Endotoxin LPS, interferon c IFNc, tumour-necrosis factor TNF and TGF-b did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maxi-mally after 12 h.. Abbreviation
Trang 1of S100A8
Farid Rahimi, Kenneth Hsu, Yasumi Endoh and Carolyn L Geczy
Inflammatory Diseases Research Unit, School of Medical Sciences, University of New South Wales, Sydney, Australia
Fibroblasts are heterogeneous stromal resident cells
that participate in wound healing, fibrosis⁄ scarring
and immune⁄ inflammatory processes [1,2] by
contri-buting to leukocyte recruitment⁄ accumulation,
angio-genesis, matrix metabolism, and protection against
oxidative damage [3,4] Numerous factors inclu-ding extracellular matrix (ECM) components, some cytokines, prostaglandins, reactive oxygen species (ROS), and growth factors [5] modulate fibroblast function
Keywords
FGF-2; fibroblasts; interleukin-1b; S100A8
gene; TGF-b
Correspondence
C Geczy, Inflammatory Diseases Research
Unit, School of Medical Sciences, The
University of New South Wales, Sydney,
NSW 2052, Australia
Fax: + 61 293851389
Tel: + 61 293851599
E-mail: c.geczy@unsw.edu.au
Website: http://www.med.unsw.edu.au/
(Received 3 March 2005, revised 28 March
2005, accepted 5 April 2005)
doi:10.1111/j.1742-4658.2005.04703.x
Growth factors, including fibroblast growth factor-2 (FGF-2) and transform-ing growth factor-b (TGF-b) regulate fibroblast function, differentiation and proliferation S100A8 and S100A9 are members of the S100 family of Ca2+ -binding proteins and are now accepted as markers of inflammation They are expressed by keratinocytes and inflammatory cells in human⁄ murine wounds and by appropriately activated macrophages, endothelial cells, epithelial cells and keratinocytes in vitro In this study, regulation and expression of S100A8 and S100A9 were examined in fibroblasts Endotoxin (LPS), interferon c (IFNc), tumour-necrosis factor (TNF) and TGF-b did not induce the S100A8 gene in murine fibroblasts whereas FGF-2 induced mRNA maxi-mally after 12 h The FGF-2 response was strongly enhanced and prolonged
by heparin Interleukin-1b (IL-1b) alone, or in synergy with FGF-2⁄ heparin strongly induced the gene in 3T3 fibroblasts S100A9 mRNA was not induced under any condition Induction of S100A8 in the absence of S100A9 was confirmed in primary fibroblasts S100A8 mRNA induction by FGF-2 and IL-1b was partially dependent on the mitogen-activated-protein-kinase pathway and dependent on new protein synthesis FGF-2-responsive elements were distinct from the IL-1b-responsive elements in the S100A8 gene promoter FGF-2-⁄ heparin-induced, but not IL-1b-induced responses were significantly suppressed by TGF-b, possibly mediated by decreased mRNA stability S100A8 in activated fibroblasts was mainly intracytoplas-mic Rat dermal wounds contained numerous S100A8-positive fibroblast-like cells 2 and 4 days post injury; numbers declined by 7 days Up-regulation
of S100A8 by FGF-2⁄ IL-1b, down-regulation by TGF-b, and its time-dependent expression in wound fibroblasts suggest a role in fibroblast differentiation at sites of inflammation and repair
Abbreviations
ActD, actinomycin D; BCS, bovine calf serum; BM, bone marrow; BMF, bone-marrow-derived fibroblast-like cells; C ⁄ EBP, CCAAT ⁄ enhancer binding protein; CM, culture medium; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modified Eagle’s medium; DPBS, Dulbecco’s phosphate-buffered saline; DTT, dithiothreitol; ECM, extracellular matrix; ERK, extracellular signal-regulated kinase; FBS, fetal bovine serum; FGF, fibroblast growth factor; Hepes, N-2-hydroxyethylpiperazine-N¢-2-ethanesulfonic acid; HPRT, hypoxanthine phosphoribosyl-transferase; HRP, horseradish-peroxidase; IFNc, interferon c; IL-1b, interleukin-1b; JNK, c-Jun N-terminal kinase; LDL, low-density lipoprotein; LPS, endotoxin; mS100A8, murine S100A8; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; mOxS100A8, HOCl-oxidized murine S100A8; PKC, protein kinase C; ROS, reactive oxygen species; SPF, splenic ‘primary’ fibroblast-like cells; TGF-b, transforming growth factor-b; TNF, tumor-necrosis factor.
