Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway RALF BAUMANN,* CARMEN CASAULTA,† DAGMAR SIMON,# SE´BASTIEN CONUS,* SHIDA YOUSEFI,* AND HANS-UWE SIMON*,1 Departments of *Pharmacology, †Pediatrics, and #Dermatology, University of Bern, Bern, Switzerland Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine known to activate macrophages and T cells. In this study, we demonstrate that recombinant MIF delays apoptosis of neutrophils in vitro. MIF action is dose and time dependent as well as specific since it was abolished with a neutralizing anti-MIF antibody. MIF, like G-CSF, delayed cleavage of the proapoptotic members of the Bcl-2 family Bid and Bax in neutrophils, suggesting that MIF inhibits apoptosis pathways proximal to mitochondria activation. Indeed, MIF also prevented release of cytochrome c and Smac from the mitochondria and subsequent activation of the critical effector caspase-3 in these cells. Moreover, we observed increased MIF plasma levels in patients with cystic fibrosis, a heterogeneous recessive genetic disorder associated with bacterial infections and delayed neutrophil apoptosis. In conclusion, MIF is a survival cytokine for human neutrophils, a finding with potential pathologic relevance in infectious diseases.—Baumann, R., Casaulta, C., Simon, D., Conus, S., Yousefi, S., Simon, H.-U. Macrophage migration inhibitory factor delays apoptosis in neutrophils by inhibiting the mitochondria-dependent death pathway. FASEB J. 17, 2221–2230 (2003)
ABSTRACT
Key Words: inflammation 䡠 MIF 䡠 mitochondria Neutrophils are important players within the innate immune system. Neutrophil numbers are controlled by rates of generation and apoptosis (1). Apoptosis is the most common physiologic cell death of neutrophils in vitro (2) and in vivo (3). This form of cell death prevents the release of inflammatory mediators from the dying cell (4). Clearly, any failure in the process of neutrophil apoptosis would result in the initiation of an inflammatory response and/or in the maintenance of an already existing inflammation. Therefore, studying neutrophil apoptosis under normal and inflammatory conditions seems important. Delayed neutrophil apoptosis has been associated with several acute and chronic inflammatory diseases and appears to be largely mediated by overexpression of granulocyte colony-stimulating factor (G-CSF) and granulocyte–macrophage (GM) -CSF (5), which are potent survival factors for these cells (2, 6, 7). The 0892-6638/03/0017-2221 © FASEB
induction of neutrophil apoptosis during the resolution of a neutrophilic inflammatory response can be mimicked in vitro by culturing the cells in the absence of sufficient amounts of survival factors, a process called spontaneous apoptosis. Spontaneous neutrophil apoptosis can be enhanced by Fas receptor stimulation (8). Similar observations have been made in eosinophil in vitro cultures. Eosinophils undergo spontaneous apoptosis, which can be inhibited by survival cytokines such as interleukin 5 (IL-5) or GM-CSF (9). Eosinophils also express functional Fas receptors that accelerate in vitro cell death (10, 11). The intracellular mechanisms that delay neutrophil apoptosis mediated by survival factors have been partially identified. Survival factors appear to induce the expression of antiapoptotic genes and to reduce or inactivate proapoptotic molecules of the Bcl-2 family (12). Although neutrophils were reported to have only a few mitochondria (13), these data indicate that mitochondria-mediated mechanisms play an important role in the regulation of neutrophil apoptosis. Besides mitochondria, which might be activated via Bax (5), caspases are crucial elements of apoptotic pathways. Active caspase-3 appears to be critical for the induction of apoptosis in neutrophils (14 –16). Despite the progress in our understanding of the molecular control of neutrophil apoptosis, it is still unclear how apoptosis is triggered in the absence of survival factors, and the exact mechanisms of how survival factors delay apoptosis remain to be identified. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine secreted by monocytes/macrophages and T cells (17). As the name indicates, it inhibits the random migration of macrophages (18, 19). Functional studies became possible after the molecular characterization of MIF (20) and its subsequent production as a bioactive, recombinant protein (21). Studies using neutralizing antibodies indicated that MIF plays important roles in T cell activation (22) and as a counter-regulator of glucocorticoid action (23). 1 Correspondence: Department of Pharmacology, University of Bern, Friedbu¨hlstrasse 49, CH-3010 Bern, Switzerland. E-mail:
[email protected] doi: 10.1096/fj.03-0110com
