytes (CXCL8).


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Innate immune responses are the first line of defence to pathogens that have crossed natural epithelial barriers. They are initiated by resident macrophages and dendritic cells through the recognition of pathogen products and result in the production of cytokines/chemokines mediating local inflammation through the recruitment of neutrophils and monocytes to sites of infection. The role of this cellular infiltrate is to identify, ingest and destroy the pathogens. Once this is achieved, the neutrophils undergo apoptosis and are then recognized and phagocytosed by local macrophages [1]. At the same time, the tissue damage is healed through angiogenic processes [2].

Soon after recognition of a pathogen, macrophages and dendritic cells produce interleukin (IL)-12 and IL-18, and these in turn stimulate the induction of interferon (IFN)-γ secretion by T and natural killer (NK) cells [3]. However, the precise role of IL-12 plus IL-18 in innate immune responses, where the effector cells are principally neutrophils and monocytes, is more elusive. Certainly, infiltrating monocytes and macrophages are likely to find themselves in an IL-12/IL-18 rich environment as, besides macrophages, IL-12 is also produced by neutrophils [4], while IL-18 is also produced by keratinocytes, stromal cells and damaged endothelial cells [5].

There is increasing evidence that supports the controversial autocrine role for IL-12 and IL-18 on monocytes and macrophages [6–9]. As a first requirement, cells of the myeloid lineage do express the receptors for IL-12 and IL-18 in mouse and humans [5,7,10,11] and several authors have described that macrophages and monocytes can respond to IL-12 and IL-18 by producing IFN-γ[12,13]. Furthermore, IL-12 and IL-18 up-regulate intercellular adhesion molecule-1 (ICAM-1) expression on human monocyte cultures [14], and can activate the signal transducer and activator of transcription 4 (STAT 4) pathway in mice [15].

In this study we present data that further support the concept that IL-12 plus IL-18 can have multiple effects on monocytes; specifically, prevention of monocyte apoptosis, induction of monocyte differentiation and stimulation of the secretion of CXC chemokine ligands (CXCL)8, CXCL9 and CXCL10. The former chemokine activates the chemokine receptors CXCR1 and CXCR2 expressed on granulocytes, while CXCL9 and CXCL10 are ligands for CXCR3, present in a variety of cells including monocyte and activated T cells [16–18]. Interestingly, these chemokines are also involved in angiogenic (CXCL8) and angiostatic processes (CXCL9, CXCL-10) [19,20].

Finally, as innate immune responses to bacterial infections take place commonly in the lung, we demonstrated an enormous stimulation of CXCL9 and CXCL10 secretion by bronchoalveolar lavage (BAL) macrophages upon stimulation with IL-12 plus IL-18.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of monocytes

Peripheral blood cells from 12 healthy blood donor volunteers were provided by the Centre de Transfusions i Banc de Teixits and obtained after approval from the Ethical Committee of the Centre. The procedures followed in the study were in accordance with the Helsinki Declaration in 1975, as revised in 1983. Peripheral blood mononuclear cells were obtained from a standard Ficoll density gradient purification. Monocytes were purified by negative selection by magnetic beads labelled with antibodies to CD2, CD3, CD19, CD41, CD56, CD66b and glycophorin A and separated according the manufacturer’s instructions (Stemcell Technologies Inc., Vancouver, BC, Canada), or by positive selection using Macs CD14 microbeads (Miltenyi Biotech SL, Madrid, Spain).

In order to confirm the absence of T, B and NK cells and the purity of the CD14+ population, isolated monocytes were stained with a combination of antibodies to CD14, CD3, CD19 and CD56 and (Becton Dickinson, Madrid, Spain) (Fig. 1). No platelets were present in our cultures as they were removed during monocyte purification with an antibody to CD41 and this was confirmed by microscopy. Furthermore, to exclude the possibility that our results were due to a minute proportion of contaminating cells, we removed monocytes from total mononuclear cell cultures by incubating the mononuclear cells in the presence of carbonyl iron for 30 min at 37°C followed by magnetic depletion. The monocyte-depleted populations were adjusted to 1 × 106 cells/ml and stimulated with IL-12 and IL-18 for a week and the levels of chemokines released in the supernatants were measured as described below and compared to the levels observed in the monocyte-stimulated cultures.

image

Figure 1. Purity of isolated monocytes. Peripheral blood mononuclear cells (left panels) and purified monocytes isolated by positive (middle panels) or negative selection (right panels) with immunobeads were stained with anti-CD14, anti-CD3, anti-CD19 and anti-CD56 for the presence of monocytes, T cells, B cells and natural killer cells.