Trang 2S100 Ca2+-binding proteins may have evolved from
a calmodulin ancestor [6] and are implicated in
numer-ous intra⁄ extracellular processes and can act as Ca2+
sensors [7] Some (e.g S100A6 (calcyclin) [8], S100A4
[9] and S100A11 [10,11]) have been implicated in
fibro-blast growth and differentiation S100A12 acts as
a pro-inflammatory ‘cytokine’ [12,13] and S100A12
mRNA is induced by interleukin-1a (IL-1a) and tumor
necrosis factor (TNF) in bovine corneal fibroblasts
[14] S100B is also expressed by fibroblasts [15] and
may be involved in regulation of growth arrest and
apoptosis [16], stimulation of cell proliferation [17]
and protection against apoptosis [18] Other S100
proteins also have proliferative and anti-apoptotic
effects [8,9,19]
S100A8 and S100A9 (calgranulins A and B; MRP8
and MRP14) have been associated with leukocyte
dif-ferentiation, inflammation and wound healing [20,21]
They are proposed to be involved in reorganization of
the keratin cytoskeleton and differentiation of
kera-tinocytes and in antibacterial or antioxidant defense in
the wounded or normal epidermis [20–22] Some
func-tions are dependent on S100A8–S100A9 heterodimers,
e.g arachidonic acid binding, antimicrobial defense
and regulation of NADPH-oxidase activity (reviewed
in [20]) S100A9 and the S100A82–S100A9 complex
(calprotectin) stimulate IL-8 production by airway
epi-thelial cells, hence potentially amplifying neutrophilic
inflammation in chronic pulmonary disease [23]
Murine S100A8 is a potent leukocyte chemoattractant
[24] and intracellular S100A8 and S100A9 essentially
regulate phagocyte migration by integrating the
cal-cium and mitogen activated protein kinase (MAPK)
transduction signals thereby controlling reorganization
of the phagocyte microtubular system [25] Expression
of S100A8 in the absence of S100A9 in activated
mu-rine macrophages [26,27] and keratinocytes [22], and
presence of S100A8 without S100A9 in the low density
lipoprotein (LDL) proteome [28] is strong evidence
that S100A8 does not depend on S100A9 for structural
stability [29] and strengthens the proposal for
inde-pendent function We showed that S100A8 scavenges
O2 and hypochlorite, suggesting a role in oxidative
defense [22,30,31] The S100A8 gene is up-regulated by
anti-inflammatory mediators [27], corticosteroids [32]
and by oxidative stress [22] indicating a protective
function Other S100 proteins are also implicated in
cellular responses to oxidative stress In particular in
keratinocytes, S100A2 oxidation and translocation
were proposed as early markers of oxidative stress and
were markedly attenuated in malignant keratinocytes,
favoring a role in oxidant defense rather than in tumor
proliferation [33]
Here we show that factors important in wound heal-ing regulate S100A8, but not S100A9, in fibroblasts Fibroblast growth factor-2 (FGF-2) and IL-1b strongly induced S100A8 via a MAPK-dependent pathway Responses to FGF-2 were amplified by hep-arin and there was strong synergy between FGF-2 and IL1b The protein was cytoplasmic TGF-b suppressed S100A8 induction by FGF-2 but not by IL-1b, sug-gesting important regulatory differences, and promoter analysis confirmed different enhancer elements regula-ting induction by IL-1b and FGF-2 In a rat incisional wound, immunohistochemical studies showed S100A8 expression in fibroblast- and macrophage-like cells, keratinocytes and neutrophils in the incision area We propose that S100A8 may be involved in pathways regulating fibroblast growth and differentiation, pos-sibly by regulating intracellular redox, at sites of inflammation and⁄ or repair ⁄ remodeling
Results
FGF-2 induces S100A8 mRNA in 3T3 fibroblasts Initially, induction of murine S100A8 (mS100A8) mRNA in 3T3 fibroblasts was variable, suggesting that, like microvascular endothelial cells [34], cell–cell contact may be important Fibroblast monolayers grown to various states of confluence, were stimulated with FGF-2 ± heparin and harvested after 24 h Unstimulated confluent (C, Fig 1) or subconfluent (not shown) 3T3 cells had little detectable S100A8 mRNA whereas the bone-marrow (BM) RNA was positive (BM, Fig 1) FGF-2 (1.5 nm) weakly induced the S100A8 gene and responses gradually increased from 21–51% of maximum in cells grown from
30–80% confluence; levels were maximal at conflu-ence (Fig 1) Heparin modulates FGF-2-receptor bind-ing and activity [35] and although it had no direct influence (Fig 6), S100A8 mRNA increased approxi-mately two-fold in confluent 3T3 cells stimulated with FGF-2 and heparin compared to FGF-2 alone Poten-tiation was minimal in cells grown to 80% conflu-ence (Fig 1) indicating that cell–cell contact is important for optimal induction of S100A8 mRNA expression in 3T3 fibroblasts Real-time RT-PCR con-firmed that S100A9 was not induced in confluent cells stimulated with FGF-2 with⁄ without heparin
Kinetics of induction of S100A8 mRNA
in 3T3 fibroblasts mS100A8 mRNA induction in FGF-2-heparin-activa-ted fibroblasts was dose- and time-dependent Up to
