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and TNF Regulate Macrophage Expression of the Chemotactic S100 Protein S100A81
Cytokine Research Unit, School of Pathology, Faculty of Medicine, University of New South Wales, Sydney, Australia
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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,
and TNF were distinct from the C-C chemokines monocyte chemoattractant
protein-1 (MCP-1), macrophage-inflammatory protein-1
(MIP-1), and
RANTES. Monomeric S100A8 was predominantly secreted. IFN substantially
increased S100A8 mRNA levels after 1 h with optimal induction
after 12 h; induction by TNF was slower and more sustained. TNF
did not up-regulate MCP-1 and MIP-1 mRNA in these cells. Luciferase
reporter assays confirmed that LPS and IFN induce S100A8 gene
transcription and mRNA in LPS-treated Mac showed little decay over
16 h, whereas transcripts induced by IFN and TNF were markedly
less stable. Newly synthesized proteins may be required for mRNA
transcription and stabilization in response to LPS. S100A9 associates
with A8 in neutrophils, but was not coinduced with S100A8. S100A8 gene
induction in Mac stimulated with LPS and IFN may be modulated by
mobilization of intracellular Ca2+ concentration from
distinct intracellular stores and/or the extracellular compartment and
by distinct pathways involving protein kinase C and leading to
activation of mitogen-activated protein kinase. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mS100A8 stimulates myeloid cell chemotaxis in vitro and sustains leukocyte recruitment, with monocytes following an early influx of PMN, and kinetics similar to that of a delayed-type hypersensitivity (DTH) response in vivo (2, 8, 9, 10). Moreover, mS100A8 and A9 are associated with granuloma formation and injection of agarose-bound complexes into mice causes severe infiltration of neutrophils and Mac over 7–14 days (11). S100A8 is a more potent chemoattractant than most chemokines (8) and does not activate degranulation, enzyme release, or provoke an oxidative burst (12). S100A8 and A9 may influence leukocyte margination and transmigration into tissues (13) by increasing leukocyte deformability (12) and integrin-mediated adhesion (14). Expression of the proteins by microvascular endothelial cells activated by IL-1 and TNF (5) may facilitate these processes.
Mac recruited by mS100A8 in vivo have a particular phenotype which would favor bacterial clearance by virtue of increased levels of scavenger receptor, Fc receptor, and phagocytosis (10). Our recent evidence that S100A8 can efficiently scavenge hypochlorite anions produced by activated neutrophils (15) suggests that it can promote and modulate inflammatory responses. TGF-ß1 and mS100A8 share apparently paradoxical functions in immune and inflammatory processes. TGF-ß1 is also chemotactic at picomolar levels but fails to activate leukocytes and both are implicated in embryogenesis. Deletion of the S100A8 gene is lethal to embryos at mid-gestation when it is expressed by migrating trophoblasts (16).
Although there is increasing interest in S100A8/A9 in inflammatory disease, little is known concerning their regulation by appropriate mediators. S100A8/A9 are not expressed by tissue Mac (17, 18) whereas Mac in inflammatory lesions may do so (19, 20, 21, 22) and, although not always coordinately expressed (21, 22), S100A8/9 +ve Mac produce high amounts of TNF and IL-1, suggesting a particular proinflammatory phenotype (23).