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Isolation of alveolar macrophages

Alveolar macrophages were obtained from BAL fluid of five otherwise healthy individuals who underwent flexible bronchoscopy to rule out lung cancer in peripheral pulmonary lesions. In none of the patients studied were malignant cells observed in the BAL. Alveolar macrophages were obtained from BAL fluid of five patients who underwent flexible bronchoscopy. The patients gave written consent and the study was approved by the Ethics Committee of the Hospital Germans Trias i Pujol. BAL was performed using three 50-ml aliquots of sterile saline. The liquid recovered after instillation of the first aliquot was discarded, so that the fluid analysed was not contaminated with bronchial cells. The total cell count was obtained using a Neubauer’s chamber and differential counts were performed on May–Grünwald–Giemsa (Merck, VWR, Barcelona, Spain) stained cytospins by counting 500 cells. Samples with > 2% of lymphoid-appearing cells were excluded from the study. Furthermore, we also depleted BAL of macrophages and restimulated the cultures with IL-12 and IL-18; the levels of all detectable chemokines decreased dramatically, indicating that the chemokines were not produced by cells other than the macrophages.

Monocyte and macrophage cultures

Monocytes (1 × 106 cells/ml) and macrophages (0·5 × 106 cells/ml) were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS) in the presence or absence of IL-18 (usually 100 ng/ml, Bionova, Madrid, Spain), IL-12 (usually 100 ng/ml), IFN-γ (100 ng/ml), granulocyte–macrophage colony-stimulating factor (GM-CSF, 65 ng/ml), IL-1β (50 ng/ml), IFN-α (100 ng/ml) and IFN-β (25 ng/ml) (R&D Systems, Abingdon, Oxon, UK). Supernatants were collected daily for a week and stored at −80°C. All the commercial cytokines and the fetal calf serum were guaranteed to have endotoxin or chemokine levels lower than 0·01 ng/µg or 0·01 ng/ml, respectively. For blocking experiments, neutralizing monoclonal antibodies to IL-12 or IFN-γ (R&D Systems), IL-18 (MBL, Barcelona, Spain) and ICAM-1 (kindly donated by Dr M Joan) were added to the cultures at the same time that IL-12 and IL-18 were added. Isotype controls were used as negative controls (DakoCytomation, Barcelona, Spain).

Cell survival and morphology

The monocyte cultures were photographed at low magnification ( × 100) every 24 h for a week. The monocytes from three representative fields for each culture condition were counted, and the mean cell count was plotted against the original input. The cells were then harvested, cytocentrifuged, stained with May–Grünwald–Giemsa (DakoCytomation) and analysed for morphological changes and for the presence of contaminating lymphocytes. There was no proliferation or an increase in the number of cells with lymphoid morphology in these cultures with time (data not shown)

The proportion of living cells was estimated by flow cytometry after determining the mitochondrial membrane potential (MMP) and plasma membrane integrity. Briefly, cells were stained (37°C, 15′) with 40 nm of the mitochondrial probe DiOC6 (Molecular Probes, Leiden, the Netherlands) and 1 µg/ml of propidium iodide (PI; Sigma, Madrid, Spain). To control for differential cell recovery, stained cells were mixed with a fixed number of fluorescent beads (Perfectcount cytometer beads; Cytognos, Salamanca, Spain) and counted in a FACScalibur flow cytometer. Cells and beads were identified by morphological parameters. Living cells were defined as DIOC bright/PI low cells [21].