Trang 30.15 nm FGF-2 with 1 IUÆmL)1heparin did not induce
detectable mRNA (Fig 2A), whereas 1.5 nm FGF-2
and heparin induced strong responses that were
maxi-mal with 3 nm FGF-2 (Fig 2A); 6 and 15 nm FGF-2
induced mS100A8 mRNA levels that were 80 and
60% of maximal expression, respectively (Fig 2A),
suggesting production of a suppressor Heparin
gener-ated maximal responses at 1 and 10 IUÆmL)1(Fig 2B)
whereas higher amounts (50 and 100 IUÆmL)1) reduced
responses to 72 and 48% of maximum, respectively,
in cells costimulated with 1.5 nm FGF-2 (Fig 2B)
possibly due to soluble heparin-mediated inhibition of
FGF-2-receptor binding [35] For subsequent
experi-ments, 1.5 nm FGF-2 with 1 IUÆmL)1 heparin were
used to stimulate confluent fibroblasts
S100A8 mRNA induction in 3T3 fibroblasts
activa-ted with FGF-2 was evident after 8 h and in the
pres-ence of heparin, mRNA levels increased in parallel up
to 12 h when the response to FGF-2 was maximal and
gradually declined over 36 h (Fig 2C) Potentiation by
heparin was most apparent at 18 h, when mRNA
lev-els were approximately double those with FGF-2 alone
at 12 h In FGF-2-heparin-stimulated cells, S100A8
mRNA levels were maintained for up to 36 h and
declined to20% of maximum by 48 h (Fig 2C)
Because the mS100A8 gene in elicited macrophages and other cells [26,34] is induced by particular pro-inflammatory mediators, endotoxin (LPS, 250–
Fig 1 S100A8 mRNA induction in fibroblasts depends on
ence 3T3 cells grown to approximately 30, 80 and 100%
conflu-ence were stimulated with FGF-2 (1.5 n M ) in the presence (+) or
absence (–) of heparin (1 IUÆmL)1) for 24 h Total RNA was
exam-ined by Northern blotting using an S100A8 riboprobe and a rat 18S
rRNA oligoprobe as indicated BM indicates murine bone-marrow
RNA (positive control) and C, the negative control (unstimulated
cells) The graph indicates percentage maximum of the normalized
response Similar results observed in at least three experiments.
A
B
C
Fig 2 Dose- and time-dependence of induction of S100A8 mRNA
by FGF-2 and heparin in confluent 3T3 cells (A) Confluent 3T3 cells treated with FGF-2 (1.5 p M -15 n M ) and heparin (1 IUÆmL)1) for 24 h and total RNA analyzed by Northern blotting; percentage maximum response is given in the bar graph; C and BM indicate unstimulated fibroblasts’ and bone-marrow RNA, respectively (B) 3T3 cells trea-ted with 1.5 n M FGF-2 were costimulated with increasing amounts
of heparin (0.01–100 IUÆmL)1) (C) Kinetics of induction of S100A8 mRNA 3T3 cells treated with FGF-2 (1.5 n M ) or FGF-2 ⁄ heparin (1 IUÆmL)1) were harvested at the times indicated and Northern analysis performed Similar results observed in three different experiments.
Trang 41000 ngÆmL)1), interferon c (IFNc, 500 UÆmL)1), or
TNF (30 ngÆmL)1) were tested No S100A8 mRNA
was detected in 3T3 cells stimulated for 24 h although
mRNA was substantially augmented with a
combina-tion of LPS, IFNc, and TNF and confluence
influ-enced levels (not shown) Induction was maximal after
24 h and decreased to 47% of maximum by 48 h
(not shown)
IL-1b-stimulated 3T3 fibroblasts express S100A8
mRNA
IL-1b is a strong inducer of the S100A8 gene in
micro-vascular endothelial cells [34] IL-1b induced S100A8
mRNA in confluent 3T3 fibroblasts in a dose- and
time-dependent manner As little as 1 UÆmL)1 was
effective and responses were maximal with 10–
20 UÆmL)1(Fig 3A); 10 UÆmL)1IL-1b were used
rou-tinely mRNA was detected after 8 h, gradually
increased to 28% of maximum after 24 h, was
max-imal at 32 h and declined to 30% of maximum after
48–52 h (Fig 3B)
S100A8 mRNA levels in 3T3 cells stimulated with
FGF-2⁄ heparin almost tripled when costimulated with
0.1 UÆmL)1 IL-1b and were augmented 7.5-fold
with 2 UÆmL)1 Maximal up-regulation was with
10 UÆmL)1 IL-1b which generated a 14.2-fold increase compared to 10 or 20 UÆmL)1 IL-1b alone, or FGF-2 and heparin without IL-1b (Fig 3C) To quantitate this more accurately, relative S100A8 mRNA levels were analyzed by real-time RT-PCR Table 1 shows that IL-1b (10 or 20 UÆmL)1) induced mRNA levels similar to those induced by FGF-2 and heparin The magnitude of synergy was more apparent, with 580-fold more S100A8 mRNA in cells costimulated with FGF-2, heparin and IL-1b compared to those stimula-ted with FGF-2 and heparin
D
C
Fig 3 Effects of IL-1b on S100A8 mRNA induction (A) Northern analysis of mRNA from confluent 3T3 cells stimulated with the given doses of IL-1b for 24 h Results repre-sent three experiments (B) 3T3 cells stimu-lated with 10 UÆmL)1IL-1b were harvested
at the times indicated The line graph indi-cates normalized levels of S100A8 mRNA Similar results observed in two experi-ments (C) Northern blot analysis of conflu-ent 3T3 cells stimulated for 24 h with FGF-2 (F, 1.5 n M ) and heparin (H, 1 IUÆmL)1) in the presence of increasing doses of IL-1b; data representative of at least three different experiments (D) Confluent SPF stimulated with FGF-2 and heparin and IL-1b at the doses indicated The bar graph indicates normalized levels of S100A8 mRNA.