Leukocyte recruitment is mediated by a variety of chemoattractants, and specific temporal and differential induction may regulate the composition of inflammatory exudates. Activated Mac are a primary source of chemokines that bear no amino acid sequence similarities and that are located within different chromosomal clusters (murine chromosome 5 for CXC and 11 for CC chemokines (reviewed in Ref. 24)) to the S100 chemoattractant proteins (murine chromosome 3 (Ref. 25)). In addition, S100A8 has a single Cys at position 41 (2) and, unlike the chemokines, binds calcium with high affinity. Here, we present evidence that mS100A8, but not mS100A9, is up-regulated by some key mediators of Mac function, including IFN, IL-1, and TNF and compare induction with some C-C chemokine genes considered important in monocyte/Mac recruitment in cell-mediated immune responses.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RPMI 1640 from Life Technologies (Grand Island, NY) was
supplemented with 126.6 µg/ml penicillin, 126.6 µg/ml streptomycin
(Sigma, St. Louis, MO), and 2% heated (56°C, 30 min) bovine calf
serum (HyClone, Logan, UT), hereafter referred to as culture medium
(CM). Plastic flasks and plates were obtained from Falcon (Lincoln
Park, NJ). Thioglycolate broth (TG) and LPS (E. coli 055:B5)
were purchased from Difco (Detroit, MI). IFN- was obtained from
Genzyme (Cambridge, MA; endotoxin content <0.01 ng/µg; sp. act.,
1.14 x 107 U/mg) or Genentech (San
Francisco, CA; 0.032 endotoxin units/mg; sp. act., 0.5 x
107 U/mg). TNF was purchased from Genzyme (0.51
endotoxin units/mg; sp. act., 1.2 x 107
U/mg) or Sigma (
0.1 EU ng/µg; L929 cell inhibition
EC50: 0.04 ng/ml). IL-1ß (sp. act., 3.5 x
107 U/mg), IL-6, IL-12, and recombinant human
IFN- were obtained from Genzyme. Neutralizing hamster
anti-murine TNF Ab (10–100 ng neutralized 1 U mouse TNF in vitro)
was a generous gift from Dr. Ian Clarke (Australia National University,
Canberra, Australia). Human IL-1 receptor antagonist (sp. act., 1000
U/ml) was purchased from Boehringer Manneheim (Manneheim, Germany).
Native murine S100A8 and S100A9 from bone marrow were purified in our
laboratory by Dr. Mark Raftery.
Sodium periodate was purchased from BDH Laboratory Supplies (Merck, Kilsyth, Victoria, Australia). Calcium ionophore A23187, PMA, and EGTA were obtained from Sigma. 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA-AM), SB202190, PD98059, U73122, TMB-8 hydrochloride, and thapsigargin were obtained from Calbiochem (La Jolla, CA). For inhibition of mRNA synthesis, 5 mg/ml actinomycin D (ActD; Calbiochem) in ethanol was diluted into CM. Cycloheximide (CHX; Sigma) was used as protein synthesis inhibitor. H-7 (Sigma) and calphostin C (Calbiochem) were used to inhibit protein kinase (PK) C, and H-98 (Calbiochem) and Rp-cAMP (Calbiochem) to inhibit PKA. All reagents and media had <50 pg/ml endotoxin assayed using the chromogenic Limulus amoebocyte lysate assay (Associates of Cape Cod, Woods Hole, MA).
Mac isolation and culture
Quakenbush-Swiss mice (6–8-wk old) were maintained under
conventional clean conditions and were injected i.p. with 2 ml 5 nM
NaIO4 or 10% TG broth and peritoneal cells were
lavaged with cold HBSS containing 0.38% sodium citrate (15 ml) 4 days
later. Washed cells (5 x 106) in 60-mm
tissue culture plates were incubated for 1 h at 37°C in an
atmosphere of 5% CO2 in air, washed three times
with warmed HBSS to remove nonadherent cells, and equilibrated in CM
for 18 h. CM (3 ml) was replenished before activation and
populations contained >98% Mac (
98% viable by trypan blue
exclusion) and <0.3% neutrophils. Mac were stimulated for up to
96 h with the agents indicated.
Northern blot analysis
Total cellular RNA (from 5 x 106
Mac) (4) was size fractionated on a 1% agarose-2.2 M
formaldehyde gel and transferred onto Hybond N+
membrane (Amersham, Buckinghamshire, U.K.) with alkali fixing in 0.05 M
NaOH (4). Hybridizations were performed for 16 h at
58°C for riboprobes and at 36°C for the oligoprobe in
formamide-containing buffer. S100A8 and A9 riboprobes and an 18S
rRNA oligoprobe were used as described previously
(5). pBluescript containing murine MCP-1, RANTES, and
MIP-1 (kindly provided by Dr. T. Yoshimura, National Cancer
Institute, Frederick, MD) were used to produce riboprobes. Membranes
were washed twice at 48°C for 10 min in 2x standard saline citrate
phosphate/EDTA with 0.1% SDS and then twice with 0.1x standard saline
citrate phosphate/EDTA with 0.1% SDS at 65°C for 30 min. Phosphor
imager analyses were performed with a BAS 1000 bio-Imaging analyzer
(Fujix, Tokyo, Japan) using the MacBas program or Bio-Rad
MultiAnalyst/Macintosh version 1.0 software with the Bio-Rad Molecular
Imager GS-525 system (Bio-Rad, Richmond, CA). The relative magnitude of
expression for each gene was determined using software packages and
normalized to the level of 18S RNA on the same blot. Blots were
stripped according to the manufacturer’s instructions.
