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Published ahead of print on March 27, 2003, doi:10.1165/rcmb.2002-0158OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 29, pp. 367-374, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0158OC

Interleukin-4–Induced Apoptosis Entails Caspase Activation and Suppression of Extracellular Signal–Regulated Kinase Phosphorylation

Andrea I. Doseff, Jon H. Baker, Jr., Trevor A. Bourgeois and Mark D. Wewers

The Dorothy M. Davis Heart and Lung Research Institute, and Divisions of Pulmonary and Critical Care and Molecular Genetics, Ohio State University, Columbus, Ohio

Address correspondence to: A. I. Doseff, 201 Davis Heart and Lung Research Institute, Molecular Genetics, Ohio State University, 473 West 12th, Columbus, OH 43210. E-mail: doseff-1{at}medctr.osu.edu or wewers.2{at}osu.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Monocytes are important components of the innate immune response. The number of circulating monocytes is controlled in part by apoptosis. We have previously shown that monocyte apoptosis requires the activation of caspase-3, a central component of the apoptotic machinery, and that several stimulatory signals, including endotoxin (lipopolysaccharide [LPS]), induce monocyte survival, by the inhibition of caspase-3. We hypothesized that the Th2 anti-inflammatory cytokine, interleukin (IL)-4, may also influence monocyte life span by modulating the apoptotic cascade and the kinases known to be activated by LPS. Here, we show that the IL-4–dependent killing of LPS-treated monocytes reactivates the apoptotic cascade blocked by endotoxin, evidenced by the activity of the effector caspase-3 and the upstream caspases-8 and -9. IL-4 did not affect the activity of caspase-3 or the fragmentation of DNA in nonstimulated monocytes, suggesting that the induction of the apoptotic cascade by IL-4 is specific for stimulated monocytes. In addition, we show that the ability of IL-4 to induce apoptosis is associated with the dephosphorylation of the extracellular signal–regulated kinase, but not with changes in TLR4 expression. Together, these findings suggest a molecular mechanism by which the anti-inflammatory cytokine IL-4 modulates the life span of monocytes at least in part by an extracellular signal–regulated kinase–dependent pathway.

Abbreviations: aminotrifluoromethylcoumarin assay, afc • dimethyl sulfoxide, DMSO • extracellular signal–regulated kinase, ERK • interleukin, IL • lipopolysaccharide, LPS • mitogen-activated protein kinase, MAPK • phosphate-buffered saline, PBS • phycoerythrin, PE


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Monocytes play a pivotal role in the innate immune response. They are produced in the bone marrow and circulate in the bloodstream for 24–48 h (1) before undergoing spontaneous monocyte apoptosis. However, during sepsis or chronic inflammation, lipopolysaccharide (LPS), a main component of Gram-negative bacterial cell walls, binds to the Toll receptor (2). Binding of LPS to its receptor triggers a signal transduction cascade that includes the activation by phosphorylation of extracellular signal–regulated kinase (ERK), a member of the mitogen-activated protein kinase (MAPK) family (3). In addition, we have previously shown that LPS promotes prolonged monocyte survival by blocking the activation of the apoptotic pathway (4). Prolonged monocyte survival in the presence of differentiating stimuli has been associated with disease pathogenesis and progression of a number of cardiopulmonary diseases such us atherosclerosis, sarcoidosis and pulmonary fibrosis (5–8). Thus, understanding the biochemical pathways that regulate monocyte life span may allow controlling monocyte accumulation, providing new targets for the more effective therapeutic intervention of patients with these diseases.

Apoptosis is a central homeostatic process in regulating the number of circulating monocytes. Caspases are highly conserved cysteine proteases essential to apoptosis (9) that are constitutively expressed as inactive precursors. In response to an apoptotic stimulus, the precursors are converted into active enzymes (10). Some, like caspase-8 (FLICE) and caspase-9 (LAP6), are called initiators caspases because they transmit the apoptotic signals to the downstream, effector caspases (11). Caspase-3 (cpp-32) is an effector caspase into which many apoptotic signals converge to execute apoptosis. In this context, we have recently shown that caspase-9 is involved in monocyte apoptosis and that the activation of caspase-3 is central to spontaneous monocyte apoptosis (4, 12).