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MIF has also been described as an inhibitor of p53dependent apoptosis in macrophages (24), and a potential intracellular MIF receptor has been identified (25). Here we demonstrate that recombinant MIF delays apoptosis of neutrophils in vitro. To understand how MIF exerts its antiapoptotic function in these cells, intracellular events associated with apoptosis were studied. We report that Bid cleavage occurs during early, and Bax cleavage at later, stages of spontaneous neutrophil apoptosis. Both potential proapoptotic processes are delayed in neutrophils exposed to MIF or G-CSF. Consequently, these two survival factors are able to delay activation of the critical effector caspase-3 and apoptosis in these cells. MATERIALS AND METHODS Antibodies Goat anti-human MIF, control goat IgG, and goat anti-human Bid antibodies were purchased from R&D Systems (distributed by Bu¨ hlmann AG, Basel, Switzerland). Anti-Fas agonistic monoclonal antibody (CH11) was obtained from LabForce (Nunningen, Switzerland) and mouse anti-human -actin from Sigma (Buchs, Switzerland). Rabbit polyclonal anti-human Bax and anti-human caspase-3 antibodies were from Becton Dickinson Biosciences (Basel, Switzerland). Mouse and rabbit horseradish peroxidase (HRP) -conjugated secondary antibodies were purchased from Amersham Pharmacia Biotech (Du¨ bendorf, Switzerland) and goat HRPconjugated secondary antibody was from DAKO (Zug, Switzerland). Anti-cytochrome c monoclonal antibody was obtained from Becton Dickinson Biosciences, anti-Smac polyclonal antisera from Alexis Cooperation (La¨ ufelfingen, Switzerland), and anti-voltage-dependent anion channel (VDAC) monoclonal antibody was from Calbiochem-Novabiochem Corp. (La Jolla, CA, USA). TRITC and FITC-conjugated secondary antibodies were from Jackson ImmunoResearch Laboratories (Milan Analytica, La Roche, Switzerland). AntiCD16 microbeads for eosinophil isolation were from Miltenyi Biotec (Bergisch-Gladbach, Germany). Recombinant MIF MIF protein was produced as a maltose binding protein (MBP) fusion protein in BL21 Escherichia coli cells, which were a kind gift of Dr. D. Beach, Institute of Child Health, London, UK, as described previously (26). MIF-MBP fusion protein was affinity purified by amylose chromatography using amylose resin (New England Biolabs, Allschwil, Switzerland). MIF was cleaved from MBP by the endoprotease factor Xa (New England Biolabs) at room temperature for 48 h. Efficiency of cleavage was checked by Coomassie gel staining. Since MBP or factor Xa had no effect in the assays used in this study, experiments with recombinant MIF were performed immediately after cleavage without further purification. MIF concentrations presented here are total protein concentrations of the mixed MIF/MBP solution. Approximately 25% of the protein concentration in this solution represents MIF. MIF bioassay The functional activity of recombinant MIF was tested using a monocyte migration assay. Migration was investigated using 2222
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48-well chemotaxis chambers with 5 mm pore diameter poly(vinylpyrrolidone)-free polycarbonate membranes as described previously (27). Briefly, wells in the bottom plate were filled with 28 L of RPMI 1640 medium in the presence or absence of the indicated concentrations of recombinant MIF. For MIF inhibition, goat anti-human MIF antibody (20 g/ mL) was preincubated with MIF for 20 min before adding to the well. Peripheral blood mononuclear cells (PBMC) (28) (50 L of 1.8⫻106 cells/mL) were placed in the top wells and incubated for 3 h in humidified air with 5% CO2 at 37°C. Membranes were removed, air-dried, and stained with DiffQuik. Migration of monocytes was evaluated microscopically by counting 10 randomly chosen 400⫻ fields. Cells Blood neutrophils were isolated from healthy individuals as well as patients with cystic fibrosis (CF), and blood eosinophils were purified from patients with atopic dermatitis associated with mild eosinophilia. PBMC were separated by centrifugation on Ficoll-Hypaque (Seromed-Fakola AG, Basel, Switzerland). The lower phase, mainly granulocytes and erythrocytes, was treated with erythrocyte lysis solution (155 mmol/L NH4Cl, 10 mmol/L KHCO3 , and 0.1 mmol/L EDTA, pH 7.3). The resulting cell populations contained mostly neutrophils (29). Eosinophils were further isolated by negative selection using anti-CD16 mAb-labeled microbeads and a magnetic cell separation system (MACS, Miltenyi Biotec) using a column type C attached to a 21-gauge needle in the field of a permanent magnet (9, 29). Cell purity was assessed by staining with Diff-Quik (Baxter, Du¨ dingen, Switzerland) and light microscopy analysis. The resulting populations contained at least 95% neutrophils or 98% eosinophils. Jurkat