Characterization of mS100A8 protein
TG-elicited Mac (5 x 105) were cultured in 24-well plates with or without stimuli and S100A8 in supernatants or cell lysates was quantitated with a double-sandwich ELISA and rabbit polyclonal anti-S100A8 IgG, as described elsewhere (4, 26), using recombinant S100A8 (0.1–50 ng/ml) as standard.
Reporter plasmids
The S100A8-luciferase construct pCP-178/+465 was produced by deletion of a PCR-amplified 781-bp fragment spanning nucleotides -316 to +465 subcloned into the BglII site of a luciferase reporter construct (pGL2-Basic; Promega, Madison, WI). PCR was performed as described previously (3) except that Pfu DNA polymerase (Stratagene, La Jolla, CA) was used. The insert sequence was confirmed to be the same as the genomic on both strands.
Transient transfections
Luciferase reporter gene plasmids were transiently transfected into RAW264.7 cells using DEAE-dextran (Sigma) as described previously (27). Briefly, cells were seeded at 2 x 105/well in 12-well plates 24 h before transfection, and then 0.5 µg reporter luciferase plasmid or 0.1 µg reference plasmid (pRL-TK; Promega) was transfected in the presence of DEAE-dextran (300 µg/ml). After 24 h, cells were stimulated with LPS (500 ng/ml), IFN (300 U/ml), or TNF (25 ng/ml) for 20 h and firefly and Renilla luciferase activities were assayed with 20 µl extract using Promega reagents according to the manufacturer’s instructions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because of endogenous levels of S100A8 mRNA and protein in the
small numbers of neutrophils contaminating elicited Mac populations,
Mac were enriched by two adhesion steps and cultured for 18 h
before stimulation. This generally yielded cells expressing
low-negligible endogenous mRNA (Figs. 1
and 2) although low basal levels of
cell-associated protein were always detected. TG-elicited Mac were
initially stimulated for 24 h, the time for maximal induction of
S100A8 by LPS in RAW cells (4). IFN and TNF markedly
increased the steady-state levels of mRNA whereas induction by IL-1ß
was weak (Fig. 1A) and increased 2- to 3-fold in Mac primed
with LPS or IFN (Fig. 1B). Anti-TNF antiserum inhibited the
TNF-induced response and weakly suppressed (<10%) that stimulated by
LPS. TNF and IL-1 were not involved in induction by IFN because the
anti-TNF or IL-1 receptor antagonist did not alter S100A8 mRNA
induction (data not shown). IFN-, IL-6, IL-12 (Fig. 1A),
and mS100A8 and/or S100A9 (10-8 M) did not
induce S100A8 mRNA (data not shown). There were no obvious differences
in responses of periodate- or TG-elicited Mac. IFN-primed Mac did not
exhibit enhanced responses to LPS whereas cells primed with suboptimal
amounts of LPS showed elevated responses to IFN; priming with either
mediator did not influence TNF-induced mRNA levels (Fig. 1B).
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) with 10 U/ml; maximal expression occurred at 50 U/ml with little
further increase with up to 500 U/ml. TNF-induced S100A8 mRNA was first
apparent with 0.1–0.5 ng/ml and maximal at 10 ng/ml (Fig. 2).
Different sources of the stimulants produced similar results (Fig. 1
A).
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mRNA stimulated with predetermined optimal amounts
of LPS, IFN, and TNF. S100A8 mRNA was evident 6 h after addition
of LPS, peaked at 24–28 h, and declined slowly over 96 h (Fig. 3A), whereas the chemokines
exhibited high mRNA levels 3 h after onset of stimulation (data
not shown). MCP-1 mRNA declined more rapidly than S100A8 mRNA but
RANTES was sustained over 96 h. Constitutive expression of
MIP-1 mRNA was always obvious and the high levels induced between 3
and 12 h slowly declined thereafter. IFN substantially increased
S100A8 mRNA after only 1 h, with optimal induction after 12
h, and gradually decreased over 48 h. Fig. 3A
emphasizes the rapid induction by IFN compared with the later and more
sustained responses to LPS and TNF. MCP-1 and MIP-1 mRNA induced by
IFN was maximal within 3 h and not detected after 48 h,
whereas RANTES increased slowly to maximal levels at 24 h (data
not shown). Major differences in induction patterns were evident with
TNF. S100A8 mRNA induction was strong and followed a time course
similar to that provoked by LPS (Fig. 3A). In marked
contrast, constitutive levels of MCP-1 and MIP-1 mRNAs
were barely altered by TNF and RANTES mRNA was optimal only after
48 h (Fig. 3B).
