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* Departments of Pharmacology,
Pediatrics, and
# Dermatology, University of Bern, Bern, Switzerland
1Correspondence: Department of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland. E-mail: hus{at}pki.unibe.ch
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: inflammation • MIF • mitochondria
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. 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 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
15
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)
. MIF has also been described as an inhibitor of p53-dependent 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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 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 µL 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 µg/mL) was preincubated with MIF for 20 min before adding to the well. Peripheral blood mononuclear cells (PBMC) (28)
(50 µL of 1.8x106 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 Diff-Quik. Migration of monocytes was evaluated microscopically by counting 10 randomly chosen 400x 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, Düdingen, 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 x 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 µg/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 µg/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 µM. If not stated otherwise, MIF was used at 7.5 µg/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 µmol/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-like activity was measured in 10 µL 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 (1x106/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 (14,000xg, 4°C, 15 min). Equal amounts of cell lysates were mixed with running buffer, boiled, and subjected to gel electrophoresis on Tris-glycine 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-ß-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.
Confocal laser scanning microscopy
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 FITC-conjugated 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.
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.
Reverse transcriptase polymerase chain reaction (RT-PCR)
Total RNA was isolated from 10 x 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 ß-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 ß-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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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. 1
A, MIF delayed the spontaneous neutrophil death in a dose- and time-dependent manner. Maximal inhibition of neutrophil death was reached with 7.5 µg/mL; higher concentrations had no further effect. The EC50 of the recombinant MIF used for these experiments was 
2.4 µg/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 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
).
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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. 3
A). 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.
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MIF inhibits caspase-3 activation in neutrophils
Caspase-3 is a critical effector caspase in neutrophil apoptosis (14
15
16)
. We investigated caspase-3 activation by immunoblotting and an enzymatic assay. Freshly isolated blood neutrophils expressed the 32 kDa proform of caspase-3 (Fig. 4
A). 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
). 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.
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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. 5
A), 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)
. 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.
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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 degradation 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 fluorescence immunostaining and microscopic analysis. Cytochrome c and Smac colocalized to mitochondria and a punctate pattern was observed in freshly isolated neutrophils (Fig. 6
A). 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.
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Neutrophils express MIF mRNA and store endogenous MIF
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. 7
A). 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)
.
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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. 8
A), 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
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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
35
36)
. Early cleavage of Bid and its inhibition by a caspase-8 inhibitor are consistent with previous findings from our laboratory suggesting a critical 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 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 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 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 µg/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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication January 30, 2003. Accepted for publication August 5, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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