Interleukin (IL)-4 is an anti-inflammatory cytokine produced by activated T-lymphocytes, which has pleiotropic biological effects in leukocytes (13). For example, IL-4 has been shown to induce cell death in peripheral blood eosinophils (14) but to delay apoptosis in neutrophils (15). The therapeutic effects of IL-4 as an anti-inflammatory and antitumor cytokine in animal models of arthritis and nonobese diabetic mice are well known (13). Gene delivery of IL-4 in mice models of rheumatoid arthritis results in a significant reduction in swelling (16). IL-4, when added together with granulocyte-macrophage colony–stimulating factor (GM-CSF),induces survival and differentiation of monocytes into dendritic cells (17). In contrast, IL-4 is sufficient to induce DNA fragmentation of LPS- or IL-1ß–stimulated monocytes (18).

Despite our extensive knowledge of the receptors and the signaling cascade activated by LPS (19), the possible connections between these stimulatory factors and the apoptotic machinery involved in monocyte life span remains unknown. This study identifies some of the molecular mechanisms used by IL-4 to induce cell death of LPS-treated monocytes. We show here that IL-4 does not affect the activity of caspase-3 in monocytes undergoing spontaneous cell death. However, IL-4 reactivates specifically the apoptotic pathway blocked in LPS-stimulated monocytes. We demonstrate that this novel effect of IL-4 requires the upstream activators, caspases-8 and -9. Finally, we found that IL-4 induces changes in the levels of phosphorylated ERK but not of p38 kinase, and that the IL-4 effect is independent of the level of TLR4 expression. Taken together, our results suggest that the ability of IL-4 to antagonize specifically the prolonged survival effect of inflammatory stimuli uses a crosstalk between the apoptotic pathway and the signal transduction cascade, providing an important molecular balance between these two pathways to control the life span of monocytes.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Monocyte Purification and Culture Conditions
Human monocytes were purified by clumping, as previously described (4). The population of monocytes obtained was on average 70–80% pure as estimated by flow cytometry using an anti-CD14 marker (Becton Dickinson, San Jose, CA). For flow cytometry experiments, monocytes were purified by a CD14-positive selection system using magnetic beads (Miltenyi Biotec, Auburn, CA). Purified monocytes were resuspended in RPMI 1640 (BioWitthaker, Walkersville, MD) to a final concentration of 3 x 106 cells/ml and cultured for 20 h at 37°C in 5% CO2. Human recombinant IL-1ß and IL-4 were obtained from R&D (Minneapolis, MN), prepared as recommended by the manufacturer, and used at the concentration of 1 ng/ml for IL-1ß and 10 ng/ml for IL-4, except where otherwise indicated. Lipopolysaccharide (LPS from E. coli strain 0127:B8; Difco, Detroit, MI) was used at 1 ng/ml. Caspase inhibitors DEVD-fmk, LETD-fmk, LEHD-fmk, and zVAD-fmk were obtained from Enzyme System Products (Livermore, CA) and used at 100 µM in dimethylsulfoxide (DMSO). YVAD-cmk was from Calbiochem (La Jolla, CA). For all the experiments involving caspase inhibitors, controls contained an equivalent amount of the diluent DMSO (Sigma, Milwaukee, WI). The MEK inhibitor PD98059 was dissolved in DMSO and added to the cells 1 h previous to the addition of LPS at 10 and 50 µM (Calbiochem).