Detection of chemokines

The amounts of the inflammatory chemokines, CXCL8, CXCL9, CXCL10, CCL3, CCL4 and CCL5 were estimated by cytometric bead array (Becton Dickinson) following the manufacturer’s instructions. The detection range was 5–1000 pg/ml. When necessary the supernatants were diluted to ensure that the values were within the linear range of the standards. The chemokines were already detectable between 24 and 48 h of culture, and the levels of chemokines were accumulative. The data refer to the production of chemokines at day 7 of culture.

Detection of phosphorylated STAT4 by immunoblotting

Purified monocytes were incubated in the presence of IL-12 plus IL-18 for 0, 45 or 120 min. Cells were lysed, clarified by centrifugation and the protein concentration of the lysates was measured by BCA protein assay kit (Pierce, Barcelona, Spain). The proteins were separated by sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE) (Invitrogen, Madrid, Spain) and transferred to a nitrocellulose transfer membrane (Schleicher & Schuell Bioscience, Dassel, Germany). The membrane was incubated with a primary antibody to pSTAT4(E2) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or to actin (Chemicon, Barcelona, Spain). Phosphorylated STAT4 and total actin were revealed with peroxidase-coupled anti-mouse antibodies and enhanced chemiluminiscence (ECL) (Amersham). Light was captured in non-saturating conditions in a Kodak Gel Logic 440 digital imaging system. Raw digital images were then used to quantify specific bands using molecular imaging software (Kodak). The ratio between P-STAT4 and actin net band volumes was then calculated for each sample. Fold increase values are relative to the control untreated sample.

Phagocytosis

Fluorescent latex beads (Serva, Heidelberg, Germany) at 108/ml were added to the cultures for 2 h and the cells were thoroughly washed, harvested and evaluated by flow cytometry to detect ingested fluorescent latex particles.

Statistical methods

Data were analysed with the prism statistical package and, if not stated otherwise, was normally distributed and expressed as mean ± s.e.m. (95% confidence interval). When comparing three or more groups, we used the one-way analysis of variance (anova) Kruskal–Wallis test. When comparing two groups we used the t-test or paired t-test if the results were obtained from the same samples. All tests of significance were two tailed.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Isolation of monocytes

Particular care was taken over the purification and characterization of monocytes, as this is a critical consideration for the interpretation of our results. First, the purity of the monocyte preparations was assessed by staining the isolated cells with a combination of antibodies to CD14, CD3, CD19 and CD56 in order to discard the presence of T, B and NK cells and to confirm the purity of the CD14+ population. As can be seen in Fig. 1, CD14+ monocytes accounted for > 95% of the total cells in monocyte preparations using either positive or negative selection. Secondly, the examination of the morphology of cytocentrifuge preparations of the purified monocytes revealed an absence of platelets and < 0·5% of contaminating granulocytes (see Material and methods).

The effect of IL-12 and IL-18 on monocyte survival and differentiation

Purified monocytes were cultured in medium alone, or in the presence of IL-12, IL-18, IL-12 plus IL-18, and with known anti-apoptotic factors (IFN-γ, GM-CSF, IL-1β, IFN-β or IFN-α) (Fig. 2a). The number of cells was counted daily and expressed as the percentage of the initial input (Fig. 2b). In control, IL-12- or IL-18-stimulated cultures, the number of cells decreased rapidly with time. However, in the presence of IFN-α, IFN-β or IL-1 β the number of monocytes at day 3 was 50% of the original input, but at day 7 the number of cells was as low as in the non-stimulated cultures and significantly (P < 0·001) less than at day 0. In contrast, cultures containing both IL-12 and IL-18 had similar cell numbers at day 0 and day 7, as did the positive control treated with GM-CSF or IFN-γ (Fig. 2b). Strikingly, monocytes cultured with both IL-12 and IL-18 survived over a month in culture without reduction of cell numbers.