EC and HeLa cells were both obtained from ATCC (Rockville, MD, USA). Cell cultures Cells were cultured at 1 ⫻ 106/mL in the presence or absence of cytokines and/or antibodies for the indicated times using complete culture medium (RPMI 1640 containing 10% FCS and 200 IU/mL penicillin/100 g/mL streptomycin, all from Life Technologies, Basel, Switzerland). GM-CSF was used at 50 ng/mL, G-CSF (R&D Systems) at 25 ng/mL, and anti-Fas antibody (CH11) at 1 g/mL. The caspase inhibitors (Alexis) N-benzyloxycarbonyl (z)-Val-Ala-Asp (VAD)-fluormethylketone (fmk) and z-Ile-Glu-Thr-Asp (IETD)-fmk were used at 50 M. If not stated otherwise, MIF was used at 7.5 g/mL. For MIF inhibition, goat anti-human MIF antibody was preincubated with MIF for 20 min before stimulation. Determination of cell death and apoptosis Cell death of human neutrophils and eosinophils was assessed by uptake of 1 mol/L ethidium bromide and flow cytometric analysis (FACS Calibur, Becton Dickinson, Heidelberg, Germany) (9, 29, 30). To confirm that cells underwent apoptosis, annexin V staining (31) and DNA fragmentation were analyzed (32). Enzymatic caspase-3 assay Neutrophils were cultured at the conditions indicated, washed with cold PBS, and subsequently lysed in cell lysis buffer (50 mM HEPES pH 7.4/0.1% Chaps/5 mM DTT/0.1 mM EDTA) using a Teflon glass homogenizer (VWR International, Ismaning, Germany) on ice for 10 min. Caspase-3-
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like activity was measured in 10 L cell lysate as enzymatic conversion of the colorimetric substrate Ac-DEVD-pNA at 405 nm according to the manufacturer’s instructions (QuantiZyme caspase 3 cellular activity assay kit; Biomol, Plymouth Meeting, PA, USA). Immunoblotting Neutrophils (1⫻106/mL) were cultured, washed with PBS, and lysed by using RIPA buffer (PBS supplemented with 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS in PBS) supplemented with a protease inhibitor cocktail (Sigma, Buchs, Switzerland) with frequent vortexing on ice for 30 min. In the Bid experiments, the inhibitor cocktail was additionally added to the cell cultures just before harvesting to prevent cleavage during cell lysate preparation. Insoluble material was removed by centrifugation (14,000⫻g, 4°C, 15 min). Equal amounts of cell lysates were mixed with running buffer, boiled, and subjected to gel electrophoresis on Trisglycine gels or NuPage-Gels (Novex, Invitrogen Corp., Groningen, Netherlands). Separated proteins were electrotransferred onto PVDF membranes (Immobilion-P, Millipore, Volketswil, Switzerland). The filters were incubated with the primary antibodies in TBS/0.1% Tween-20/3–5% nonfat dry milk at room temperature for 2 h. The primary antibodies were anti-Bid (1/1000), anti-Bax (1/1000), anti-caspase-3 (1/1000), or anti-MIF (1/1000). For loading controls, stripped filters were incubated with anti--actin antibody (1/15,000). Filters were washed in TBS/0.1% Tween-20 for 30 min and incubated with the appropriate HRP-conjugated secondary antibody (1/2000) at room temperature for 1 h. After another washing step, filters were developed by an ECL technique (ECL-Kit, Amersham) according to the manufacturer’s instructions.
Reverse transcriptase polymerase chain reaction (RT-PCR) Total RNA was isolated from 10 ⫻ 106 freshly purified neutrophils from two control individuals and HeLa cells (positive control) using TRIzol solution (Invitrogen, Basel, Switzerland) according to the manufacturer’s instructions. First-strand synthesis was performed using total RNA, oligo (dT) 15 primer (Promega, distributed by Catalys AG, Wallisellen, Switzerland), and Superscript Reverse Transcriptase (Invitrogen). To determine MIF gene expression, a PCR technique (33) using oligonucleotides that recognize specific sequences with exon 1 and exon 3 was used. Primers for MIF (5⬘-AGT GGT GTC CGA GAA GTC AGG-3⬘ and 5⬘-GCG AAG GTG GAG TTG TTC CAG-3⬘) and -actin (5⬘-CCC CTT CAT TGA CCT CAA CTA C-3⬘ and 5⬘-GAG TCC TTC CAC GAT ACC AAA G-3⬘) amplifications were synthesized (MWG-Biotech AG, Ebersberg, Germany). The MIF primers were designed to span two introns within the cytokine gene. Therefore, amplification of any residual genomic DNA would appear as an unexpected larger PCR product. The cycling parameters for MIF cDNA amplification were as follows: 40 cycles of 94°C for 30 s, 55°C for 60 s, and 72°C for 30 s, followed by 7 min at 72°C. MIF (510 bp) and -actin (450 bp) PCR products were separated on 1% agarose gels and visualized by ethidium bromide staining. Statistical analysis Statistical analysis was performed by using the ANOVA test. If mean levels are presented, se and the number (n) of independent experiments are indicated in each case. A probability value of ⬍0.05 was considered statistically significant.