Extract Preparation
Extracts used to determine activated caspases were prepared as described previously (4). Briefly, 3 x 106 human monocytes were collected by centrifugation and washed with KPM buffer (50 mM KCl, 50 mM PIPES, 10 mM EGTA, 1.92 mM MgCl, pH 7.0, 1 mM DTT, 0.1 mM PMSF, 10 µg/ml of cytochalasin B, and 2 µg/ml of protease inhibitors: chymostatin, pepstatin, leupeptin, antipain). Cells were snap-frozen in liquid nitrogen and lysed by four cycles of freeze-thawing. Extracts were then centrifuged for 20 min at 14,000 x g in a microcentrifuge. Supernatants were aliquoted and kept at -70°C for future use. Whole cell extracts were prepared by the addition of 2x Laemmli buffer containing 1% ß-mercaptoethanol (BioRad, Hercules, CA) directly to the cells and boiling for 5 min before loading onto gels.

Enzymatic Caspase Activity
The presence of active caspases was determined by the aminotrifluoromethylcoumarin assay (afc), as previously described (4). Lysates were incubated with DEVD-afc to determine the presence of active caspase-3 or LEHD-afc for caspase-9 in a cyto-buffer (10% glycerol, 50 mM Pipes, pH 7.0, 1 mM EDTA) containing 1 mM DTT and 20 µM tetrapeptide substrate. Caspase-9 activity was measured after pre-incubation for 15 min. Caspase-8 activity was determined using LETD-afc in a cyto-buffer (100 mM Hepes, pH 7.5, 20% glycerol, 0.5 mM EDTA) containing 1 mM DTT and 100 µM tetrapeptide substrate after 30 min of pre-incubation. All the tetrapeptides were obtained from Enzyme Systems Products (Livermore, CA). Release of free afc was determined using a Cytofluor 4,000 fluorimeter (Filters: excitation; 400 nm, emission; 508 nm; Perseptive Co., Framingham, MA).

DNA Fragmentation Assays
The presence of DNA fragmentation was determined using 4 x 106 human monocytes for each condition. Cells were collected, washed once with KPM buffer, and resuspended in resuspension buffer provided by the suicide kit from Oncogene (Boston, MA). Samples were kept at -20°C in resuspension buffer or processed as described by the manufacturers. DNA was resolved by electrophoresis in 1.8% agarose. The gel was subsequently stained with a 1:10,000 dilution of Syber Green (Molecular Probes, Eugene, OR) in 1x TAE buffer for 30 min to 1 h. The DNA ladders were imaged using a gel imaging system (Bio-Rad). A 123–base pair DNA marker (Gibco BRL, Carlsbad, CA) was included.

Antigenic Detections
In experiments performed to detect ERK and p38 kinase, 0.5 x 106 cells were loaded in each lane. Gels were transferred to nitrocellulose and Western blots were performed using manufacturer specifications (Cell Signaling, Boston, MA). Secondary antibodies linked to HRP were from Amersham (Arlington Heights, IL). Proteins were visualized by enhanced chemiluminescence (ECL; Amersham).

Flow Cytometry
Monocytes were purified by a CD14-positive selection and cultured for different lengths of time as described above. Cells were rinsed once with phosphate-buffered saline (PBS) and blocked for 15 min on ice with 200 µg /ml of human IgG and 1% heat inactivated fetal bovine serum in PBS. The cells were then stained with phycoerythrin (PE)-conjugated TLR4 (PE-TLR4 clone HTA125; eBioscience, San Diego, CA) for 30 min on ice. IgG conjugated with PE was used as control for labeling monocytes. After staining, the cells were washed once with cold PBS. Samples were read on a Becton Dickinson FACS Calibur using Cell Quest version 3.3 software. PE-stained was read in FL-2, excitation, 480, emission; 578 nm. WinMDI version 2.8 was used to generate histogram plots which were normalized to number of events.

