(Received for publication, October 25, 1996, and in revised form, January 10, 1997)
From the Institute of Experimental Dermatology and
§ Department of Pediatrics, University of Münster,
48129 Münster, Germany and
Department of Dermatology,
University of Würzburg, 97080 Würzburg, Germany
Myeloid-related protein (MRP) 8 and MRP14, two
members of the S100 family expressed in myelomonocytic cells, have been
ascribed some extracellular functions, e.g.
antimicrobial, cytostatic, and chemotactic activities. Since S100
proteins lack structural requirements for secretion via the classical
endoplasmic reticulum/Golgi route, the process of secretion is unclear.
We now demonstrate the specific, energy-dependent release
of MRP8 and MRP14 by human monocytes after activation of protein kinase
C. This secretory process is not blocked by inhibitors of vesicular
traffic through the endoplasmic reticulum and Golgi, and comparative
studies on tumor necrosis factor- and interleukin-1
indicate that
MRP8 and MRP14 follow neither the classical nor the
interleukin-1-like alternative route of secretion. Inhibition
by microtubule-depolymerizing agents revealed that MRP8/MRP14 secretion
requires an intact tubulin network. Accordingly, upon initiation of
MRP8/MRP14 secretion, immunofluorescence microscopy showed a
co-localization of both proteins with tubulin filaments. Release of
MRP8 and MRP14 is associated with down-regulation of their de
novo synthesis, suggesting that extracellular signaling via
MRP8/MRP14 is restricted to distinct differentiation stages of
monocytes. Our data provide evidence that the S100 proteins MRP8 and
MRP14 are secreted after activation of protein kinase C via a novel
pathway requiring an intact microtubule network.
Myeloid-related protein-8 (MRP8;
S100A8)1 and MRP14 (S100A9) are two
calcium-binding proteins of the S100 family (1-3), which has grown to
be one of the largest subfamilies of EF-hand proteins (4). Members of
this family are defined by their homologies to two calcium-binding
proteins highly enriched in nervous tissue, S100 and S100
. S100
proteins are characterized by the presence of two calcium-binding sites
of the EF-hand-type, the N-terminal of which differs from the conserved
EF motive by two additional amino acids. They have a relatively small
molecular mass of around 10 kDa and, in contrast to other
calcium-binding proteins such as calmodulin, show tissue-specific
expression patterns. S100 proteins play a role in cell differentiation,
cell cycle progression, regulation of kinase activities, and
cytoskeletal-membrane interactions (4, 5). In addition, extracellular
functions have been reported for distinct S100 proteins; S100
, the
prototypic member of this family, can be found as an extracellular
protein inducing neurite extension (5, 6). S100L (S100A2) is
chemoattractive for eosinophils (7), whereas psoriasin (S100A7)
exhibits chemotactic activity for neutrophils and CD4+
lymphocytes (8).
MRP8 and MRP14 are expressed at high concentrations by granulocytes and
during early differentiation stages of monocytes but are absent in
lymphocytes and mature tissue macrophages (9-12). Down-regulation of
MRP8 and MRP14 expression in monocytes involves a calcium-mediated
suppressor mechanism (13). Phagocytes express MRP8 and MRP14 under
multiple inflammatory conditions, e.g. during rheumatoid
arthritis, allograft rejections, or inflammatory bowel diseases (9, 14,
15). Inflammatory disorders as chronic bronchitis, cystic fibrosis, or
rheumatoid arthritis are associated with elevated serum levels of MRP8
and MRP14 (16, 17). The close correlation between serum levels and
disease activity led to the assumption that MRP8 and MRP14 are released
from leukocytes during inflammatory events (4, 5, 16), e.g.
during transendothelial diapedesis (11). The mechanism of release,
either after cell death or by an active secretory process, has yet not
been elucidated. In recent years several studies reported extracellular
functions of MRP8 and MRP14, including antimicrobial, cytostatic, or
chemotactic activities (18-20), thus favoring an active mechanism of
secretion. However, neither MRP8 and MRP14 nor any other S100 protein
has a signal sequence for secretion via the classical ER/Golgi route. They therefore resemble cytokines such as interleukin-1 (IL-1
) and basic fibroblast growth factor, which are released into the extracellular space via a so-called alternative pathway of secretion (21). However, it is not known whether all nonclassically secreted proteins use the same mechanisms of release.