image

Figure 2. Growth and morphology of monocytes cultured with interleukin (IL)-12 and IL-18. (a) Photographs of non-stimulated (NS) monocyte cultures at time 0 (top left) or after 5 days of culture (all other panels) in the presence of IL-12, IL-18, IL-12 plus IL-18, interferon (IFN)-γ, granulocyte–macrophage colony-stimulated factor (GM-CSF), IL-1β, IFN-β and IFN-α as described in Material and methods. Note the clumping effect of IL-12 plus IL-18 on cultured monocytes (middle left) (magnification × 100). (b) Growth of purified monocytes in vitro. The figure represents the number of live cells during culture related to the original input in the presence of the different anti-apoptotic stimuli. The numbers of cells of three different fields were counted every day for a period of 7 days. (c) Photographs of monocyte cultures stimulated with IL-12 plus IL-18 for 48 in the absence of presence of anti-intercellular adhesion molecule-1 (ICAM-1) at 2·5 µg/ml (d) Photographs of monocyte cultures in the presence of constant amounts of IL-12 (100 ng/ml) and increasing amounts of IL-18 (12–200 ng/ml: top panels) or constant amounts of IL-18 (100 ng/ml) and increasing amounts of IL-12 (12–200 ng/ml: bottom panels) (magnification × 100). Note that the survival and clustering of monocytes was dose dependent. (e) Photographs of monocytes cultured with IL-12 plus IL-18, IFN-γ or GM-CSF for 7 days (bottom row). The cells were harvested and cytospins were stained with May–Grünwald–Giemsa (magnification × 400). Note the morphological changes induced by the different stimuli. (f) Photographs of BAL macrophages cultured with the indicated cytokines for 7 days. Cells were harvested and cytospins were stained with May–Grünwald–Giemsa (magnification × 400). Note that no morphological changes were induced in these cells by the different stimuli.

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In addition to promoting monocyte survival, IL-12 and IL-18 also induced variable but reproducible macroscopic and microscopic changes in the cell morphology (Fig. 2a, e). Control monocytes and those treated with IL-12 or IL-18 alone were distributed evenly at the bottom of the culture wells. In sharp contrast, in the presence of both IL-12 plus IL-18, and after 24 h in culture, the monocytes clustered firmly together and were difficult to dissociate by mechanical means. The formation of these clusters was due to the known selective up-regulation of ICAM-1 by IL-12 and IL-18 on human monocytes [14], as the addition of an antibody to ICAM-1 inhibited the IL-12- and IL-18-mediated monocyte cluster formation (Fig. 2c). The combined effect of IL-12 and IL-18 was dose-dependent (Fig. 2d) and could be blocked by adding neutralizing anti-IL-12 and anti-IL-18 but not anti-IFN-γ antibodies (data not shown).

The aggregation of monocytes was not induced by other cytokines known for their anti-apoptotic effects (GM-CSF, IFN-α, IFN-β and IL-1β) [22], except for IFN-γ, where the monocytes occasionally formed smaller and dissociable clumps (Fig. 2a). These observations suggest a role for IL-12 and IL-18 in the survival and retention of newly arrived monocytes at sites of inflammation.

The presence of IL-12 plus IL-18, but not IL-12 or IL-18 alone, also induced microscopic changes consistent with monocyte activation, namely, increased size, an increased cytoplasm : nucleus ratio (Fig. 2e) and increased protein synthesis (detected by the blue colour of the cytoplasm seen in May–Grünwald–Giemsa-stained cells). The morphological changes observed in these cultures indicate that IL-12 and IL-18 play an important role in the control of the differentiation and activation of monocytes to macrophages. Consistently, no major changes were observed when in vivo matured macrophages purified from BAL were treated with IL-12 plus IL-18 (Fig. 2f).

Dose-dependent induction of the inflammatory and angiogenic/angiostatic chemokines CXCL8, CXCL9 and CXCL10 by IL-12 plus IL-18

We then tested the effect of IL-12 and IL-18, IFN-γ and GM-CSF on the production of known inflammatory and chemoattractant cytokines associated with inflammatory reactions: CXCL8, CXCL9, CXCL10, CCL3, CCL4 and CCL5. In non-stimulated cultures we detected low levels of CXCL8 (2·0 ± 1·3 ng/ml), but none of the other chemokines tested. The levels of CXCL8 were slightly up-regulated by IL-18 (3 ± 1·3 ng/ml), but not IL-12 alone (2 ± 0·6 ng/ml). In contrast, the levels of CXCL8 were fivefold higher after stimulation with both IL-12 and IL-18 (14·8 ± 6·1 ng/ml) (Fig. 3a). As a positive control, stimulation with IFN-γ resulted in the production of 717 ± 47·7 ng/ml of CXCL8 (Fig. 3a).