Statistical Analysis
All data are expressed as mean ± SEM. For comparisons that involved multiple variables and observations, ANOVA (JMP; SAS Institute, Cary, NC) was used. Having passed statistical significance by ANOVA, Tukey-Kramer test for all pair comparisons or individual comparisons were made by using the contrast method. Statistical significance is stated in the figure legends.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Caspase-3 Activity Is Not Modulated by IL-4 during Spontaneous Apoptosis
To investigate the effect of the anti-inflammatory cytokine IL-4 on monocyte life span, purified monocytes were cultured in the presence of different concentrations of IL-4. We have previously shown that in the absence of survival stimuli, monocytes undergo apoptosis, reflected by the activation of caspase-3 (4). Immediately after purification, monocytes are nonapoptotic, as evidenced by the lack of caspase-3 activity (Figure 1A, Fresh). Caspase-3 activity increases during spontaneous monocyte apoptosis over 20 h (Figure 1A). Treatment of monocytes with different doses of IL-4 does not affect their normal commitment to undergo spontaneous cell death (Figure 1A). Similarly, IL-4 does not alter the DNA fragmentation profile, a typical hallmark of apoptosis, observed in monocytes undergoing spontaneous cell death (Figure 1B). The activity of caspase-3 correlates with the DNA fragmentation patterns observed, indicating that it provides a good marker to follow apoptosis of human monocytes. These results show that IL-4 does not affect the normal program of spontaneous monocyte apoptosis.



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  		Figure 1. Spontaneous monocyte apoptosis is not modulated by IL-4. Monocytes were cultured for 20 h with increasing concentrations of IL-4 (0, 1, 10, and 100 ng/ml). (A) The presence of caspase-3 activity on monocyte extracts was determined using the fluorogenic substrate DEVD-afc. Results are expressed as the mean ± SEM from three separate experiments. Results for caspase-3 activity in the presence of different concentrations of IL-4 are not statistically significant (P = 0.99) ANOVA. (B) Monocyte oligonucleosomal DNA ladder formation was determined on samples from monocytes treated with increasing concentrations of IL-4, by agarose gel electrophoresis of cytosolic DNA.

 
IL-4 Induces Activation of Apoptosis Only in Stimulated Monocytes
During Gram-negative bacterial infection, monocytes respond to LPS by inducing inflammatory cytokines such as IL-1ß, and by recruiting monocytes to the site of inflammation (20). Consistent with the increase in monocyte numbers at inflammatory sites, we showed that LPS blocks spontaneous monocyte apoptosis (4). To test the hypothesis that IL-4 exerts its anti-inflammatory effect (13) by activating the apoptotic pathway inhibited by LPS, we examined the effect of IL-4 on LPS treated monocytes. Monocytes cultured in the presence of both LPS and IL-4 show a 3-fold increase in caspase-3 activity when compared with monocytes treated with LPS alone (Figure 2A, asterisk; 78 ± 22 pmol/min/mg LPS versus 272 ± 42 pmol/min/mg LPS+IL-4, n = 4, P < 0.001). Similar results were obtained when monocytes were treated with IL-1ß (data not shown). Consistent with our caspase-3 activity assays, we also observed a dramatic increase in DNA ladder formation in LPS- and IL-4–treated monocytes, compared with monocytes treated with LPS alone (Figure 2B).



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  		Figure 2. IL-4 activates caspase-3 in stimulated monocytes. (A) The presence of caspase-3 activity was tested using the fluorogenic substrate DEVD-afc in extracts from freshly isolated monocytes (Fresh), monocytes undergoing spontaneous apoptosis, or monocytes treated with LPS or IL-4 (10 ng/ml) alone or the combination. Results are expressed as the mean ± SEM from at least three separate experiments. Analysis using ANOVA and individual comparisons indicate that the caspase activity that resulted by the addition of IL-4 to LPS-treated cells were significantly different compared with the activity of apoptotic monocytes or IL-4–treated monocytes (P < 0.01, denoted by ). The caspase-3 activity is highly increased by the addition of IL-4 to LPS-treated monocytes (*P < 0.001). (B) DNA ladder formation was determined using agarose gel electrophoresis in freshly isolated monocytes (Fresh), monocytes undergoing spontaneous apoptosis or treated with the inflammatory and anti-inflammatory stimuli as mentioned above.