Since the pathway of S100 protein release has not yet been examined (4), we intended to elucidate the molecular mechanisms associated with the release of MRP8 and MRP14 by human monocytes. Here we provide evidence that MRP8 and MRP14 are secreted after activation of protein kinase C via a novel pathway requiring an intact microtubule network.
Human peripheral blood leukocytes were obtained from buffy coats which arise during preparation of packed red blood cell concentrates. Monocytes were isolated by Ficoll-Paque and Percoll (Pharmacia, Freiburg, FRG) density gradient centrifugation or by leukapheresis of individual donors using a cell separator CS 3000 plus Omnix (Baxter, Unterschleißheim, FRG) and were cultured for 1-3 days in Teflon bags as previously reported (9).
AntibodiesMRP8 and MRP14 were detected by
noncross-reactive affinity-purified rabbit antisera (a-MRP8, a-MRP14)
the monospecifity of which was evaluated by immunoreactivity against
recombinant proteins and transfected cell lines as described previously
(1, 22). For detection of MRP8/MRP14 heterodimers mAb 27E10 was
employed which recognizes only the heterodimers, but not MRP8 or MRP14 monomers (9, 23). Vimentin intermediate filaments were detected by
mouse mAb V9 (Dianova, Hamburg, FRG), actin filaments by FITC-labeled phalloidin (Sigma, Deisenhofen, FRG) and microtubuli by mAb TUB 2.1 against -tubulin (Sigma). mAb H21 directed against S100 protein p11
(S100A10) (24) was kindly provided by Dr. V. Gerke, University of
Münster. For controls, monoclonal mouse IgG1
(Dianova) and polyclonal rabbit IgG (Pharmacia) of irrelevant specifity
were employed. Affinity-purified goat-anti-mouse or goat-anti-rabbit secondary antibodies conjugated with either Cy3, Texas Red, or FITC
were obtained from Dianova. Protein G Sepharose 4 Fast Flow was
purchased from Pharmacia.
For stimulation of
monocytes lipopolysaccharide (1 µg/ml, Sigma), granulocyte-monocyte
colony-stimulating factor (GM-CSF; 100 units/ml; Sigma), IL-1 (10 units/ml), IL-4 (5 units/ml;), IL-6 (100 units/ml; all from Calbiochem,
Bad Soden, FRG), interferon-
(50 units/ml; Boehringer Mannheim,
Mannheim, FRG), 4
-phorbol 12-myristate 13-acetate (PMA, 1-100
nM), 4a
-phorbol 9-myristate 9a-acetate (4aPMA, 1-100
nM), 12-deoxyphorbol 13-phenylacetate 20-acetate (dPPA,
1-100 nM), pertussis toxin (100 ng/ml), cholera toxin (100 ng/ml), forskolin (100 nM), and dibutyryl cAMP
(Bt2cAMP, 10 µM, all from Sigma) were
employed.
Inhibitory effects on PMA-induced MRP8/MRP14 secretion were analyzed by concomitant application of 10 nM PMA and either staurosporine (0.2-50 nM), H7 (0.1-30 µM), HA1004 (0.1-200 µM, all from Calbiochem), cycloheximide (10 µg/ml), carbonyl cyanide chlorphenylhydrazone (CCCP, 10 µM), dinitrophenol (1 mM), monensin (10 µg/ml, all from Sigma), brefeldin A (0.5 µg/ml, Calbiochem), nocodazole (2 µg/ml), colchicine (5 µg/ml), demecolcine (1 µM), or cytochalasin B (5 µg/ml, all from Sigma). Cell viability was assayed by trypan blue exclusion staining, by propidium iodide labeling with subsequent flow cytometry, or by determination of lactate dehydrogenase activity in the medium at the end of exposure periods as described earlier (22). Viability was found to be higher than 95% in all experiments. Lactate dehydrogenase activity in the medium differed maximally up to 20% between controls and the various treatment procedures.