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and 3). S100A8 protein production by stimulated Mac
Unstimulated elicited Mac lysates contained 0.7 ng
S100A8/106 cells, possibly derived from
neutrophils originally contaminating Mac exudates. Cell-associated
protein in lysates of IFN- and TNF-stimulated cells decreased by
5–20% after a 76-h culture. The low levels of S100A8 released into
supernatants of unstimulated cells (Fig. 4) may represent amounts released by
cells dying over this period. Secreted S100A8 increased steadily over
76 h and remained elevated 96 h after stimulation with the
mediators shown. In contrast to the high mRNA levels (Fig. 3A) in LPS-activated cells, secreted S100A8 was
approximately half that produced by IFN- or TNF-activated Mac. S100A8
from stimulated supernatants enriched by immunoaffinity
(5) eluted from C4 RP-HPLC at 20.1 min, the same time as
native S100A8 standard and Western blotting confirmed monomeric S100A8
(10 KDa) as the dominant structural form. S100A9, S100A8/A9
heterodimers, or covalent S100A8 homodimers were not detected in any
sample tested (data not shown).
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To assess whether RNA stabilization contributed to mRNA
accumulation, Mac incubated with LPS for 24 h, IFN for 12 h,
or TNF for 36 h were treated with ActD for various times to block
further transcription. The half-lives of S100A8 mRNA induced by IFN or
TNF were similar (t1/2, 9
h for IFN; 7 h for TNF; Fig. 5) and
transcripts were undetectable after 16 h. S100A8 mRNA in
LPS-treated cells decayed with markedly different kinetics (Fig. 5)
with levels maintained over 16 h, with little decay.
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). In contrast,
IFN- and TNF-induced responses were reduced by 30 and 55%,
respectively, indicating different requirements for gene transcription.
Similar responses were evident with higher doses of CHX, although
superinduction made results difficult to interpret.
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). The construct pCP-178/+465 (-178 to
+465 of the S100A8 gene) was up-regulated 7-fold by LPS and 3-fold by
IFN compared with control. Levels of induction were less than those
observed by Northern blot analysis, indicating that this construct may
not contain all of the elements necessary for maximal expression.
Induction by TNF was not significantly above control levels and
preliminary Northern blot analysis indicated that these cells were
unresponsive to TNF (data not shown). Low basal expression in the
absence of stimulants indicates that pCP-178/+465 either lacks some
elements required to suppress the gene in unstimulated cells or that a
low level of expression occurs naturally in RAW cells.
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B) but strongly amplified
responses to LPS or IFN. The PKC inhibitor H-7 and the more specific
antagonist calphostin C maximally reduced induction by LPS and IFN at
the time of optimal gene expression. (Fig. 7, A and
B). In contrast, PMA strongly induced MCP-1 mRNA after
12 h, and an additive response was evident with PMA cultured with
LPS or IFN; H-7 reduced induction by the stimulants (data not shown).
H-89 and Rp-cAMP decrease cAMP and PKA activity but had little effect
on S100A8 mRNA in the presence or absence of activators. Mac activation
can result in rapid tyrosine phosphorylation via activation of the
mitogen-activated protein (MAP) kinase pathways. Fig. 7C
shows that PD98059, a selective inhibitor of MAP/extracellular
signal-related kinase 1 kinase, and the highly specific p38 kinase
inhibitor SB202190 both markedly reduced LPS- and IFN-stimulated S100A8
mRNA, suggesting involvement of members of the MAP kinase (MAPK)
subfamily in S100A8 induction in elicited Mac.
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). The
ionophore A23187 completely abolished S100A8 mRNA induced by both
stimulants whereas reduction of resting cytoplasmic
Ca2+ levels by BAPTA-AM had no effect (Fig. 8).
In contrast, MCP-1 mRNA induction was somewhat enhanced 1.5- to
1.9-fold by EGTA and reduced by changes in cytosolic
Ca2+ provoked by BAPTA-AM and A23187 (Fig. 8).