Table 1 S100A8 mRNA levels assayed in activated 3T3 cells by real-time RT-PCR 3T3 cells unstimulated or treated with IL-1b (10 UÆmL)1), FGF-2 (1.5 n M ) + heparin (1 IUÆmL)1) or their combina-tion for 24 h mS100A8, mS100A9, and HPRT mRNA levels were quantitated by real-time RT-PCR S100A8 expression was normal-ized to that of HPRT and expressed as fold-increase compared to medium control The data are means and standard errors of dupli-cate measurements S100A9 mRNA was not detected.
Treatment S100A8 induction S100A9 induction
FGF-2 + heparin + IL-1b 5907 ± 390.2 0
Trang 5Induction of S100A8 mRNA in primary murine
fibroblast-like cells
Because fibroblasts are phenotypically and functionally
heterogeneous and possess unique subpopulations even
in the same tissue [36,37] and because 3T3 fibroblasts
may not be representative of tissue fibroblasts, the
S100A8 gene regulation was assessed in different
fibro-blast populations and RNA from splenic ‘primary’
fibroblast-like cells (SPF) and bone marrow-derived
fibroblast-like cells (BMF) were analyzed Confluent
SPF contained no detectable S100A8 mRNA by
northern analysis (Fig 3D) and cells stimulated for 24 h
with IL-1b (samples 8 and 9), FGF-2 (sample 10), or
FGF-2 and heparin (samples 4 and 5) did not express
the gene IL-1b and FGF-2 combined (samples 6 and 7,
Fig 3D) weakly induced mRNA ( 38–47% of
maxi-mum), responses at 24 h were maximal with 6 nm FGF-2,
1 IUÆmL)1 heparin and 10 UÆmL)1 IL-1b (sample 3)
and half-maximal with 2 UÆmL)1 IL-1b and 1.5 nm
FGF-2 (sample 2, Fig 3D) The same pattern was
observed with BMF stimulated with FGF-2, heparin
and IL-1b (Fig 6D) RT-PCR confirmed that S100A9
was not induced in primary murine fibroblast-like cells
by FGF-2, heparin and IL-1b stimulants (not shown)
Promoter analysis in 3T3 fibroblasts
To examine mechanisms of transcriptional regulation
of the S100A8 gene by IL-1b, and FGF-2 plus hep-arin, 5¢-flanking sequences upstream of the transcrip-tion initiatranscrip-tion site, untranslated intron 1 and sequences upstream of exon 1, were used to evaluate activities of deletion constructs after transient transfection into 3T3 cells (Fig 4) Marked differences between FGF-2-heparin- and IL-1b-induced responses were seen with all constructs IL-1b did not change the luciferase activity
of any construct although northern blotting of mRNA preparations of IL-1b-stimulated cells in the same experiment was positive (not shown) Levels of luci-ferase activity after FGF-2⁄ heparin stimulation were similar for all constructs, with two- to fourfold increa-ses over luciferase activity in unactivated cells The region )94 to )34 bp contained the essential promoter because its deletion completely abrogated luciferase activity The region )178 to )94 bp is responsible for luciferase activity in unactivated cells (basal activity) because deletion strongly reduced basal activity but elements involved in FGF-2 enhancement were retained Consensus motifs for a number of transcrip-tion factors, including CCAAT⁄ enhancer binding
Fig 4 Luciferase activity of S100A8 promoter deletion constructs in fibroblasts stimulated with FGF-2 + heparin or IL-1b Various S100A8 5¢-truncated mutant constructs shown on the left were used and the transcription initiation site was at position +1 Promoterless is the par-ent vector (pGL2) and Promoter is the promoterless with an SV40 promoter (pGL2-promoter) 3T3 cells were transipar-ently transfected with var-ious constructs and a Renilla luciferase construct The cotransfected cells were left unstimulated (in CM) or treated with FGF-2 (1.5 n M ) + heparin (1 IUÆmL)1) or IL-1b (10 UÆmL)1) for 48 h The firefly luciferase activity, normalized to that of Renilla luciferase, was compared with the normalized activity of unstimulated promoter-transfected cells and expressed as normalized fold-induction The right bars represent aver-aged luciferase activities obtained from duplicates from at least three separate experiments Error bars represent standard deviation of the mean.