Enzyme-linked Immunosorbent Assay (ELISA) for MRP8/MRP14, Tumor Necrosis Factor-The MRP8/MRP14 content in the supernatants of cultured monocytes was quantified by a sandwich ELISA as described earlier (16, 22). The ELISA was calibrated with recombinant MRP14 in concentrations ranging from 1 to 1000 ng/ml. The sensitivity was less than 2.5 ng/ml.
For detection of secreted IL-1 and TNF-
, Biotrak sandwich ELISA
systems were obtained from Amersham-Buchler (Braunschweig, FRG) and
employed according to the manufacturer's instructions. The
sensitivities were less than 5 and 1 pg/ml for TNF-
and IL-1
, respectively.
Monocytes of culture day 1 were stimulated as described above and processed for flow cytometry. Immunostaining procedures were performed as reported earlier employing mAb 27E10 (9, 23). Surface expression was analyzed employing a FACScan (Becton Dickinson, Heidelberg, FRG) equipped with Lysis II software.
Metabolic Labeling and ImmunoprecipitationMonocytes of culture day 1 were harvested, washed, and preincubated in modified Eagle's medium without methionine (Life Technologies, Inc.) at a density of 5 × 107 cells/ml. Monocytes were then labeled by adding 250 µCi/ml [35S]methionine (Amersham-Buchler) to the same medium for 2 h. Medium was removed and replaced by McCoy's 5A medium supplemented with 15% fetal calf serum. Cells (2 × 107) were treated for another 4 h with either medium, 10 nM PMA, or 10 nM PMA plus 20 nM staurosporine. Thereafter, supernatants were collected, and cells were washed and lysed in phosphate-buffered saline containing 1% Nonidet P-40 and 2 mM phenylmethylsulfonyl fluoride (Sigma). Supernatants and lysates were prepared for immunoprecipitation as described earlier (13) using monospecific affinity-purified antisera against MRP8 or MRP14, mAb 27E10, mAb H21 against p11, or nonspecific isotype-matched antibodies as a control. Samples were separated by 15% SDS-polyacrylamide gel electrophoresis under reducing conditions. The relative amounts of MRP8 and MRP14 monomers were determined densitometrically by scanning of autoradiography bands using a Fast Scan supplied with Image Quant software (Molecular Dynamics, Sunny Vale, CA).
Immunofluorescence MicroscopyMonocytes cultured for 3 days
on fibronectin (Becton Dickinson)-coated Lab-Tec chamber slides (Nunc,
Wiesbaden, FRG) were either left untreated or incubated for 4 h
with 10 nM PMA. In some experiments 5 µg/ml colchicine, 2 µg/ml nocodazole, 1 µM demecolcine, or 5 µg/ml
cytochalasin B was added during the last hour of the incubation period.
Cells were washed with phosphate-buffered saline, permeabilized by 10 mM Hepes, pH 6.8, 100 mM KCl, 3 mM MgCl2, 200 mM saccharose, 1 mM
phenylmethylsulfonyl fluoride (CS buffer) containing 0.5% Triton X-100
for 2 min, washed twice in CS buffer for 5 min, subsequently fixed with
3.7% formaldehyde in phosphate-buffered saline for 4 min and methanol
for 6 min at 20 °C, and processed for single- or double-labeling
immunofluorescence as described earlier (25) using a-MRP8, a-MRP14, mAb
27E10, phalloidin-FITC, mAb TUB 2.1 against
-tubulin, and mAb V9
against vimentin as primary antibodies. For double-labeling experiments staining of MRP8 or MRP14 with a polyclonal rabbit serum was followed by detection of cytoskeletal components employing mAbs from mice. No
cross-reactivity or spillover was detected in control experiments after
omitting specific antibodies or replacing them by isotype-matched control antibodies of irrelevant specifity. Fluorescence stainings were
analyzed on a Zeiss photomicroscope.