Thapsigargin, an inhibitor of the endoplasmic reticulum
Ca2+-ATPase, dramatically inhibited S100A8 mRNA
induction by LPS but not by IFN, indicating divergent pathways of
activation. The phospholipase C (PLC) inhibitor U73122 reduced S100A8
and MCP-1 mRNAs induced by IFN but amplified the genes in cells
activated with LPS, indicating a regulatory role for PLC-mediated
pathways in the transcription of these genes.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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TG- or periodate-elicited murine Mac do not express S100A8 (4, 17) but respond directly to LPS, and rechallenge markedly
down-regulates mRNA expression (4). We chose elicited Mac
as a model because transmigration of these cells from the blood into
the peritoneal cavity may more closely resemble an inflammatory
setting. The Th1 cytokine IFN plays a central role in Mac activation
associated with bactericidal activity, Ag presentation, and
orchestration of leukocyte-endothelial cell interactions and is a key
mediator of DTH reactions (reviewed in Ref. 30). In
contrast, to the inability of IFN to up-regulate mS100A8 mRNA in the
murine microvascular cell lines (5), it was strongly
up-regulated in elicited Mac by as little as 10 U/ml IFN (Fig. 2).
Kinetics was rapid, with mRNA apparent within 1–3 h and optimal
expression between 6 and 12 h (Fig. 3A). IFN primes Mac
for a number of functional responses (31) but IFN-primed
Mac were not hyperresponsive to LPS or TNF. In contrast, responses of
LPS-primed cells were amplified by suboptimal doses of IFN (Fig. 1B). IFN-, IL-6, and IL-12 failed to induce the mS100A8
gene (Fig. 1A).
TNF and IL-1 are key mediators of inflammation and sepsis. They promote
leukocyte-endothelial cell adhesion, leukocyte migration, and localized
production of other mediators (32). IL-1 strongly induces
S100A8 mRNA in microvascular endothelial cell lines (5)
but only weakly activated mRNA expression, whereas LPS- or IFN-, but
not TNF-primed Mac, exhibited synergistic responses (Fig. 1B). S100A8 may represent another mechanism of regulating
monocyte recruitment in situations in which TNF plays a major role.
S100A8 mRNA was strongly up-regulated within 6 h by TNF in a
dose-dependent manner (Fig. 2); expression was maximal at 36 h and
slowly decreased over 96 h (Fig. 3, A and
B). Here, we demonstrate differential induction of S100A8
mRNA and MCP-1 mRNA by TNF which did not alter MCP-1 gene expression
(Fig. 3B). Moreover, TNF had relatively little effect on
endogenous MIP-1 mRNA levels, and induction of RANTES mRNA was
substantially slower than that of S100A8. In contrast, MCP-1 mRNA was
more rapidly stimulated by LPS and IFN (data not shown), although
expression was less sustained than that of S100A8. Differences in the
state of activation and/or differentiation may be important
determinants in induction of MCP-1 by TNF in Mac (33, 34)
and may determine the nature of the response.
S100 proteins have no structural sequences required for secretion by
the classical endoplasmic reticulum-Golgi pathway, but extracellular
functions are well accepted (1). The human S100A8/9
complex may be released via a novel tubulin-dependent mechanism
following leukocyte activation (35), suggesting active
secretion. We demonstrated mS100A8 in supernatants from LPS-activated
Mac cell lines (4) and from IFN and TNF-activated elicited
Mac (Fig. 4). S100A9 mRNA or protein was not induced by any mediator
tested (Figs. 1 and 3), supporting the notion that coexpression with
S100A9 is not essential for S100A8 function or secretion
(35). S100A8 protein levels secreted by elicited Mac after
stimulation with predetermined optimal amounts of IFN and TNF were
higher than those produced by LPS (Fig. 4), suggesting that these
mediators may also activate pathways involved in S100A8 release.
The high levels (
10-10 M) of S100A8 produced
8 h after stimulation with TNF or IFN were above the
chemotactically optimal dose of 10-12 M
(8). The monomer was the predominant structural form in
supernatants (data not shown). S100A8 is exquisitely sensitive to
oxidation by hypochlorite generated by activated neutrophils
(15), and, in situations where concentrations within the
range generated at the later time points are potentially liberated,
S100A8 may be protective by virtue of its ability to scavenge reactive
oxygen species. Moreover, oxidation of mS100A8 to the covalent
homodimer negates its chemotactic capacity and, along with the ability
of hypochlorite to reduce the chemotactic activity of C5a and fMLP
(36), strengthens the notion that this mechanism may be
physiologically relevant in limiting leukocyte recruitment by these
stimulants.