RESULTS Confocal laser scanning microscopy
MIF delays apoptosis in neutrophils Cytospins were prepared from freshly purified neutrophils and neutrophils were cultured in the presence or absence of MIF, G-CSF, and CH11 antibody for 11 h. Cells were fixed in 4% paraformaldehyde at room temperature for 12 min and washed four times in PBS, pH 7.4. Permeabilization of cells was performed with 0.05% saponin in blocking buffer (3% BSA in PBS) at room temperature for 5 min and with acetone at –20°C for 15 min. To prevent nonspecific binding, slides were incubated in blocking buffer at room temperature for 1 h. Indirect immunostaining for cytochrome c, Smac, and VDAC was performed at room temperature for 1 h using the following primary antibodies: anti-cytochrome c monoclonal antibody (1/100; diluted in blocking buffer), anti-Smac polyclonal antisera (1/100), and anti-VDAC monoclonal antibody (1/200). Incubation with appropriate TRITC- and FITCconjugated secondary antibodies was performed in the dark at room temperature for 1 h. The anti-fading agent Slowfade (Molecular Probes, Eugene, OR, USA) was added and the cells were covered by coverslips. The slides were analyzed by confocal laser scanning microscopy (LSM 510, Carl Zeiss, Heidelberg, Germany) equipped with Arg and HeNe lasers.
The functional activity of recombinant MIF was tested by a bioassay. The migration of monocytes was blocked by MIF in a dose-dependent manner and the inhibitory activity was reversed in the presence of a neutralizing anti-MIF antibody (Table 1). These data suggested that the generated recombinant MIF is functionally active, and we investigated its effect on the apoptosis of neutrophils in vitro. As demonstrated in Fig. 1A, MIF delayed the spontaneous neutrophil death in a doseand time-dependent manner. Maximal inhibition of neutrophil death was reached with 7.5 g/mL; higher concentrations had no further effect. The EC50 of the recombinant MIF used for these experiments was ⬃2.4 g/mL (Fig. 1B). Optimal concentrations of MIF, GM-CSF (not shown), and G-CSF had similar anti-death potencies on neutrophils (Fig. 1C). To demonstrate TABLE 1. Inhibition of monocyte migration by MIF (%)a
MIF immunoassay MIF concentrations were measured in plasma from CF patients and normal control individuals by an immunoassay (Chemicon, distributed by Juro, Lucerne, Switzerland) according to the manufacturer’s recommendations. The assay sensitivity was 1.6 ng/mL. MIF IS A NEUTROPHIL SURVIVAL FACTOR
Medium Anti-MIF a
0
MIF, 5 g/mL
MIF, 10 g/mL
MIF, 20 g/mL
0 0
0 0
39.7 ⫾ 6.2 0
100 0
Values are means ⫾ se of three independent experiments.
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Figure 1. MIF delays neutrophil death in vitro. A) Dose-dependent inhibition of neutrophil death by MIF. This panel is representative of four independent experiments. B) Concentration-effect curve of MIF in 30 h neutrophil cultures (n⫽4). Maximal anti-death effects were seen at 7.5 g/mL. Higher MIF concentrations did not further increase this effect. The EC50 for MIF was ⬃2.4 g/mL. C) MIF (7.5 g/mL, n⫽4), G-CSF (25 ng/mL, n⫽4), and GM-CSF (50 ng/mL, n⫽4; data not shown) had very similar efficacy regarding maintaining neutrophil survival. D) Neutralizing anti-MIF antibody (but not control antibody; data not shown) dosedependently abolished the anti-death effect of MIF but not of G-CSF. This panel shows data from a 27 h neutrophil culture and is representative of 3 independent experiments.