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Figure 3. Stimulation of chemokine synthesis by interleukin (IL)-12 and IL-18 in monocyte and cultures. Freshly isolated monocytes were cultured for a week with medium (NS), IL-12, IL-18, a mixture of IL-12 and IL-18 (IL-12/18), interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF) at 100 ng/ml, as described in Material and methods. Culture supernatants were assayed for the presence of CXC chemokine ligands (CXCL)8 (a), CXCL9 (b) and CXCL10 (c). The data in panels (a), (b) and (c) represent the mean ± s. e.m. of 12 different samples. The concentration of CXCL10 in the monocyte cultures stimulated with IL-12 plus IL-18 was measured daily for 7 days. The graph shows that the production of CXCL-10 was accumulative (d). Purified monocytes were cultured with increasing amounts of IL-12 (12·5–200 ng/ml) and IL-18 (12·5–200 ng/ml) (e) or in the presence of IL-12 and IL-18 and neutralizing antibodies to IL-12 (3·12–50 µg/ml) and to IL-18 (0·15–2·5 µg/ml) (f) as described in Material and methods. The y axis shows the levels of CXCL10 expressed in ng/ml, and shows that the induction of these chemokine was dose-dependent and could be blocked with neutralizing antibodies to IL-12 and IL-18. Panels (d–f) show a representative experiment.

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When compared to unstimulated cultures or cultures stimulated with IL-12 or IL-18 alone, co-stimulation with IL-12 plus IL-18 induced the production of very high levels of CXCL9 and CXCL10 (Fig. 3b, c) but not CCL3, CCL4 or CCL5 (data not shown). The production of CXCL9 and CXCL10 was accumulative (Fig. 3d). To characterize this effect further, we have studied the dose-dependence of CXCL10 induction by IL-12 and IL-18. Figure 3e shows clearly the synergistic effect of both cytokines that can be reversed dose-dependently by the addition of neutralizing antibodies directed against either IL-12 or IL-18 (Fig. 3f).

Although IL-12 and IL-18 can induce the production of IFN-γ by monocytes or T or NK cells, the possibility of IFN-γ-mediated stimulation of these chemokines was ruled out: first, the induction of these chemokines could be blocked by antibodies to IL-12 and IL-18 but not to IFN-γ (Fig. 3f and data not shown). Secondly, stimulation with exogenous IFN-γ resulted in very low levels of CXCL9 and CXCL10 compared to the levels induced by IL-12 and IL-18 (Fig. 3b, c).

Furthermore, monocyte-depleted populations were stimulated with IL-12 and IL-18 for a week and the levels of chemokines released in the supernatants were measured as described (see Material and methods). In summary, when stimulated with IL-12 and IL-18, monocyte-depleted mononuclear cells produced three logs less CXCL9 and CXCL10 than activated purified monocytes. The monocyte-depleted mononuclear cell cultures, on the other hand, produced CCL5, a chemokine undetectable in our monocyte cultures (data not shown). Therefore, the results may be attributed to the activity of monocytes and not to any other contaminating cell type.

IL-18 enhances chemokine synthesis by BAL stimulated with IL-12

We then studied the effects of IL-12 plus IL-18 on BAL macrophages as an example of mature macrophages differentiated in vivo. There were clear differences in the behaviour of monocytes and BAL macrophages in response to IL-12 and IL-18. Unstimulated BAL produced levels of the inflammatory chemokines CXCL8, CXCL9 and CXCL10 comparable to those present in IL-12- and IL-18-stimulated monocyte cultures. Because high levels of CXCL8 (Fig. 4a) were already present in non-stimulated cultures, stimulation with either IL-12, IL-18 or both had only marginal effects on the production of these two chemokines. On the other hand, the production of CXCL9 (Fig. 4b) and CXCL10 (Fig. 4c) was 10-fold higher with the addition of either IL-12 or IL-18 (P < 0·01), and 50-fold higher with the simultaneous presence of both chemokines (P < 0·01).