Experiments reported here indicate differences in transcriptional and
posttranscriptional regulation of the S100A8 gene by LPS and IFN.
Transcriptional regulation was measured using transient transfection of
luciferase reporter constructs into the RAW monocytoid cell line (Table I). Half-lives of mRNA induced by IFN and TNF were similar (9 and
7 h, respectively; Fig. 4) but markedly shorter than that induced
by LPS (>16 h). The 3' untranslated region does not contain known
AU-rich sequences which destabilize the mRNA of numerous cytokine genes
(37), and mechanisms regulating S100A8 mRNA stability may
depend on inducible factors. CHX superinduced low and variable levels
of S100A8 mRNA, but induction by IFN and TNF was relatively resistant
whereas CHX completely inhibited the LPS response (Fig. 6 and Ref.
4), suggesting regulation by an inducible component.
Results presented in Fig. 7 implicate PKC and MAP kinase pathways in
the regulation of transcriptional events leading to S100A8 gene
expression. PKC regulates human S100A8/A9 in myelocytic differentiation
(38), and H9, and the specific antagonist calphostin C
(39), inhibited induction by LPS and IFN whereas PKA
inhibitors had little effect. Although direct activation was weak, PMA
synergized with LPS and IFN to markedly elevate S100A8 mRNA (Fig. 7).
MAP kinases regulate Mac activation in response to LPS, TNF, and IL-1;
PD98059 binds to inactive MAP/extracellular signal-related kinase 1 and
SB202190 inhibits p38 activity and both pathways can cooperatively
regulate transcription of AP-1 components c-jun and
c-fos (40). Results shown in Fig. 7C
implicate both pathways in transcriptional events regulating S100A8
expression in LPS- or IFN-activated Mac. MAPK phosphorylation also
modulates STAT and NF-
B activity (40). Consensus motifs
for transcription factors located within the region of the promoter
tested (Table I) include TATA, NF1, E box, GC box, NF-IL-6, NF-B,
SPE, IRE, Ets box, Myb, and AP-1, many of which are associated with
LPS- and IFN-induced genes involved in myeloid-specific
differentiation, activation, and inflammation.
Calcium regulates transcription of a number of S100 genes (1, 18) and changes in Ca2+ mobilization via
release from intracellular stores and/or the extracellular space may
regulate S100A8 gene expression in Mac (Fig. 8). In contrast to S100A8,
elevated levels of cytosolic Ca2+ increase
c-fos, TNF, and MCP-1 expression in Mac (Ref.
41 and Fig. 8). Chelation of extracellular
Ca2+ with EGTA and inhibition of
Ca2+ release from the endoplasmic reticulum by
TMB-8 (42) suppressed LPS- and IFN-induced responses (Fig. 8). EGTA increased, but TMB-8 decreased MCP-1 mRNA and reduction of
resting cytoplasmic Ca2+ levels with BAPTA-AM
reduced MCP-1 but not S100A8 mRNA levels. A23187, which passively
transfers extracellular calcium and triggers release from intracellular
pools causing a calcium spike, was an effective inhibitor of MCP-1 and
S100A8 genes (Fig. 8). Divergent pathways of S100A8 gene expression by
LPS and IFN were indicated with thapsigargin. This causes influx of
stored Ca2+ by inhibiting microsomal ATPases
without producing inositol 1,4,5-trisphosphate and only inhibited the
LPS-induced response. IFN-induced S100A8 and MCP-1 mRNAs, but not
S100A8 mRNA induced by LPS, were suppressed by U73122 which inhibits
PLC and the resultant conversion of
phosphatidylinositol-4.5-bispospate to inositol 1,4,5-trisphosphate
and mobilization of intracellular Ca2+.
Taken together, we suggest that S100A8 gene induction in Mac stimulated
with LPS and IFN is modulated by mobilization of intracellular
Ca2+ concentration from distinct intracellular
stores and by converging pathways leading to phosphorylation of MAP
kinase, an important event in stress-induced and inflammatory
responses. LPS activation may occur via a calcium-independent PKC (
or 
) and require inducible transcriptional mediators, possibly AP-1.
c-Jun can also influence mRNA stability (43) and its
involvement in enhancing stability of LPS-induced transcripts is worthy
of testing. On the other hand, the response induced by IFN may be
mediated by Ca2+-dependent PLC to generate PKC
and activate MAPK. This response relied largely on constitutive
factors, possibly via NF-B or the JAK/STAT pathway.
