* Cytokine Research Unit and the
Inflammation Research Unit, School of Pathology, The University of New South Wales, Kensington 2052, Australia
Correspondence: Carolyn L. Geczy, Cytokine Research Unit, School of Pathology, The University of New South Wales, Kensington, New South Wales 2052, Australia. E-mail: C.Geczy{at}unsw.edu.au
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70 nM) was present in synovial fluid (SF) from
rheumatoid arthritis patients, and synovium contained A12-positive
neutrophils in the sublining and interstitial region, often surrounding
the perivasculature but rarely in the synovial lining layer, although
some macrophages were positive. The A12 gene was transiently
up-regulated in monocytes by tumor necrosis factor 
(6 h); induction
by lipopolysaccharide (LPS) was sustained (12–48 h). A12 may
contribute to leukocyte migration in chronic inflammatory
responses.
Key Words: leukocyte recruitment • rheumatoid arthritis • gene induction
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A12 has highest amino acid sequence homology with S100A9 (47%), which is expressed constitutively together with S100A8 in neutrophils. The S100A8/A9 complex, known as calprotectin [8 ], is associated with chronic inflammation including rheumatoid arthritis (RA), cystic fibrosis, Crohn’s disease, ulcerative colitis, allergic dermatitis, infection, and psoriasis [8 ]. Since our first demonstration of the potent chemotactic activity of murine S100A8 (mA8, formerly called "CP-10") for neutrophils and monocytes in vitro [9 ] and in vivo [10 , 11 ], S100A2 and S100A7 were also reported to have chemotactic activity for eosinophils and lymphocytes, respectively [12 , 13 ], suggesting a new class of chemoattractants.
Here we present additional evidence for the chemotactic properties of
human A12 (hA12) for human neutrophils and monocytes in vitro and in
vivo. A12 initiated actin polymerization accompanied by a calcium flux
in monocytoid cells. We show that A12 in monocytes was up-regulated by
LPS and tumor necrosis factor (TNF), and we report the first
evidence of expression of A12 in the synovium and SF from rheumatoid
patients, supporting the notion that A12 may contribute to the
pathogenesis of acute and chronic inflammatory responses.
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(endotoxin content, <1
EU/mg; specific activity, 6 x 107 U/mg) was a
gift from G. R. Adolf (Ernst-Boehringer Institut für
Arzneimittelforschung, Vienna, Austria). Recombinant interleukin
(IL)-1ß (specific activity, 1.87 x 105 U/µg) was
purchased from R & D Systems (Minneapolis, MN).
Cell culture
THP-1 cells (American Type Culture Collection, Manassas, VA) and
Mono Mac 6 cells (German Collection of Microorganisms and Cell
Cultures, Braunschweig, Germany) were maintained in RPMI 1640 as stated
below.
Cells were routinely cultured at 37°C in 5% CO2 in air in RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 2 mM L-glutamine (Flow Laboratories, Irvine, Scotland), 100 U/mL of penicillin, 100 µg/mL of streptomycin (Sigma, St Louis, MO), and 10% heat-inactivated bovine calf serum (BCS; Hyclone Laboratories, Logan, UT) unless otherwise stated. The medium was sterilized by filtration through Zetapor 0.2-µm membrane (Cuno, Meriden, CT) to remove contaminating traces of endotoxin. Endotoxin levels in reagents and medium were routinely monitored and used at <50 pg/mL, tested by the chromogenic Limulus amoebocyte lysate assay (Cape Cod Associates, Woods Hole, MA).
Citrated blood from normal donors (Department of Haematology, Prince of Wales Hospital, Sydney, Australia) was used as a source of neutrophils and monocytes in most experiments. Neutrophils were isolated using Mono-Poly resolving medium (Flow Laboratories) as previously described [9 ].