specificity of MIF actions, a neutralizing anti-MIF antibody was used in these assays. As shown in Fig. 1D, this antibody dose-dependently inhibited the survival effect of MIF but not of G-CSF. The anti-MIF antibody had no effect on neutrophil viability when used in the absence of cytokine stimulation (Fig. 1D) nor did it block GM-CSF-mediated neutrophil survival (data not shown). Moreover, a control antibody had no effect and heating of MIF resulted in loss of its functional activity (data not shown). MIF effects were not blocked when optimal concentrations of polymyxin B were added, excluding any potential nonspecific effect via LPS (data not shown). A similar anti-death effect of MIF was observed in eosinophils, but did not reach statistical significance and was much lower than with GM-CSF (Fig. 2). We next investigated whether the anti-death effect mediated by MIF was due to inhibition of apoptosis. MIF reduced redistribution of phosphatidyl serine (PS), a characteristic feature of apoptotic neutrophils (31), with the same efficacy as G-CSF did (Fig. 3A). Moreover, MIF inhibited DNA fragmentation as assessed by staining of DNA with propidium iodide and flow cytometric analysis (Fig. 3B). Anti-MIF antibodies neutralized MIF survival activities, further demonstrating that the observed effects were indeed MIF mediated. These data suggest that MIF delays neutrophil apoptosis in vitro.
by immunoblotting and an enzymatic assay. Freshly isolated blood neutrophils expressed the 32 kDa proform of caspase-3 (Fig. 4A). Culturing the cells for 8 h resulted in the appearance of the active 17 kDa form and cleavage of caspase-3 was accelerated in anti-Fas receptor antibody-treated neutrophils. In contrast, MIF and G-CSF prevented the occurrence of the 17 kDa form, suggesting that both survival cytokines blocked caspase-3 processing at this time point. Furthermore, both MIF and G-CSF suppressed caspase-3-like DEVDase activity in neutrophils, whereas Fas receptor activation enhanced the enzymatic activity (Fig. 4B).
MIF inhibits caspase-3 activation in neutrophils
Figure 2. MIF has only marginal effects on eosinophil death in vitro. MIF (7.5 g/mL, n⫽3) slightly inhibited spontaneous eosinophil death in 50 h cultures and its efficacy was lower compared with GM-CSF (50 ng/mL, n⫽3). *P⬍0.05; **P⬍0.01.
Caspase-3 is a critical effector caspase in neutrophil apoptosis (14 –16). We investigated caspase-3 activation 2224
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Cleavage of Bid into the 15 kDa fragment was blocked by caspase-8 and pan-caspase inhibitors (Fig. 5B). However, caspase inhibition allowed cleavage into the 18 kDa fragment, implying that Bid might also be a target of other intracellular proteases in neutrophils. Bax is another proapoptotic member of the Bcl-2 family highly expressed in neutrophils (5) and appears to be critical for the release of proapoptotic factors from mitochondria (37, 38). Spontaneous neutrophil apoptosis was associated with cleavage of Bax into an 18 kDa fragment (Fig. 5C, lower right panel), suggesting the activation of calpain in this process (39, 40). In contrast to Bid (Fig. 5C, upper left panel), cleavage of Bax was not detectable in short-term neutrophil cultures (⬍ 6 h) (Fig. 5C, lower left panel). However, Bax cleavage was consistently seen in neutrophils that had been cultured for ⬎8 h (data not shown). Whereas in 18 h neutrophil cultures the 18 kDa form of Bax was clearly visible (Fig. 5C, lower right panel), only traces of truncated Bid were detectable at this time (Fig. 5C, upper right panel), possibly due to its rapid degrada-
Figure 3. MIF delays neutrophil apoptosis in vitro. A) MIF (7.5 g/mL) reduced PS redistribution in neutrophil membranes (8 h cultures, n⫽4). Right: Representative examples of flow cytometric analysis. B) MIF reduced the formation of hypoploid DNA in neutrophils (30 h cultures, n⫽4). Right: Representative examples of flow cytometric analysis. **P⬍0.01.