 
IL-4 Acts Upstream of the Activator Caspases
Upstream activator caspases such as caspase-8 and caspase-9 can activate caspase-3 (21), although the proteolytic cascade that results in caspase-3 activation in human monocytes is still poorly defined. We examined whether the effect of IL-4 on apoptosis evidenced by the activation of caspase-3 was mediated by caspase-8 and/or caspase-9. Purified monocytes were cultured for 20 h in the presence of IL-4, LPS, or the combination of IL-4 and LPS. The reduction of apoptosis after the addition of LPS results in the blockage of caspase-8 activity, as evidenced by the reduction in the ability of cellular extracts to cleave the fluorogenic caspase-8 substrate LETD-afc (Figure 3A, noted by ; 27.5 ± 1.6 pmol/min/mg LPS versus 86 ± 5.6 pmol/min/mg untreated monocytes, n = 3, P < 0.001). The addition of IL-4 to LPS-treated monocytes results in a less than 2 fold increase in caspase-8 activity, when compared with monocytes treated with LPS alone (Figure 3A). Interestingly, however, the activity of caspase-8 induced by IL-4 on LPS-treated monocytes does not reach the levels found in untreated or IL-4–treated monocytes (Figure 3A, asterisk; 45 ± 3.7 pmol/min/mg LPS+IL-4 versus 86 ± 5.6 pmol/min/mg untreated monocytes, n = 3, P < 0.001).



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  		Figure 3. IL-4 activates both caspase-8 and caspase-9 upstream caspases. The presence of upstream caspase activity was tested using fluorogenic substrates in extracts from freshly isolated monocytes (Fresh), monocytes undergoing spontaneous apoptosis, or monocytes treated with either, LPS or IL-4 (10 ng/ml) alone or in combination with LPS. Results are expressed as the mean ± SEM from at least three separate experiments. (A) Caspase-8 activity was determined using the fluorogenic substrate LETD-afc. Analysis using ANOVA and individual comparisons indicate that the caspase-8 activity is significantly reduced in LPS-treated monocytes compared with untreated monocytes (P < 0.001, denoted by ). Caspase-8 activity is significantly reduced in IL-4– and LPS-treated cells compared with the activity of apoptotic or IL-4–treated monocytes (*P < 0.001). (B) Caspase-9 activity was determined using the fluorogenic substrate LEHD-afc. ANOVA followed by individual comparisons show that the increase in caspase-9 activity in IL-4– and LPS-treated monocyte does not reach the levels of caspase-9 activity found in spontaneous apoptosis (*P < 0.001). The caspase-9 activity is significantly reduced in LPS-treated monocytes compared with the activity found in spontaneous apoptosis (P < 0.001, denoted by ).

 
We have previously shown that caspase-9 is also activated during monocyte apoptosis (12). Thus, we investigated the effect of IL-4 on the activity of this second upstream activator caspase. As expected, the addition of LPS to monocytes (ready to enter spontaneous apoptosis) dramatically reduces caspase-9 activity (Figure 3B, noted by ; 18.5 ± 2.5 pmol/min/mg LPS versus 112 ± 2.3 pmol/min/mg untreated monocytes, n = 3, P < 0.001). The addition of IL-4 to monocytes treated with LPS results in a 2- to 3-fold increase in caspase-9 activity, when compared with monocytes treated just with LPS (Figure 3). Similarly to caspase-8, however, this activity is only ~ 50% of the caspase-9 activity observed on monocytes undergoing spontaneous cell death (Figure 3B, asterisk; 55 ± 3.4 pmol/min/mg LPS+IL-4 versus 112 ± 2.3 pmol/min/mg untreated monocytes, n = 3, P < 0.001). Similar results were obtained when IL-1ß instead of LPS was used as stimulatory signal (data not shown). These results indicate that IL-4 is able to reactivate both caspase-8 and caspase-9. However, unlike the full restoration of caspase-3 activity observed in LPS- and IL-4–treated monocytes, the levels of caspase-8 and caspase-9 are only partially recovered compared with the activity levels found in untreated monocytes.