Trang 6protein (C⁄ EBP), Ets, and E box are located within
this region Constructs not containing the 1st exon and
intron ()178–0 bp) generated positive, but relatively
weak luciferase activities but in the same proportions,
indicating that this region still contains elements
essen-tial for gene induction by FGF-2
The MAPK pathway is involved in S100A8 mRNA
induction by FGF-2 and IL-1b in 3T3 fibroblasts
The MAPK pathway is implicated in S100A8 mRNA
induction by IFNc and LPS in macrophages [26] In
3T3 cells, PD 098059 (MAPK kinase (MEK) inhibitor,
50 lm) suppressed FGF-2- and
FGF-2-heparin-induced S100A8 mRNA by 65 and 62%,
respect-ively, and SB 202190 [c-Jun N-terminal kinase
(JNK)⁄ p38 inhibitor, 10 lm] by 50% (Fig 5A)
Similarly, PD 098059 (50 or 75 lm) or SB 202190 (10
or 20 lm) reduced responses to IL-1b by 70–83%
(Fig 5B) indicating converging pathways in FGF-2-⁄
heparin- and IL-1b-induced S100A8 mRNA induction
in fibroblasts
Cycloheximide completely abrogated the S100A8
gene in 3T3 cells stimulated with FGF-2 ± heparin
(Fig 5C) or IL-1b (Fig 5D), indicating a requirement
for de novo protein synthesis
TGF-b suppresses FGF-2-induced mS100A8 mRNA
in 3T3 and primary fibroblasts
Because TGF-b modulates fibroblast function and
phe-notype [38,39], its effect on S100A8 gene expression
was tested 3T3 cells cultured with TGF-b, heparin,
or a combination of both, did not express S100A8
mRNA (Fig 6A) S100A8 mRNA induction by
FGF-2 ± heparin was almost completely abrogated in
cells simultaneously cultured with TGF-b for 24 h
(Fig 6A) Quantitative RT-PCR showed 34.3-fold
induction of S100A8 mRNA by FGF-2⁄ heparin which
decreased approximately sixfold with TGF-b; mRNA
levels induced by FGF-2 alone were halved in cells
co-incubated with TGF-b (Fig 6B) No S100A9 mRNA
was found in any of the samples tested by RT-PCR in
this experiment (not shown)
In marked contrast to its effect on the FGF-2-⁄
hep-arin-induced responses (lane 10, Fig 6C), TGF-b
(8 pm-0.8 nm) did not reduce S100A8 mRNA levels
induced by IL-1b (lanes 2–4, Fig 6C) However, the
high mRNA levels with a combination of IL-1b,
FGF-2 and heparin (lane 5, Fig 6C) were reduced only by
74% with 0.08 nm TGF-b (lane 7, Fig 6C);
concen-trations up to 0.8 nm did not increase suppression
(lane 8, Fig 6C) S100A8 mRNA induced with
FGF-2⁄ heparin and IL-1b in BMF decreased by
67% with 0.08 nm TGF-b (Fig 6D)
To clarify the mechanism of TGF-b suppression, 3T3 cells incubated for 20 h with FGF-2⁄ heparin in the presence⁄ absence of TGF-b were treated with actinomycin D (ActD) for another 20 h to inhibit fur-ther transcription Cells stimulated with FGF-2 or FGF-2⁄ heparin harvested 20 h after treatment with
A
B
C
D
Fig 5 Involvement of the MAPK pathways and de-novo protein synthesis in induction of S100A8 mRNA in fibroblasts (A) 3T3 cells stimulated (24 h) with FGF-2 (F, 1.5 n M ) or FGF-2 and heparin (H, 1 IUÆmL)1) with ⁄ without 4-h preincubation with PD 098059 (PD, 50 l M ) or SB 202190 (SB, 10 l M ) as indicated (B) 3T3 cells sti-mulated (24 h) with IL-1b (10 UÆmL)1) with ⁄ without 4-h preincuba-tion with PD 098059 (50–75 l M ) and SB 202190 (10–20 l M ) Data represent three experiments (C) 3T3 fibroblasts stimulated with FGF-2 (F, 1.5 n M ) and heparin (H, 1 IUÆmL)1) for 24 h with or without 4-h preincubation with 5 lgÆmL)1cycloheximide (CHX) as indicated.