Total RNA of monocytes was prepared according to the guanidine hydrochloride method (13). Filters were hybridized with cDNA probes specific for MRP8 or MRP14 which were labeled with 32P by a random primer method (Multiprime DNA labeling system; Amersham). Membranes were reprobed with cDNAs described above with stripping the blots in between employing 0.1% SDS at 95 °C. Autoradiographic bands were quantified by densitometrically scanning. Data obtained from MRP8 or MRP14 mRNA bands were normalized to the corresponding 18 S rRNA bands.
StatisticsThe U test according to Mann-Whitney (for values without normal distribution) was performed to determine significant differences in MRP8/MRP14 secretion. Values of p > 0.05 were considered not to be significant.
To study the regulation of MRP8 and MRP14 secretion,
monocytes were cultured for 1 day and subsequently stimulated with
GM-CSF, IL-1, IL-4, IL-6, interferon-
, or lipopolysaccharide for
4 h. Thereafter, supernatants were analyzed for MRP8/MRP14 content by ELISA. As shown in Fig. 1A, GM-CSF,
IL-1
, and lipopolysaccharide induced significant MRP8/MRP14 release
by human monocytes. MRP8 and MRP14 have earlier been demonstrated to
assemble to noncovalently linked di-, tri-, and tetraheteromeric
complexes (26, 27). A sandwich ELISA using a mAb against MRP14 does not
provide information about the stoichiometric ratio of heteromeric
complexes; therefore, the data presented refer to MRP8/MRP14
(MRP8/14). The exact molecular ratio of secreted MRP8
and MRP14 was determined by metabolic labeling (see below). To consider
nonspecific MRP8 and MRP14 release due to cell lysis, lactate
dehydrogenase activity in the supernatant was determined concomitantly.
Specific release was then presented as the ratio of MRP:lactate
dehydrogenase (nanograms/unit).
To analyze intracellular signaling pathways resulting in MRP8/MRP14
release, monocytes were exposed to PMA, Bt2cAMP,
choleratoxin, pertussis toxin, or forskolin. ELISA assays revealed a
strong induction of MRP8/MRP14 release by PMA. Modulators of
intracellular cAMP had no effect (Fig. 1B). PMA effects on
MRP8/MRP14 release could be inhibited by the protein kinase inhibitors
H-7 (inhibitory concentration (IC) 50 = 8 µM), HA-1004 (IC50 = 18 µM),
and staurosporine (IC50 = 2 nM);
IC50 of these inhibitors resembled their
Ki regarding protein kinase C, thus confirming
involvement of the latter (Fig. 2A). The PMA
analogue 4aPMA which does not exhibit intrinsic activity regarding
protein kinase C activation (28) had no effect on MRP8/MRP14 release
even at 10-fold higher concentrations (Fig. 2B). dPPA, an
agonist of the protein kinase C isotype 1 (29), the most
abundant protein kinase C isoform in monocytes (30), did not affect
MRP8/MRP14 release, indicating a protein kinase C subtype-specific
pathway of intracellular signaling. PMA- and cytokine-induced release
of MRP8/MRP14 was not associated with translocation of the latter to
the cell surface as determined by flow cytometry (data not shown).