To determine whether the caspase-8, caspase-9 and caspase-3 proteolytic pathway was the only pathway involved in the reactivation of apoptosis of stimulated monocytes by IL-4, we investigated the effect of various caspase inhibitors on monocytes cultured in the presence of LPS and IL-4 (Figure 4). Caspase-3 activity was measured to determine the effect of the inhibitors on the IL-4-apoptotic effect. The broad caspase inhibitor z-VAD-cmk, the caspase-3 inhibitor DEVD-fmk, the caspase-8 inhibitor LETD-fmk, and the caspase-9 inhibitor LEHD-fmk, all blocked completely the ability of IL-4 to induce apoptosis in monocytes stimulated with LPS. These results are in agreement with the activation of caspase-8 and caspase-9 observed in stimulated monocytes treated with IL-4 (Figures 3A and 3B). Interestingly, the caspase-1 inhibitor YVAD-cmk partially blocks the reactivation of caspase-3 in cells treated with LPS and IL-4 (Figure 4A). This effect of YVAD suggests an unexpected participation of caspase-1 in the caspase-3 apoptotic machinery. Consistent with our caspase-3 activity assays, we observed similar results with the DNA fragmentation patterns in monocytes cultured in the presence of LPS, IL-4, and the caspase inhibitors (Figure 4B). Whereas caspase-3, -8, -9, or the broad inhibitor z-VAD-cmk block DNA fragmentation, YVAD-cmk had very little effect on preventing the apoptotic program initiated by IL-4. Together, these results show that the main effect of IL-4 in inducing apoptosis of LPS-stimulated monocyte is by activating the caspase-8, -9, and -3 proteolytic cascade, and suggest that caspase-3 might be induced as well through a caspase-1–dependent mechanism.



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  		Figure 4. Caspase inhibitors block the IL-4–dependent caspase-3 activity. Purified human monocytes were cultured for 20 h with the inflammatory stimuli, LPS (1 ng/ml), in the presence or absence of IL-4 (10 ng/ml) and caspase inhibitors (100 µM). The effects of YVAD-fmk a caspase-1, a broad inhibitor z-VAD-fmk, a caspase-3 inhibitor DEVD-fmk, a caspase-8 LETD-fmk and a caspase-9 inhibitor LEHD-fmk, were tested. (A) Caspase-3 activity was determined using the fluorogenic substrate DEVD-afc. Results are expressed as the mean ± SEM from three separate experiments. (B) Monocyte oligonucleosomal DNA ladder formation was determined by agarose gel electrophoresis of cytosolic DNA.

 
IL-4 Does Not Change the Expression of TLR4
LPS binds to the TLR4 and thus stimulates a signal transduction cascade, which leads to the transcription of inflammatory cytokines (22, 23). To test whether IL-4 induces cell death by affecting the levels of TLR4, and therefore reverting the stimulatory effect of LPS, we investigated the levels of the TLR4 in monocytes untreated or treated with LPS, IL-4, or both, using an antibody against the TLR4 protein. Flow cytometry analyses showed the increase of TLR4 expression in monocytes treated with LPS when compared with untreated or IL-4–treated monocytes. Similar analyses using monocytes treated with LPS and IL-4 show that IL-4 does not have an effect on the levels of Toll receptor in LPS-treated monocytes (Figure 5). These results indicate that IL-4 must function at a different level than LPS, probably downstream of the TLR4.



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  		Figure 5. IL-4 does not change the level of expression of TLR4 in LPS-treated monocytes. Human freshly isolated human monocytes (Fresh), monocytes undergoing spontaneous apoptosis or monocytes treated with either LPS, IL-4 (10 ng/ml) alone, or IL-4 in combination with LPS for 4 h were labeled using PE-TLR4 and analyzed by flow cytometry.