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Figure 4. Stimulation of chemokine synthesis by interleukin (IL)-12 and IL-18 in monocyte and bronchoalveolar lavage cultures. Freshly isolated bronchoalveolar lavage macrophages (a–c) were cultured for a week with medium (NS), IL-12, IL-18, a mixture of IL-12 and IL-18 (IL-12/18), interferon (IFN)-γ and granulocyte–macrophage colony-stimulating factor (GM-CSF) at 100 ng/ml, as described in Material and methods. Culture supernatants were assayed for the presence of CXC chemokine ligands (CXCL)8 (a), CXCL9 (b) and CXCL10 (c). The data in panels (a), (b) and (c) represent the mean ± s.e.m. of five different samples.

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Monocytes stimulated with IL-12 plus IL-18 retain phagocytic activity

To verify the phagocytic activity of stimulated monocytes, we evaluated the capacity of control and IFN-γ- or IL-12- and IL-18-treated monocytes to phagocytose fluorescein isothiocyanate (FITC)-labelled latex beads or carbonyl iron particles. Our results confirmed that phagocytic activity was retained after stimulation with IFN-γ or a combination of IL-12 plus IL-18, suggesting that the resulting activated macrophages are able to resolve the inflammatory process.

Activation of STAT4 during IL-12 and IL-18 stimulation

Induction of IFN-γ in T, NK cells and activated murine monocytes by IL-12 and IL-18 depends on activation of the STAT4 pathway and of nuclear factor-kappaB (NF-κB) through IL-1 receptor-associated kinase (IRAK), respectively [15,23]. Under our conditions of stimulation with IL-12 plus IL-18, we have confirmed that STAT4 is activated. Specifically, a two- and fivefold increase in phosphorylated STAT4 was observed in monocytes cultured in the presence of IL-12 and IL-18 for 45 and 120 min of culture, respectively, when compared to time 0 (detected by immunoblotting (n = 3) (a representative experiment is shown in Fig. 5).

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Figure 5. The combined effect of interleukin (IL)-12 and IL-18 increases the levels of phosphorylated signal transducer and activator of transcription 4 (STAT 4). Monocytes were cultured in the presence of medium or medium supplemented with IL-12 and IL-18 for 0, 45 or 120 min. Cells were analysed for human phosphorylated STAT 4 and β-actin by immunoblotting. Phosphorylated STAT4 and total actin were revealed with peroxidase-coupled anti-mouse antibodies and enhanced chemiluminiscence (ECL) (Amersham) as described in Material and methods. The ratio between STAT4 and actin net band volumes was then calculated for each sample. Fold increase values are relative to time 0. The data are from one of three experiments with similar outcome.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In this paper we present new data that support the autocrine role of IL-12 and IL-18 in human monocytes and macrophages, which in turn suggest a possible role of IL-12 and IL-18 in T and NK independent innate immune responses. We show that a combination of IL-12 and IL-18 but not each cytokine alone inhibited spontaneous apoptosis of monocytes in cell culture. Monocytes are prone to apoptosis through the CD95 CD95L pathway [24], but this can be prevented by cytokines that stimulate differentiation [25,26]. The combination of IL-12 and IL-18 also induced monocyte survival in a dose-dependent manner. Monocytes treated with IL-12 and IL-18 form large clusters due to the selective up-regulation of ICAM-1 [14] induced by these cytokines, as the presence of an anti-ICAM 1 antibody totally prevented the aggregation of these cells. Furthermore, the formation of these clusters is due to the direct effect of IL-12 and IL-18 and not to IFN-γ secreted by monocyte, T or NK cells, as the clusters were not observed in cultures supplemented with IFN-γ. In addition, aggregated cells were not formed in cultures with other cytokines known for their anti-apoptotic effects (GM-CSF, IFN-α, IFN-β and IL-1β[26–28]. These data support and suggest a role for IL-12 and IL-18 in the survival and retention of newly arrived monocytes at sites of inflammation. It is tempting to propose that the increase in ICAM-1 [14] may play a role in the formation of granulomas in vivo, where there is an accumulation of macrophages. In non-pathogenic conditions macrophages are usually scattered throughout the tissues, rather than forming aggregates [29]. Furthermore, high levels of both IL-12 and IL-18 have been found in BALs from patients with sarcoidosis compared to healthy subjects, and both are known to be expressed by the epithelial cells of the granulomas [30–32].