Thus, the presence of the 17 kDa fragment within neutrophil lysates correlated well with an increased enzymatic caspase activity and both were blocked by MIF, further suggesting that this cytokine represents a novel antiapoptotic factor for neutrophils. Bid and Bax cleavage during neutrophil apoptosis: inhibition by MIF Additional experiments were performed to elucidate the mechanisms of MIF-mediated inactivation of caspase-3 in neutrophils. The proapoptotic Bcl-2 family member Bid demonstrated a detectable 15 kDa cleavage product in neutrophils after a short-term culture of 4.5 h (Fig. 5A), implicating caspase-8 activation and the involvement of mitochondria in the process of spontaneous apoptosis (34, 35). An intermediate 18 kDa fragment was also visible. MIF and G-CSF prevented, and stimulation with agonistic anti-Fas receptor antibodies accelerated, Bid cleavage at this time point. In these experiments, Fas-sensitive and -activated Jurkat cells served as positive controls. We observed that full-length neutrophilic Bid migrates in Western blots as a doublet. The same observation was made when mouse Bid was overexpressed in mouse cell lines (36). MIF IS A NEUTROPHIL SURVIVAL FACTOR
Figure 4. MIF blocks caspase-3 processing and activation in cultured neutrophils. A) Neutrophils were cultured in the presence or absence of cytokines for 8 h. Spontaneous apoptosis (Medium) was associated with the occurrence of a 17 kDa fragment of caspase-3. MIF and G-CSF prevented, whereas Fas receptor activation (CH11) accelerated this process. This immunoblot is representative of 4 independent experiments. B) Inhibition of the enzymatic activity was detectable in neutrophils cultured with MIF or G-CSF compared with cells cultured in medium without cytokine support (8 h cultures). In contrast, Fas receptor activation resulted in a clear increase of caspase-3-like activity. n⫽5; *P⬍0.05; **P⬍0.01. 2225
fluorescence immunostaining and microscopic analysis. Cytochrome c and Smac colocalized to mitochondria and a punctate pattern was observed in freshly isolated neutrophils (Fig. 6A). Cell culturing of normal neutrophils for 11 h demonstrated evidence for cytochrome c and Smac release (⫽diffuse pattern) in a subgroup of cells. MIF and G-CSF preserved the punctate pattern in the majority of the cells, whereas Fas receptor-activated neutrophils revealed a diffuse cytosolic staining pattern of the two proteins in almost all cells. Same results were observed in 8 h neutrophil cultures (data not shown). No difference was observed when cytochrome c and Smac stainings were compared. A statistical analysis of the experiments is given in Fig. 6B. MIF and G-CSF had the same capacity to block the transition into a diffuse cytosolic staining pattern associated with neutrophil apoptosis. Neutrophils express MIF mRNA and store endogenous MIF
Figure 5. MIF prevents Bid and Bax cleavage in cultured neutrophils. A) Bid cleavage into its active 15 kDa fragment was associated with early spontaneous and Fas receptormediated neutrophil apoptosis (4.5 h cultures). MIF and G-CSF prevented Bid cleavage. B) The caspase inhibitors z-VAD-fmk and z-IETD-fmk also prevented Bid cleavage (5 h cultures). C) In contrast to Bid, Bax was not cleaved in 6 h neutrophil cultures. In 18 h cultures, however, spontaneous apoptosis was associated with the occurrence of an 18 kDa fragment of Bax. Cleavage of Bax was prevented by MIF and G-GSF but accelerated by Fas receptor activation (CH11). In the case of Bid, a slightly higher band was maintained in CH11-treated cells, whereas the truncated 15 kDa fragment was no longer seen at this time point. Results are representative of at least 3 independent experiments.
tion by the ubiquitin proteolytic system (41). MIF and G-CSF partially blocked Bax cleavage. In contrast, Fas receptor stimulation resulted in accelerated cleavage of 21 kDa Bax (Fig. 5C, lower right panel). MIF inhibits the mitochondrial release of cytochrome c and Smac Activation of Bid and Bax during spontaneous and Fas receptor-mediated apoptosis suggested that the intracellular death pathways in neutrophils involve mitochondria. We therefore analyzed the mitochondrial release of two proapoptotic factors in cultured neutrophils in the presence and absence of MIF or G-CSF by 2226
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We examined the expression of MIF by RT-PCR in freshly purified blood neutrophils from three healthy individuals. Detectable amounts of MIF mRNA were seen in all neutrophil populations as well as in HeLa cells (Fig. 7A). To determine whether human blood granulocytes contain MIF protein, lysates of freshly purified neutrophils and eosinophils were used for immunoblotting. Blood neutrophils and eosinophils expressed MIF protein (Fig. 7B). Recombinant MIF was resolved in the same gel and served as a positive control. Moreover, cell lysates from HeLa cells served as positive controls (25). Increased MIF levels in blood and delayed neutrophil apoptosis associated with CF MIF levels in plasma were measured in 6 CF patients with permanent bacterial infections and 11 healthy control individuals. Mean MIF levels were ⬃23-fold higher in patients with CF than in control persons (Fig. 8A), suggesting that neutrophils are exposed to increased MIF concentrations in this particular disease. Moreover, in agreement with previously published work (5), we observed delayed apoptosis of blood neutrophils derived from CF patients in vitro (Fig. 8B).