 
IL-4 Downregulates ERK Phosphorylation
The results described above suggest that the apoptotic effect of IL-4 on LPS-stimulated monocytes is likely to occur downstream of the TLR4. Because of the reported effect of LPS in stimulating the phosphorylation of the ERK kinase, which results in an increased ERK kinase activity, we decided to investigate whether IL-4 functions upstream or downstream of this kinase to stimulate the apoptosis of LPS-stimulated monocytes. We investigated the effect of LPS on ERK phosphorylation in the presence and absence of IL-4, using a combination of antibodies specific for phosphorylated ERK, or an antibody that recognizes phosphorylated and nonphosphorylated ERK protein (Figure 6A). As expected from our previous results, the treatment of monocytes with IL-4 alone does not result in a significant increase of ERK phosphorylation. In contrast, LPS induced a dramatic increase in phospho-ERK without a significant change in protein accumulation, consistent with other reports (3). Interestingly, the treatment of LPS-stimulated monocytes with IL-4 results in a decrease in the levels of phosphorylated ERK to levels comparable, yet not identical, to those found in untreated monocytes. Treatments of LPS-stimulated monocytes with IL-4 however, did not change the levels of phosphorylated p38 other member of the MAP kinase family (data not shown). Because dephosphorylation of ERK appears to be an important factor on the IL-4–apoptotic inducing pathway of LPS-treated monocytes, we next hypothesized that inhibition of ERK on LPS-treated monocytes may be sufficient to induce apoptosis. ERK activation is mediated by the upstream MAPK family member, MAPK kinase/MEK/MAPKK (24). We found that the addition of the MEK inhibitor PD98059 on LPS-treated monocytes resulted in significant activation of caspase-3 activity (Figure 6B). Consistent with the findings, the addition of the ERK inhibitor also induces DNA fragmentation on LPS-treated monocytes (Figure 6C). Together, these results suggest that the apoptotic effect of IL-4 on LPS-stimulated monocytes happens by a pathway that at least in part uses the ERK-dependent signaling cascade.



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  		Figure 6. IL-4 induces cell death on LPS-treated monocytes by an ERK-dependent pathway. (A) Extracts from freshly isolated human monocytes (Fresh), monocytes undergoing spontaneous apoptosis or monocytes treated with either, LPS, IL-4 (10 ng/ml) alone or in combination with LPS were separated by SDS-PAGE. The membranes were blotted with anti–phospho-ERK and total ERK antibodies and developed using chemoluminescence. Monocytes were preincubated with the MEK inhibitor PD98059 (10 or 50 µM) for 1 h. The cells were then treated with LPS (1 ng/ml) or left untreated for 20 h. (B) Caspase-3 activity was determined using the fluorogenic substrate DEVD-afc. Results are expressed as the mean ± SEM from three separate experiments. (C) Monocyte DNA ladder formation was determined by gel electrophoresis of cytosolic DNA.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Monocytes are important components in the persistence and resolution of inflammation. In chronic inflammation, monocytes escape their normal apoptotic commitment and are recruited from the circulatory system to inflamed tissues, where they differentiate. Here, we investigated the effect of IL-4 in restoring the apoptosis of monocytes stimulated by LPS. Our studies demonstrate that IL-4 functions by reactivating the apoptotic pathway blocked by LPS and decreasing the levels of phospho-ERK. Our findings support and provide a molecular mechanism to explain prior studies (18) that suggested that in the presence of LPS, monocytes treated with IL-4 undergo apoptosis. We show here that IL-4 induces apoptosis only when monocytes were stimulated with inflammatory molecules, such as LPS (Figure 2) and IL-1ß (data not shown). In the absence of these signals, IL-4 has no significant effect on the spontaneous apoptosis of monocytes (Figure 1). We established that IL-4 induces apoptosis of stimulated monocytes by reactivating the apoptotic pathway blocked by LPS. Interestingly, we identified an unexpected participation of caspase-1 in the activation of caspase-3 in LPS- and IL-4–treated monocytes. Although we cannot rule out the possibility that YVAD inhibits another enzyme besides caspase-1, it is tempting to speculate that caspase-3 might be a target of caspase-1 under the conditions tested here. Indeed, caspase-1 is expected to be induced by LPS resulting in the release of IL-1ß (25, 26), a key component in the response of monocytes to LPS (Figure 5). Previous studies have shown that, in vitro, caspase-3 can be cleaved and activated by caspase-1 (27). The complete absence of caspase-3 activity in the presence of the caspase-8 and caspase-9 inhibitors suggests that, if caspase-1 participates in the activation of caspase-3, this effect is minor and is obscured by the more significant contribution of caspase-8 and -9 in caspase-3 activation.