The morphological changes observed in IL-12- and IL-18-treated monocytes point to these cytokines as important factors in control of the differentiation and activation of monocytes to macrophages, and the subsequent production of chemokines. Furthermore, IL-12 and IL-18 selectively induced the production of the monocyte or neutrophil attracting cytokines CXCL8, CXCL9, CXCL10, but not CCL3, CCL4 and CCL5 (although these cytokines can also attract monocytes at sites of inflammation). One striking difference between these two groups of cytokines is that only the former have known angiogenic (CXCL8) or angiostatic (CXCL9 and CXCL10) properties [2] and therefore, as well as their capacity to attract monocytes or neutrophils at the site of inflammation, they are also likely to contribute to the wound-healing processes associated with an inflammatory response. Finally, IL-12 and IL-18 did not decrease the phagocytic capacity of monocytes in culture. This is a significant observation, as the retention of this capacity is crucial to eliminate the residual apoptotic neutrophils needed to resolve the inflammatory reaction [1].

The synergistic effects of IL-12 and IL-18 on the induction of chemokines highlights the need for a two-signal control as a versatile and sensitive mechanism connecting different transmission pathways, such as T cell activation through the T cell receptor and CD28. Similarly, IL-12 and IL-18 belong to two different families of cytokines, with IL-12 signalling through the STAT4 pathway and IL-18 activating NF-κB through IRAK [23]. However, it is intriguing whether the described synergy may occur at physiologically relevant concentrations of IL-12 and IL-18. In vitro, the optimal concentrations of IL-12 and IL-18 to induce monocyte differentiation and chemokine production was 100 ng/ml, interestingly within the same concentration range as that recommended for IFN-γ induction in human T cells [33] but higher than the concentrations used in other studies [9]. However, in vivo these cytokines are produced locally where cells are in direct contact and this, in itself, may well be an important factor that controls the effectiveness of cytokines in immune responses.

In contrast to monocytes, freshly isolated BAL macrophages constitutively produced chemokines and the levels were up-regulated in response to IL-12 or IL-18 alone. Upon co-stimulation with IL-12 plus IL-18 the levels of CXCL9 and CXCL10 also increased even further. Macrophages constitute the most prominent population in the lung, a compartment with few lymphocytes and NK cells [34]. Therefore, the fact that macrophages could produce chemokines upon IL-12 and or IL-18 stimulation is directly relevant to the control of pulmonary infections. The production of IFN-γ by BAL macrophages has been described [35,36]. Thus, the detectable levels of IL-12 and IL-18 found in the lung might be responsible for the activation of local macrophages and hence production of IFN-γ, a suggestion that is consistent with the finding that patients with deficiencies in the IFN-γ or IL-12 pathways frequently have intracytoplasmic mycobacterial infections [37].

In conclusion, the fact that monocytes and macrophages can produce inflammatory and angiogenic chemokines upon IL-12 plus IL-18 co-stimulation implies that these cytokines could also play an important role in the initiation, recruitment, effector and resolution phases of NK-independent innate immune responses, and in the resolution of respiratory bacterial infections, where macrophages and neutrophils are the main components of lung inflammatory reactions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

This work was supported partially by Fundació Irsicaixa, FIS Red Temática de Investigación en SIDA (RG GO3/173) and projects FIS 02/0879 and MEC BFI-2003–00405.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References