C, mRNA from unstimulated cells; BM, murine bone-marrow RNA (D) 3T3 cells stimulated with IL-1b (10 UÆmL)1) for 24 h with or with-out 4-h preincubation with cycloheximide (5 and 10 lgÆmL)1) as indi-cated Similar results observed in four experiments.
Trang 7ActD had 126% of the mRNA levels of untreated
cells, indicating somewhat increased mRNA
stabil-ity⁄ accumulation In the presence of TGF-b, mRNA
levels from cells stimulated with FGF-2 or
FGF-2⁄ heparin and treated with ActD were reduced
by 49 and 66%, respectively, indicating reduced mRNA stability Similar results were obtained in two experiments (not shown)
S100A8 protein in activated 3T3 fibroblasts S100A8 was not detected in unprocessed lysates or supernatants of 2.5–3· 106 stimulated⁄ unstimulated confluent 3T3 cells by western blot analyses or by dou-ble-sandwich ELISA (detection limit¼ 0.03 nm [40]) Pull-down experiments, to concentrate the protein from a larger number of cells (20–25· 106), showed
no S100A8 in supernatants or lysates of unstimulated cells (S1 and L1, respectively, Fig 7A) Low levels were present in supernatants (S2, Fig 7A) but S100A8 was mainly cell-associated in cells stimulated for 30 h with FGF-2⁄ heparin and IL-1b (L2, Fig 7A) Separ-ation was initially performed in the presence of dithio-threitol (DTT) to increase the yield by reducing disulfide-linked complexes and the characteristic mono-meric mass of 10 kDa (rA8; Fig 7A) was confirmed
In an alternative approach, supernatants and lysates
of activated cells were concentrated using an affinity
A
B
C
D
Fig 6 TGF-b regulates S100A8 mRNA induction by FGF-2 (A)
Nor-thern analysis of mRNA from 3T3 cells stimulated with FGF-2
(F, 1.5 n M ), heparin (H, 1 IUÆmL)1), TGF-b (T, 0.08 n M ) alone or in the
combinations indicated for 24 h; C, unstimulated cells (B) RT-PCR
analysis of the RNA samples used in (A) Levels of S100A8 mRNA
were compared to those of HPRT in the corresponding samples and
ratios of S100A8 mRNA ⁄ HPRT are indicated for each sample (C)
Cells stimulated with FGF-2 (F, 1.5 n M ) ⁄ heparin (H, 1 IUÆmL)1) with
or without IL-1b (10 UÆmL)1) in the presence or absence of
increas-ing concentrations of TGF-b (8 p M -800 p M ) as indicated BM, total
bone-marrow RNA (D) Confluent BMF treated for 24 h with the
indi-cated doses of FGF-2, heparin, IL-1b and TGF-b.
A
B
Fig 7 Expression of S100A8 protein in 3T3 cells (A) 20–25 · 10 6
confluent 3T3 cells unstimulated (S1, L1) or stimulated with 1.5 n M
FGF-2 and 1 IUÆmL)1heparin with (S2, L2) or without 10 UÆmL)1 IL-1b (S3, L3) for 30 h Total cell-associated (L) and secreted (S) protein was immunoprecipitated, SDS ⁄ PAGE was in the presence
of DTT (100 m M ), and western analysis using anti-mOxA8 IgG Recombinant murine S100A8 (4, 40 and 100 ng) was the positive control Results obtained in two experiments (B) Approximately 20–25 · 10 6 cells were either unstimulated (lane 1) or stimulated (lane 2) for 30 h; cell lysates and supernatants (600 lL) were sub-jected to immunoaffinity purification through an anti-mS100A8 affin-ity column and C4 RP-HPLC The collected fractions were then analyzed by SDS ⁄ PAGE and western blotting in the absence of DTT Recombinant mS100A8 (25 ng) was used as the positive control.