Identification of MRP8 and MRP14 Subunits in Monocyte Supernatants
To study potential complex assembly of released
MRP8/MRP14, supernatants of monocytes exposed to
[35S]methionine and stimulated with PMA were analyzed by
immunoprecipitation, subsequent SDS-polyacrylamide gel
electrophoresis, and autoradiography. Using a-MRP8, a-MRP14,
or mAb 27E10 against the complex of MRP8/MRP14, all antibodies
precipitated a similar pattern of MRP8 and MRP14, indicating that
complexes of both represent the predominant extracellular form (Fig. 3,
A-D). Quantification of precipitated MRP8
and MRP14 by densitometrical scanning revealed a ratio of MRP8:MRP14 of 1:3 which reflects the relative methionine content of both proteins (2 in MRP8 and 6 in MRP14). Furthermore, there was no difference between
the intracellular and extracellular MRP8:MRP14 ratio, indicating that
both are released at similar rates. To exclude nonspecific release of
MRP8 and MRP14 into the supernatant, parallel immunoprecipitation
experiments were performed for p11, another member of the S100 family
expressed by myelomonocytic cells (4). As demonstrated in Fig.
3E, p11 expression was induced by exposure to PMA. In
contrast to the strong [35S]methionine incorporation into
intracellular p11, no p11 could be detected in the supernatant of
PMA-treated monocytes. This observation adds to the evidence that
PMA-stimulated cells were viable and that MRP8 and MRP14 were
selectively released.
Molecular Mechanisms of MRP8/MRP14 Secretion
In the next set
of experiments we analyzed molecular mechanisms involved in PMA-induced
MRP8/MRP14 release. To evaluate dependence of PMA-induced MRP8/MRP14
secretion on de novo protein synthesis, monocytes were
exposed to cycloheximide. Treatment with this protein synthesis
inhibitor did not affect the amount of MRP8 and MRP14 in the
supernatant (Fig. 4A). In contrast,
concomitant incubation with PMA and either one of the two inhibitors of
cellular energy metabolism, dinitrophenol or CCCP, led to a significant
inhibition of MRP8/MRP14 release, indicating MRP secretion as being an
energy-dependent active process (Fig. 4A). The
intracellular route of MRP8 and MRP14 is independent from the classical
ER/Golgi pathway as demonstrated by the inability of monensin and
brefeldin A to inhibit PMA-induced release of these proteins (Fig.
4A). Inhibition of tubulin polymerization by nocodazole,
colchicine, or demecolcine, however, significantly suppressed release
of MRP8/MRP14 into the supernatants (Fig. 4B). In contrast,
perturbation of the actin filament system by cytochalasin B had no
effect (Fig. 4B). To confirm the latter observations, we
performed experiments using immunofluorescence microscopy. In
nonstimulated cells, MRP8 and MRP14 show a diffuse staining pattern,
whereas treatment with PMA resulted in a filamentous MRP8/MRP14
distribution, which resembled the tubulin network (Fig. 5,
A, B, and D). As a control, a
mAb against the intermediate filament vimentin was employed. Vimentin
generally shows a thinner filamentous network that was most prominent
perinuclear but sparse at submembraneous areas (Fig. 5C). In
contrast, antibodies against tubulin as well as against MRP8 and MRP14
stained cytoskeletal filaments of larger diameter, which extended out
to the cell periphery. Actin filaments were visualized by
phalloidin-FITC and showed a completely different pattern (data not
shown). Furthermore, double-labeling experiments were performed that
revealed a clear co-localization of MRP8 and MRP14 with the tubulin
network in PMA-treated monocytes (Fig. 5, E and
F). Double-labeling with vimentin revealed that the
cytoskeletal network stained by a-MRP8 or a-MRP14 was stronger and
clearly more extended to the cell periphery (Fig. 5, G and
H). Depolymerization of microtubules by demecolcine (Fig. 6,
A-C), nocodazole or colchicine (data not shown) resulted in a completely different staining pattern: MRP8 and
MRP14 were then found to be diffusely dispersed over all the cytoplasm
(Fig. 6A), whereas tubulin was focally condensed (Fig. 6B), implying that MRP8 and MRP14 bound preferentially to
filamentous microtubules. The intermediate filament system was
moderately affected by this treatment, leading to a more condensed
perinuclear pattern, but still presented as a clear filamentous network
(Fig. 6C).