DISCUSSION We investigated apoptotic pathways in granulocytes after stimulation with survival and death factors. The following new findings are reported. 1) MIF is a survival factor for neutrophils, which also express this cytokine. 2) Spontaneous and Fas receptor-mediated neutrophil apoptosis is associated with Bid and Bax cleavage, presumably leading to the mitochondrial release of the proapoptotic factors cytochrome c and Smac and subsequent caspase-3 activation. 3) MIF and G-CSF appear to exert their antiapoptotic functions in neutrophils by
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Figure 6. MIF prevents the mitochondrial release of cytochrome c and Smac. Freshly isolated neutrophils from 11 h cultures were investigated by confocal microscopy. A) Cytochrome c and Smac colocalized with VDAC, demonstrating that they are mitochondrial proteins in freshly isolated neutrophils. MIF and G-CSF preserved the punctate pattern in cultured neutrophils, whereas we observed diffuse staining in the absence of cytokine support or after CH11 treatment. No detectable staining was observed by using isotype-matched control antibodies (data not shown). The bars in the two upper panels represent 10 m. Results are representative of 3 independent experiments. Same results were observed in 8 h cultures. B) Quantitative and statistical analysis of the experiments shown in panel A. **P⬍0.01.
inhibiting proapoptotic events proximal to mitochondria. Bid is a BH3-only proapoptotic protein of the Bcl-2 family that transduces death stimuli from the cell surface to the central death machinery. Bid is cleaved by caspase-8 (34, 35), an event that is critical for death receptor-mediated apoptosis, at least in cells in which the mitochondrial death pathway is required. The 15 kDa carboxyl-terminal fragment of Bid is believed to translocate to mitochondria and to trigger cytochrome c release (34 –36). Early cleavage of Bid and its inhibition by a caspase-8 inhibitor are consistent with previous findings from our laboratory suggesting a critical MIF IS A NEUTROPHIL SURVIVAL FACTOR
role of caspase-8 in the process of spontaneous neutrophil apoptosis in the absence of death receptor stimulation (16). However, it remains to be determined how caspase-8 is activated in neutrophils undergoing spontaneous apoptosis. In Fas receptor-activated neutrophils, the formation of truncated Bid is accelerated, presumably due to additional caspase-8 activation. Moreover, after Fas receptor stimulation we detected a slightly higher band in the Bid immunoblot that appeared to be resistant to cleavage. This band likely represents phosphorylated Bid, which was reported in cells after experimental Bid overexpression (36). Our observation in neutrophils is the first evidence that 2227
Figure 7. Neutrophils express MIF. A) RT-PCR. Three neutrophil populations derived from normal control individuals demonstrated evidence for MIF mRNA expression. Positive control was cDNA from HeLa cells. Negative control was done without template cDNA. B) Immunoblotting. Freshly isolated blood neutrophils and eosinophils from different donors were analyzed by immunoblotting. As controls, recombinant MIF (125 ng) and lysates from HeLa cells were electrophoresed and transferred.
endogenous Bid exists as doublet in primary and nontransfected cells. Bax has been identified as a target of calpain in HL-60 (39) and Jurkat cells (40). Whereas in HL-60 cells Bax cleavage into an 18 kDa fragment occurred several hours after cleavage of poly(ADP-ribose) polymerase (PARP) and retinoblastoma protein (RB), the results reported in Jurkat cells suggested that the generation of the 18 kDa Bax fragment is an early event required for the induction of apoptosis via mitochondria. We observed Bax cleavage in neutrophil populations undergoing apoptosis, suggesting a potential role of calpain in proximal apoptosis pathways in these cells. Bax cleavage correlated with the numbers of apoptotic cells, and Fas activation resulted in increased amounts of truncated Bax, which may have enhanced proapoptotic potential (40). We are currently investigating the functional importance of Bax cleavage. It remains to be determined whether Bid or Bax or both molecules together are functionally relevant for mitochondrial activation in neutrophils. The question of whether Bid cleavage somehow triggers truncation of Bax or, alternatively, whether both events are initiated in a rather parallel manner, needs to be addressed in future studies. There is evidence that both spontaneous and Fas receptor-mediated neutrophil apoptosis require an intact mitochondrial pathway (6, 42). Moreover, the antiapoptotic effect of G-CSF on neutrophils was recently investigated and G-CSF was characterized as an agent that prevents translocation of Bax from the 2228