Trang 8support and bound protein subjected to C4
reverse-phase HPLC Recombinant mS100A8 elutes as a single
peak at 19.8–20 min (not shown) Because no major
peak were obtained (possibly due to low levels),
fractions were collected between 17.25 and 21.25 min
(covering the expected retention-time range for native
mS100A8 monomer and dimer), and lyophilized No
mS100A8 was detected in fractions from supernatants
(not shown) or lysates (lane 1, Fig 7B) of
unstimu-lated cells Western blotting of the three fractions from
lysates of stimulated cells collected over 18.25–20.25 min
(lane 2, fraction collected at 18.25–19.25 min, Fig 7B)
contained components of molecular mass 20 kDa, with
the same migration profile as dimeric mS100A8,
contained in the positive control (lane 3, Fig 7B) No
mS100A8 monomer (10 kDa) was detected This was
unexpected as the same conditions have yielded
mono-meric and complexed forms of mS100A8 in other
stimulated cell types [34,41]
No S100A8 was found in unstimulated 3T3 cells stained with preimmune IgG or an antibody against HOCl-oxidized mS100A8 (mOxS100A8) (Fig 8A,B, respectively); 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei were evident Similarly, stimulated cells stained with the nonimmune IgG were unreactive (Fig 8C) Approximately 10–30% of 3T3 cells stimula-ted with FGF-2⁄ heparin ⁄ IL-1b showed bright cyto-plasmic fluorescence (Fig 8D) When costimulated with TGF-b, S100A8-positive cells dropped to5% of total (Fig 8E) Confocal microscopy clearly showed localization of S100A8 (red fluorescence) in the cyto-plasm with no obvious associations with cytoskeletal structures (Fig 8F)
S100A8 expression in rat dermal wounds
To assess in vivo expression of S100A8 in fibroblasts, rat dermal wound tissue was used Examination of
Fig 8 Immunofluorescent detection of
S100A8 Confluent 3T3 cells grown in slide
chambers were unstimulated (A, B) or
stimulated for 30 h with 1.5 n M FGF-2,
1 IUÆmL)1heparin and 10 UÆmL)1IL-1b in
the absence (C, D, F) or presence of
0.08 n M TGF-b (E) Permeabilized fixed cells
stained with nonimmune rabbit IgG (A, C) or
mOxA8 (B, D, E, F) followed by
anti-rabbit IgG-Alexa-Fluor-568 and the nuclear
stain, DAPI, were analyzed by
fluores-cent ⁄ confocal microscopy A high-power
image of a representative S100A8-positive
fibroblast stimulated with FGF-2, heparin
and IL-1b is shown (F) Similar results were
obtained in two experiments
Magnifica-tions: (A–E) 400·; (F) 600·.
Trang 9wounded rat skin 2 days post injury showed an
inten-sely anti-S100A8-immunoreactive scab, containing
S100A8-positive neutrophils (Fig 9Aa), impinging on
the injured, and surrounding the normal epidermis In
normal epidermis, the superficial more differentiated
keratinocytes reacted with anti-mS100A8 more
inten-sely than those in the stratum basale which contains
the proliferating keratinocytes In the dermis, small
dilated microvessels containing intensely stained
S100A8-positive neutrophils were evident, representing the in situ positive controls (Fig 9Ab) Based on morphology, extravascular S100A8-positive cells were identified as macrophages or extravasating neutro-phils Some spindle-shaped fibroblast-like cells closely apposed to, and aligned with collagen fibers were also relatively intensely mS100A8-positive (Fig 9Ab) although staining was heterogeneous These were iden-tified as fibroblast-like cells, based on morphology,
Fig 9 Immunohistochemical localization of S100A8 in rat dermal wounds (A) Immunostaining of rat dermal wound 2 days after injury (Aa) Low-power (200 ·) view of the wounded dermis beneath the neutrophil-rich S100A8-positive scab (S) Black arrows indicate S100A8-positive fibroblast-like cells apposed to collagen fibers indicated by C Neovessels and sebaceous glands are indicated by V and SG, respectively Delineated inset in (Aa) corresponds to (Ab) (400 ·) where neutrophils and macrophage-like cells are indicated by red and blue arrows, respectively Black arrows point to S100A8-positive fibroblast-like cells (B) Staining of rat wound 4 days after injury Anti-S100A8 IgG (Ba, Bb) and nonimmune IgG (Bc) were used (Ba) The scab (S) is evident along the wounded epidermis and encroaching normal uninjured keratinocytes (K) Some hair follicles (H), small vessels (V) and sebaceous glands (SG) surrounded by collagen (C) fibers are evident (Bb) An area of granulation tissue showing macrophage-like (blue arrows) and fibroblast-like cells (black arrows) around neovessels (V) were S100A8-positive Some S100A8-negative fibroblast-like cells are indicated by asterisks (Bc) Immunostaining of the 4-day wound with nonimmune IgG (C) Anti-mS100A8 immunostaining of rat wound 7 days after injury (Ca) Low-power view of an area of mature granulation tissue and scar formation rich in collagen fibers and fibroblasts stained with anti-mS100A8 IgG The epidermal keratinocytes are evident to the right of the wounded area (K) The inset shown in (Ca) corresponds to (Cb) where S100A8-negative fibroblast-like cells are indicated by arrows.