For isolation of monocytes and lymphocytes, erythrocytes were removed by sedimentation in 0.5% dextran T500 (Pharmacia, Uppsala, Sweden), 0.5% D-glucose (BDH Laboratory Supplies, Poole, England) at room temperature for 45 min. Leukocytes were collected from the plasma by centrifugation at 600 g for 10 min, washed twice in Ca2+, Mg2+-free Dulbecco’s phosphate-buffered saline (PBS, pH 7.3) (Sigma) supplemented with 0.38% sodium citrate, and then suspended in PBS. The cells were then layered on Lymphoprep (Nycomed, Oslo, Norway) and centrifuged at 700 g for 30 min at room temperature. Mononuclear cells were collected from the interface, washed twice with PBS, and suspended in RPMI 1640 containing 2% BCS at 5 x 106/mL, and monocytes were left to adhere onto tissue culture plates (Falcon; Becton Dickinson Labware, Lincoln Park, NJ). Nonadherent cells, which comprise >95% lymphocytes, were removed after incubation at 37°C for 30 min. Adherent monocytes were washed twice with warmed medium and detached from the surface after incubating in RPMI containing 10% BCS overnight.
Isolation of hA12
Erythrocytes remaining in leukocyte suspensions from whole blood
by dextran sedimentation were removed by lysis in ice-cold isotonic
ammonium chloride (155 mM NH4Cl, 10 mM NaHCO3,
and 0.1 mM EDTA) for 10 min. Cells were washed with PBS and centrifuged
at 600 g for 5 min at 4°C. The neutrophil pellet was lysed
in 0.2 mM Tris-HCl–0.1% Triton X-100 (Sigma), pH 8.0, containing
protease inhibitors (Complete Protease Inhibitor Cocktail Tablet;
Boehringer Mannheim, Mannheim, Germany). Cell debris was removed by
centrifugation at 20,000 g for 10 min at 4°C, and the
supernatant was dialyzed against 25 mM NaOAc, 25 mM NaCl, pH 4.5, at
4°C, followed by centrifugation at 20,000 g for 10 min.
The dialysate was fractionated by cation-exchange chromatography on a Mono S HR 5/5 column (Pharmacia) equilibrated with 25 mM NaOAc, 25 mM NaCl, pH 4.5. Elution was performed over 25 min at a flow rate of 1 mL/min with a linear concentration gradient of 0.025–1.0 M NaCl in 25 mM NaOAc, pH 4.5. Fractions of 1.0 mL each were collected and analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by silver staining and Western blot analysis. A12 was identified using polyclonal anti-A12 rabbit immunoglobulin G (IgG). Fractions containing A12 (eluted in 0.35–0.42 M NaCl) were pooled and further fractionated on a C4-RP-HPLC Vydac 300 Å, 5 µm, 250 mm by 4.6 mm column (Separations Group, Hesperia, CA) with a gradient of 35–50% acetonitrile–0.1% trifluoroacetic acid at 1 mL/min over 35 min. A12 eluted in 45% acetonitrile; purity was confirmed by SDS-PAGE, silver staining, and Western blot analysis. The protein stored in acetonitrile was lyophilized and resuspended in sterile saline immediately before use. In A12 preparations used for in vitro and in vivo studies, endotoxin levels were <2 pg/10 µg of A12.
A12 expression plasmid and isolation of recombinant protein
Single-stranded cDNA was synthesized from human bone marrow
polyadenylate-RNA using a SuperScriptTM Preamplification System
(GIBCO). hA12 DNA was amplified from the cDNA mix with two
oligonucleotides 5'-CGGGATCCACAAAACTTGAAGA GCATCTGGAGGG-3'
and 5'-CGGGATCCCTACTCTTTGTGGGTGTGG-3'. PCR-amplified
fragments were digested with BamHI and cloned into the
Glutagene pGEX2T bacterial expression vector (Pharmacia) to produce the
recombinant expression plasmid.
The glutathione-S-transferase–A12 fusion protein was induced and
affinity purified essentially as previously described
[14
]. The fusion protein was extracted from the
bacterial lysate using glutathione-agarose (Sigma), and bound fusion
protein was eluted with 25 mM glutathione. Recombinant A12 (rA12) was
cleaved from the fusion protein with bovine -thrombin (Sigma) in 25
mM Tris-HCl (pH 7.5), 250 mM NaCl, 5 mM EDTA, and 0.5% Triton X-100
incubated at 37°C for 24 h. Preparative C8-RP-HPLC (Aquapore, 10
mm by 100 mm; Applied Biosystems Inc., Foster City, CA) was used to
separate rA12 from cleaved fusion protein in a 20-min gradient of
35–65% acetonitrile–0.1% trifluoroacetic acid at 3 mL/min. rA12 was
further purified on C4-RP-HPLC as described above, and purity was
confirmed by SDS-PAGE and Western blot analysis. A12, stored in
acetonitrile at -80°C, was lyophilized just before use and
resuspended in saline for testing. Endotoxin levels were <2 pg/10 µg
of A12.