Effects of Brefeldin A, Monensin, and Microtubule-depolymerizing Drugs on Secretion of IL-1
We then compared the
mode of MRP8/MRP14 release with that of IL-1 and that of TNF-
,
representatives of an alternative and the classical pathways of
secretion, respectively. Brefeldin A and monensin, known to block the
vesicular traffic at the ER and Golgi level, significantly inhibited
PMA-induced TNF-
secretion, whereas IL-1
release was up-regulated
or unaffected, respectively (Fig. 7). Concomitant
treatment with microtubule-depolymerizing agents, such as demecolcine
and nocodazole, resulted in a decrease of PMA-induced TNF-
release.
In contrast, IL-1
release was slightly increased by these agents.
Thus, MRP8 and MRP14 secretion is different from both the IL-1-like
alternative and from the classical pathways of secretion (Fig. 7).
Correlation of MRP8/MRP14 Secretion and mRNA Expression
Employing Northern blot technique, we analyzed
regulation of MRP8 and MRP14 expression at the mRNA level.
Monocytes cultured for 1 day were incubated for 4 h with 10 nM PMA or medium as control. Activation of protein kinase C
led to down-regulation of both MRP8 and MRP14 mRNA. The time course
of MRP8 and MRP14 accumulation into the supernatant was closely
paralleled by down-regulation of MRP8 and MRP14 mRNA (Fig.
8A). Accordingly, an inverse
concentration-response relationship regarding MRP8/MRP14 release and
mRNA expression was observed over a range of 1 to 100 nM PMA (Fig. 8B).
Earlier studies reported different extracellular functions of S100 proteins but did not provide any information on the mechanisms of their release (4, 5, 31). This is quite remarkable since neither MRP8 and MRP14 nor any other member of the S100 family comprise signal sequences that would determine their secretion via the classical ER/Golgi route.
We now demonstrate that MRP8 and MRP14 are released from monocytes during inflammatory activation via a novel secretory pathway. Mechanisms leading to MRP8/MRP14 release were shown to be energy-dependent and to involve protein kinase C activation. Employing metabolic labeling, both MRP8 and MRP14 were demonstrated to be secreted at similar rates and as complexes.
Potential nonspecific release of cytoplasmatic MRP8/MRP14, secondary to toxic effects of PMA, could be excluded at several levels. (i) Cell viability did not change significantly during the experimental procedures. (ii) Concomitant treatment with PMA and several potentially toxic agents inhibited release of MRP8 and MRP14, which is incompatible with a mere nonspecific release due to toxic cell damage. (iii) p11, another member of the S100 family, did not appear in the supernatant, despite intracellular up-regulation after PMA treatment.
The blockade of vesicular traffic through the ER and Golgi did not affect MRP8 and MRP14 release, thus ruling out involvement of the classical secretory pathway (21). Secretion is dependent on an intact microtubule network since disruption by depolymerizing agents inhibited MRP8 and MRP14 release. The latter observation is in accordance to morphological data demonstrating a clear co-localization of MRP8 and MRP14 with microtubules during the process of secretion.
Properties of MRP8 and MRP14 during secretion resemble in some aspects
those of IL-1, which is supposed to be released via an alternative
pathway of secretion (21). IL-1
secretion is not inhibited by
monensin or brefeldin A as well (32). Moreover, there is no association
of IL-1
with ER, Golgi apparatus, or secretory vesicles, whereas a
co-localization with the microtubule network during the secretory
process has been reported (33-35). However, secretion of IL-1
is
not inhibited by microtubule-depolymerizing drugs (36) (Fig. 7), which
is in contrast to our data for MRP8 and MRP14. Furthermore, uncouplers
of oxidative phosphorylation increased levels of secreted IL-1
(32),
whereas MRP8 and MRP14 release was significantly inhibited by CCCP and
dinitrophenol. Thus, IL-1
and MRP8 and MRP14 display clear
differences, suggesting that they do not share a common mechanism of
release. MRP8 and MRP14 therefore appear to follow neither the
classical nor the alternative secretory pathway of the
IL-1
-type.