These studies indicate that S100A8 is a major secreted product of elicited murine Mac activated with IFN, TNF, and IL-1. The different induction and regulation patterns of MCP-1 and S100A8 mRNAs indicate that environmental factors, particularly TNF and those provoking changes in Ca2+ levels, may determine the nature of the chemoattractant induced. Cytosolic free Ca2+ also regulates cytoskeletal structure and motility and the differences in responses of S100A8 and MCP-1 may regulate motility by coordinating substrate-specific attachment/detachment processes via different responses to calcium transients (44). The low levels of S100A8 produced in the early inflammatory phase may be chemotactic, whereas higher amounts produced during peak responses may protect the host against oxidative damage (15). We propose that S100A8 is an important constituent of cell-mediated immune reactions mediated by activated macrophages.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Carolyn Geczy, Cytokine Research Unit, School of Pathology, Faculty of Medicine, University of New South Wales, Sydney, NSW 2052, Australia.
3 Abbreviations used in this paper: mS100A8, murine S100A8; Mac, macrophage; CM, culture medium; TG, thioglycolate broth; BAPTA-AM, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl)ester; ActD, actinomycin D; CHX, cycloheximide; PK, protein kinase; MCP-1, monocyte chemoattractant protein-1; MIP-1, macrophage-inflammatory protein-1; MAP, mitogen-activated protein; MAPK, MAP kinase; PLC, phospholipase C.
Received for publication April 19, 2000. Accepted for publication February 17, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, and interleukin-1ß. Am. J. Respir. Cell & Mol. Biol. 12:104.[Abstract]
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 O. H. Mortensen, K. Andersen, C. Fischer, A. R. Nielsen, S. Nielsen, T. Akerstrom, M.-b. Aastrom, R. Borup, and B. K. Pedersen Calprotectin is released from human skeletal muscle tissue during exercise J. Physiol., July 15, 2008; 586(14): 3551 - 3562. [Abstract] [Full Text] [PDF]
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R. T. Sasmono, A. Ehrnsperger, S. L. Cronau, T. Ravasi, R. Kandane, M. J. Hickey, A. D. Cook, S. R. Himes, J. A. Hamilton, and D. A. Hume Mouse neutrophilic granulocytes express mRNA encoding the macrophage colony-stimulating factor receptor (CSF-1R) as well as many other macrophage-specific transcripts and can transdifferentiate into macrophages in vitro in response to CSF-1 J. Leukoc. Biol., July 1, 2007; 82(1): 111 - 123. [Abstract] [Full Text] [PDF]
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 Q. Su, Y. Zhou, and R. A. Johns Bruton's tyrosine kinase (BTK) is a binding partner for hypoxia induced mitogenic factor (HIMF/FIZZ1) and mediates myeloid cell chemotaxis FASEB J, May 1, 2007; 21(7): 1376 - 1382. [Abstract] [Full Text] [PDF]
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 J. Vakkila, R. Jaffe, M. Michelow, and M. T. Lotze Pediatric cancers are infiltrated predominantly by macrophages and contain a paucity of dendritic cells: a major nosologic difference with adult tumors. Clin. Cancer Res., April 1, 2006; 12(7): 2049 - 2054. [Abstract] [Full Text] [PDF]
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 M. M. McCormick, F. Rahimi, Y. V. Bobryshev, K. Gaus, H. Zreiqat, H. Cai, R. S. A. Lord, and C. L. Geczy S100A8 and S100A9 in Human Arterial Wall: IMPLICATIONS FOR ATHEROGENESIS J. Biol. Chem., December 16, 2005; 280(50): 41521 - 41529. [Abstract] [Full Text] [PDF]
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G. Srikrishna, O. Turovskaya, R. Shaikh, R. Newlin, D. Foell, S. Murch, M. Kronenberg, and H. H. Freeze Carboxylated Glycans Mediate Colitis through Activation of NF-{kappa}B J. Immunol., October 15, 2005; 175(8): 5412 - 5422. [Abstract] [Full Text] [PDF]