Trang 10location in the granulation tissue and alignment with
collagen fibers as no appropriate specific marker to
detect rodent fibroblasts was available Similarly, in
a mouse model of delayed-type hypersensitivity
responses, about 10% of fibroblast-like cells were
S100A8-positive (S Leong and C.L Geczy;
unpub-lished data)
In 4-day wounds, granulation tissue (macrophages,
fibroblasts, neovessels and collagen) was
well-estab-lished and most spindle-shaped fibroblast-like cells and
macrophages stained positively with anti-mS100A8
(Fig 9Ba and b) Underneath the scab containing
intensely stained S100A8-positive neutrophils, the
uninjured and proliferating keratinocytes were
S100A8-positive; superficial cornified keratinocytes were more
intensely positive, compared to controls (Fig 9Ba
and c) In 7-day wounds, healing was evident, with
little inflammatory component, decreased⁄ no
vascu-larity and many fibroblasts aligned along collagen
fibers (Fig 9Ca) Fibroblast-like cells were
S100A8-negative and keratinocytes were weakly positive
(Fig 9Cb)
Discussion
Fibroblasts are ubiquitous sentinel cells that
partici-pate in local inflammatory processes by directly
inter-acting with infiltrating leukocytes Leukocytes residing
in, or recruited into tissues are surrounded by a
mesh-work of fibroblasts and both cell types produce, and
are influenced by mediators that regulate
inflamma-tion, wound healing, fibrosis, oxidative processes and
vascular remodeling This study provides the first
evi-dence that S100A8 is regulated in fibroblasts by
partic-ular growth factors, further supporting its role in
inflammatory processes
FGFs stimulate proliferation of many cell types
involved in wound healing including endothelial cells,
fibroblasts and keratinocytes and are essential for
sur-vival, replication, differentiation and migration of
var-ious cell types during embryonic and fetal development
[42,43] Interestingly, S100A8 has roles in cell
migra-tion [24,25] and S100A8, but not S100A9 [29,44], is
essential for embryonic development [45] FGF-2
up-regulated S100A8, but not S100A9, mRNA in 3T3
and primary fibroblasts A fragment of human S100A8
(S100A821)45) is chemotactic for fibroblast-like
perio-dontal ligament cells [46] and this may be a function
of S100A8 released as a consequence of wound healing
and in an inflammatory environment S100A8
induc-tion was confluence-dependent (Fig 1), a requirement
similar to its induction in microvascular
endothel-ial cells [34] Although fibroblasts do not normally
establish contacts in vivo, in culture they establish gap junctions [37] indicating metabolic interdependence Cell–cell⁄ cell–ECM contacts may provide additional signals for optimal induction of the S100A8 gene
in vitro and because of the potentiation exhibited by heparin, ECM heparan sulfate may be important FGFs bind heparin and heparan sulfates in positively charged pockets that facilitate formation and stabiliza-tion of FGF-2 dimers and higher oligomers, resulting
in a more stable FGF–FGFR signaling complex [47] Heparin also protects FGF against glycosylation [48] and proteolysis and FGF binding to ECM increases its immediate availability and durability of action [47] Responses to FGF-2 were strongly up-regulated and prolonged by heparin (Figs 1–3 and 6), but heparin alone had no effect (Fig 6A), implying facilitation at the level of the FGF–FGFR interaction
IL-1b has high amino-acid homology with FGF-2, and is a potent inducer of the S100A8 gene in micro-vascular endothelial cells [34] and weak inducer in macrophages [26] IL-1b up-regulated S100A8 mRNA
in fibroblasts (Fig 3) and exceptionally strong synergy was observed when in combination with FGF-2⁄ hep-arin, obvious at the mRNA (Table 1) and protein level (Figs 7 and 8) In contrast, LPS, IFNc and TNF, which induce S100A8 in macrophages [26] and endo-thelial cells [34] only induced S100A8 mRNA when used in combination, suggesting cooperative pathways IL-1b synergistically increased S100A8 mRNA in pri-mary fibroblasts costimulated with FGF-2⁄ heparin (Figs 3D and 6D) Because of functional and pheno-typic heterogeneity [36,37], fibroblasts from various sources were tested and because responses in primary cells were relatively weak, quantitative RT-PCR was used to detect the S100A8 gene and to assess S100A9 gene coinduction The stability of S100A8 protein in neutrophils is suggested to be dependent on S100A9 coexpression [29], but, like the situation in activated murine macrophages [26,27] and keratinocytes [22], S100A9 was not coinduced with S100A8 in any fibro-blast type tested, strongly supporting our proposal that S100A8⁄ S100A9 coexpression is not mandatory for the function or stability of murine S100A8 This may not
be the case with the human proteins that are generally coexpressed [49] although S100A8 alone was detected
in the human LDL proteome by mass spectrometry and peptide mass fingerprinting [28]
Negligible S100A8 was secreted in response to IL-1b and FGF-2⁄ heparin (Fig 7A) This is in stark contrast with activated macrophages which secrete high levels
in response to various stimulants, particularly LPS with IL-10 or prostaglandin E2 [26,27], but similar to IL-1-activated microvascular endothelial cells [34] and