Earlier reports described elevated serum levels of MRP8 and MRP14 during the course of a number of inflammatory diseases (2, 16, 17). Immunohistological data provided indirect evidence that monocytes release MRP8 and MRP14 during endothelial diapedesis at sites of inflammation (11). The complex of MRP8 and MRP14 shows antimicrobial activities, especially against Candida albicans (18). The MRP14 subunit seems to be responsible for this antimicrobial effect (37). Furthermore, MRP8·MRP14 complexes exhibit growth-inhibitory activities against murine bone marrow cells, macrophages, and mitogen-stimulated lymphocytes (38), which appears to depend on inhibition of casein kinase II (19). In addition, murine MRP8, but not MRP14, shows chemotactic activity for granulocytes (20). Another recently reported function of MRP8 and MRP14 refers to an antiinflammatory property; systemic application of MRP8/MRP14 mitigated the course of murine experimental arthritis (39). This picture of pleiotropic extracellular activities may reflect different functions of monomeric and complexed MRP8 and MRP14. Such a hypothesis is supported by the observation that MRP8 and MRP14 are differentially expressed in defined monocyte subpopulations under various inflammatory conditions (15).
Most S100 family proteins appear to play an intracellular role during calcium-dependent signaling (4, 5). They interfere with cell cycle progression, inhibit phosphorylation reactions, or modulate membrane/cytoskeleton interactions. MRP8 and MRP14 are supposed to be involved in intracellular signaling pathways during calcium-dependent activation of monocytes. They assemble to noncovalently associated complexes (26, 27) that are translocated to membrane structures and intermediate filaments upon elevation of intracellular calcium levels by calcium ionophore A23187 (22). The latter event correlated with inflammatory activation of monocytes and neutrophils, thus implicating a role of these proteins for membrane/cytoskeleton interactions (22, 23, 40). Secretion into the supernatant, however, did not coincide with the preceding translocation of MRP8/MRP14 to the cell membrane, indicating that these phenomena are independent events.
Other members of the S100 protein family exhibit both intra- and
extracellular functions as well. For example, S100 interferes with
calcium-dependent modulation of cytoskeletal structures
(41, 42), but also functions as neurite extension factor in the
extracellular space (6, 43, 44). S100
furthermore increases
intracellular calcium levels and up-regulates protooncogene expression
(31) and nitric oxide synthetase activity in neuronal cells (45). Thus,
activities of distinct S100 proteins are not restricted to either the
intra- or extracellular space. Structural properties of S100 proteins
support such observations. Their N-terminal EF-hand exhibits a
significantly lower affinity to calcium than the C-terminal EF domain.
It has therefore been supposed that the N-terminal EF-hand binds
calcium only at high calcium concentrations as they predominate in the
extracellular environment (5).
Induction of MRP8 and MRP14 release is associated with down-regulation of de novo synthesis of these proteins at the mRNA level, thus limiting an extracellular function of these proteins to distinct stages of inflammatory reactions. Furthermore, secretion of MRP8/MRP14 is linked to a marked differentiation step in monocytes, since MRP8 and MRP14 account for up to 30% of the calcium-binding capacity of EF-hand proteins in these cells, whereas both molecules cannot be found after down-regulation of de novo synthesis in mature macrophages (13, 46).
To date, extracellular functions have been ascribed to five members of
the S100 family. We now for the first time provide data elucidating the
mechanism of release of two of these proteins, MRP8 and MRP14, which
follow neither the classical nor the IL-1-like alternative pathway
of secretion. Whether such a novel route may also account for release
of other S100 family members remains to be elucidated in future
studies.
The authors thank A. Erpenbeck, H. Hater, and D. Kortevoß for excellent technical assistance and B. Scheibel for secretarial help.