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K. Hsu, R. J. Passey, Y. Endoh, F. Rahimi, P. Youssef, T. Yen, and C. L. Geczy Regulation of S100A8 by Glucocorticoids J. Immunol., February 15, 2005; 174(4): 2318 - 2326. [Abstract] [Full Text] [PDF]
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T. A. Skwor, H. Cho, C. Cassidy, T. Yoshimura, and D. N. McMurray Recombinant guinea pig CCL5 (RANTES) differentially modulates cytokine production in alveolar and peritoneal macrophages J. Leukoc. Biol., December 1, 2004; 76(6): 1229 - 1239. [Abstract] [Full Text] [PDF]
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 K. M. Spach, L. B. Pedersen, F. E. Nashold, T. Kayo, B. S. Yandell, T. A. Prolla, and C. E. Hayes Gene expression analysis suggests that 1,25-dihydroxyvitamin D3 reverses experimental autoimmune encephalomyelitis by stimulating inflammatory cell apoptosis Physiol Genomics, July 8, 2004; 18(2): 141 - 151. [Abstract] [Full Text] [PDF]
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 T. Radaelli, A. Varastehpour, P. Catalano, and S. Hauguel-de Mouzon Gestational Diabetes Induces Placental Genes for Chronic Stress and Inflammatory Pathways Diabetes, December 1, 2003; 52(12): 2951 - 2958. [Abstract] [Full Text] [PDF]
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K. Vandal, P. Rouleau, A. Boivin, C. Ryckman, M. Talbot, and P. A. Tessier Blockade of S100A8 and S100A9 Suppresses Neutrophil Migration in Response to Lipopolysaccharide J. Immunol., September 1, 2003; 171(5): 2602 - 2609. [Abstract] [Full Text] [PDF]
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S. Berthier, M.-H. Paclet, S. Lerouge, F. Roux, S. Vergnaud, A. W. Coleman, and F. Morel Changing the Conformation State of Cytochrome b558 Initiates NADPH Oxidase Activation: MRP8/MRP14 REGULATION J. Biol. Chem., July 3, 2003; 278(28): 25499 - 25508. [Abstract] [Full Text] [PDF]
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Y. Xu, J. C. Clark, B. J. Aronow, C. R. Dey, C. Liu, J. L. Wooldridge, and J. A. Whitsett Transcriptional Adaptation to Cystic Fibrosis Transmembrane Conductance Regulator Deficiency J. Biol. Chem., February 21, 2003; 278(9): 7674 - 7682. [Abstract] [Full Text] [PDF]
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 K. BURKHARDT, M. RADESPIEL-TROGER, H. D. RUPPRECHT, M. GOPPELT-STRUEBE, R. RIESS, L. RENDERS, I. A. HAUSER, and U. KUNZENDORF An Increase in Myeloid-Related Protein Serum Levels Precedes Acute Renal Allograft Rejection J. Am. Soc. Nephrol., September 1, 2001; 12(9): 1947 - 1957. [Abstract] [Full Text] [PDF]
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Z. Yang, T. Tao, M. J. Raftery, P. Youssef, N. Di Girolamo, and C. L. Geczy Proinflammatory properties of the human S100 protein S100A12 J. Leukoc. Biol., June 1, 2001; 69(6): 986 - 994. [Abstract] [Full Text] [PDF]
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K. Xu, T. Yen, and C. L. Geczy IL-10 Up-Regulates Macrophage Expression of the S100 Protein S100A8 J. Immunol., May 15, 2001; 166(10): 6358 - 6366. [Abstract] [Full Text] [PDF]
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M. J. Raftery, Z. Yang, S. M. Valenzuela, and C. L. Geczy Novel Intra- and Inter-molecular Sulfinamide Bonds in S100A8 Produced by Hypochlorite Oxidation J. Biol. Chem., August 31, 2001; 276(36): 33393 - 33401. [Abstract] [Full Text] [PDF]
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), IFN-
), and TNF (
),
determined by densitometry and normalized to 18S rRNA content, of
autoradiograms of Northern blots. The unstimulated control (•) was
compared with maximum induction of S100A8 mRNA by LPS to give 100%
maximal response. B, mRNA induction of S100A8, MCP-1,
RANTES, and MIP-1
)-treated cells
(B) are expressed as amounts relative to 18S rRNA signal
intensities. Results are representative of three experiments.
) and levels of mRNA of CHX-treated
samples relative to this value were calculated. Results represent three
experiments.