The effect of the inhibitors of caspase-8 and caspase-9 on the activity of caspase-3 in stimulated monocytes treated with IL-4 (Figure 4) further supports a mechanism (Figure 7) in which the stimulatory signals provided by LPS acts upstream of caspase-8, believed to be the first protease in the apoptotic pathway (28). In most cell types, the apoptotic pathway is activated by the recruitment of caspase-8 to the Fas/TNF receptor (29). In the case of monocytes, treatment with an antibody that blocks the Fas receptor prevents spontaneous apoptosis suggesting the involvement of Fas in the activation of monocyte apoptosis (30, 31). Thus, our results suggest that LPS or a signal induced by inflammatory stimuli inhibits either the recruitment of caspase-8 to the receptor or its subsequent activation. Therefore, the effect of IL-4 would be to block these effects of LPS. In this context, it has recently been demonstrated that the expression of the Toll receptor decreases during IL-4 treatment (32). Our studies show no difference in the levels of TLR4 protein as a consequence of the treatment with IL-4. These results strongly indicate that the effect of IL-4 to reactivate the cell death pathway is not mediated, at least at the shorter time frame of our experiments, by the regulation of the levels of this receptor.



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  		Figure 7. Model of crosstalk between the inflammatory and anti-inflammatory pathway with the apoptotic machinery. IL-4 induces apoptosis of LPS-treated monocytes by reactivating the caspase cascade blocked by the stimulatory signals (e.g., LPS/IL-1ß). Inhibition of the inflammasome function by ERK dephosphorylation with PD98059 mirrors the effect of IL-4.

 
To further establish the effect of IL-4 in the LPS-inducing pathway, we looked at some of the molecules that are activated by LPS. Obvious targets were kinases known to form part of the signalosome, as several members of this pathway have been shown to modulate survival or death depending on the cell type (33). Our results show that one dramatic effect of IL-4 is to turn off the signal transduction cascade triggered by LPS by decreasing the levels of the phosphorylated form of the ERK kinase (Figure 6A). The effect of the MEK inhibitor PD98059 on LPS-treated monocytes recapitulates the effect of IL-4 (Figures 6B and 6C), further suggesting that the IL-4–induced apoptotic pathway is at least in part mediated by ERK.

Together, our results establish provocative links between the Toll/IL-1ß receptor and the apoptotic pathway formed by caspase-8, caspase-9, and casapase-3 (Figure 7). Although the direct target of this connection has not been yet identified, based on our results it is clear that a component downstream of the ERK kinase in the Toll signal transduction pathway must modulate the function of a factor upstream of caspase-8, the first enzyme committed to the apoptotic machinery.


   Acknowledgments
 
The authors thank P. Mark Sadler for technical assistance. They thank Drs. E. Grotewold, A. Elssner, and C. Anderson for the critical reading of this manuscript. This study was funded by NIH (ROI HL40871 to M.D.W., and ALA (RG-044-N) and OSU (SG-101105) to A.I.D.

Received in original form August 19, 2002

Received in final form February 18, 2003


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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