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cytosol to the mitochondria (6). In agreement with this study, we localized the antiapoptotic effect of G-CSF proximal to mitochondria. Like G-CSF, MIF prevented Bid and Bax truncation, suggesting that both cytokines exert their antiapoptotic effects in neutrophils by the same or very similar molecular mechanisms, which may involve negative regulatory effects on caspase and calpain functions. Bid and Bax can independently (43) or synergistically (44) trigger the mitochondrial membrane, leading to the release of cytochrome c into the cytosol. Cytochrome c forms a complex with Apaf-1 and caspase-9 that, in the presence of dATP, leads to the activation of caspase-3 (45). Smac is another proapoptotic factor released from mitochondria that inactivates members of the inhibitors of apoptosis protein (IAP) family (46, 47). We observed both events in neutrophils undergoing either spontaneous or Fas receptor-mediated apoptosis, demonstrating the presence and importance of mitochondria in these cells. Consistent with the assumption that MIF and G-CSF act proximal to mitochondria, both cytokines inhibited the release of cytochrome c and Smac from mitochondria. Whether cytochrome c and Smac act independently or synergistically in the formation of active caspase-3 in neutrophils has yet not been determined. Nevertheless, since MIF or G-CSF blocked the release of both potential
Figure 8. Increased MIF plasma levels are associated with delayed neutrophil death of CF blood neutrophils. A) Plasma MIF levels were measured by an immunoassay. Increased plasma MIF levels from CF patients (n⫽6) were observed compared with normal control individuals (n⫽11). B) Blood neutrophils of CF-patients (n⫽10) and control individuals (n⫽7) were cultured for 24 h and cell death determined. Delayed death of CF neutrophils was also seen in 48 h and 72 h cultures (data not shown). **P⬍0.01.
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caspase-3 activators, it is clear that both survival factors inhibited caspase-3 activation by preserving intact mitochondria. In this study, we characterized MIF as a novel survival factor for neutrophils and eosinophils. Although there are other studies where MIF was used in concentrations up to 10 g/mL (48), it should be noted that the concentrations needed to achieve optimal anti-death effects were ⬃10-fold higher than other reported biological MIF activities (26). Since we required similar high MIF concentrations for blocking of monocyte migration in an in vitro bioassay and since we generated MIF in a bacterial and not in an eukaryotic host, it is possible that our recombinant MIF was not fully functionally active. Nevertheless, the effects of MIF were specific and dose dependent and were demonstrated in different assays, leaving no doubt that MIF is a novel antiapoptotic factor for granulocytes. In experimental models, anti-MIF neutralizing antibodies protected from lethality in endotoxic shock and reduced the accumulation of neutrophils in the lung (49). Similarly, MIF⫺/⫺ mice were resistant to endotoxic shock and demonstrated markedly reduced neutrophil numbers in the lungs after instillation of bacteria compared with wild-type mice (50). The findings of this study suggest a role for MIF within the pathogenesis of several human inflammatory disorders. For instance, delayed apoptosis of blood neutrophils from CF patients was associated with 23-fold increased levels of MIF in plasma, pointing to the possibility that MIF, in addition to G-CSF and GM-CSF (5), acts as a survival factor for neutrophils in these patients. Similar mechanisms may occur in patients with sepsis (51) and acute respiratory distress syndrome (52). Moreover, we observed that neutrophils constitutively store endogenous MIF, implying that under certain conditions neutrophils promote their own survival via MIF release. Besides neutrophils, monocytes and macrophages are known to contain large amounts of MIF, which is released upon Toll-like receptor activation, e.g., due to LPS stimulation (53). Taken together, MIF is released from cells of the innate immune system in bacterial infectious diseases and may contribute to the accumulation of neutrophils at inflammatory sites by inhibition of apoptosis. We are grateful to Dr. D. Beach, Institute of Child Health, London, UK, for E. coli expressing the MBP-MIF fusion gene. We also thank Drs. P. Kerai and A. Carnieiro (Wolfson Institute, University of London, London) for valuable hints regarding the purification of MIF, Dr. A. Walz (Theodor Kocher Institute, University of Bern) for help with the MIF bioassay, Dr. C. Mu¨ ller (Institute of Pathology, University of Bern) for discussions during the study, and Dr. S. Russmann (Institute of Clinical Pharmacology, University of Bern) for the organization of blood samples from healthy individuals. Dr. M. H. Scho¨ ni (Department of Pediatrics, University of Bern) supported the study by providing blood samples from patients with CF. This work was supported by grants from the Swiss National Science Foundation (grant no. 31-58916.99 and 31-68449.02), the Bernische Krebsliga, Bern, and OPOFoundation, Zurich, Switzerland. MIF IS A NEUTROPHIL SURVIVAL FACTOR
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Received for publication January 30, 2003. Accepted for publication August 5, 2003.
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