Production of polyclonal anti-A12 antibodies
New Zealand White rabbits were immunized by multiple intradermal
dorsal injections with 50 µg of pure human native A12 in 0.9% NaCl
mixed in complete Freund’s adjuvant (Sigma) and boosted 2 and 6 weeks
later in incomplete Freund’s adjuvant (Sigma), containing the same
amount of protein. Antisera were collected at 2-week intervals
thereafter and tested for reactivity to S100A8, -A9, and -A12 on
Western blots. IgG from prebleed rabbit serum and from anti-A12 sera
was purified by protein A affinity chromatography (Pharmacia). The
anti-A12 polyclonal IgG cross-reacted weakly with S100A8 and S100A9,
and reactivity was removed by affinity absorption with recombinant A8
and A9 (prepared according to protocols described elsewhere
[43
]) coupled to CNBr-Sepharose (Pharmacia).
RNA extraction and semiquantitative reverse transcriptase-PCR
Total RNA was extracted using the single-step guanidinium
thiocyanate-phenol-chloroform method [15
]. To analyze
expression of A12 mRNA, oligonucleotides corresponding to the
N-terminal and C-terminal amino acids of the protein
(5'-ACAAAACTTGAAGAGCATCTGGAGGG-3' and 5'-CTACTCTTTGTGGGTGTGG-3') were
used and yielded a 273-bp fragment. Typically, 0.5 µg of total RNA
was reverse-transcribed to the first-strand cDNA using the
SuperScriptTM preamplification system (GIBCO). The resulting cDNA mix
(1/20 of cDNA reaction volume) was amplified using 10 pmol of each A12
primer and 2.5 U of Taq DNA polymerase (Perkin-Elmer, Branchburg, NJ).
cDNA concentrations of the samples were monitored by the expression of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH), using the two primers
5'-CCACCCATGGCAAATTCCATGGCA-3' and 5'-TCTAGACGGCAGGTCAGGTCCACC-3'),
which yielded an amplification product of 600 bp. PCR was performed in
a GeneAmp PCR System 2400 (Perkin-Elmer); cycling parameters of 95°C
for 2 min followed by 30 cycles of 95°C for 45 s, 60°C for 1
min, and 72°C for 1 min were used.
Detection of A12 protein
A double-sandwich enzyme-linked immunosorbent assay (ELISA) was
developed. Flat-bottom 96-well microtiter plates (Maxisorp; Nunc,
Roskilde, Denmark) were coated at 50 µL/well with 5 µg/mL of
anti-A12 IgG in 0.05 M sodium carbonate buffer, pH 9.6; incubated for
2 h at 37°C; washed four times with 20 mM Tris-HCl, 150 mM NaCl,
and 0.01% Tween 20, pH7.4 (wash buffer) and blocked with 0.1%
ovalbumin (Sigma) in wash buffer for 1 h at 37°C. Plates were
washed four times before samples were added. Samples diluted in RPMI
1640 containing 0.1% BSA (100 µL/well) were incubated overnight at
4°C. Recombinant A12 (0.0625–2.5 nM) diluted in RPMI 1640 and 0.1%
BSA was included as the standard. After four washes, biotinylated
anti-A12 IgG (4 µg/mL, 100 µL/well) was added, incubated for 1 h at 37°C, and washed four times with wash buffer. Plates were
incubated with streptavidin-horseradish peroxidase conjugate (1:1,000
dilution; Amersham, Buckinghamshire, England) for 1 h at 37°C.
After washing, plates were incubated with ABTS
[2,2'-azinobis(3-ethylbenzthiazoline sulfonic acid)] and
H2O2 (Kirkegaard & Perry, Gaithersburg, MD) for
1 h at room temperature, the reaction was stopped with 1% SDS,
and absorbency at 405 nm was measured (Titertec Multiscan MCC/340;
Labsystem, Helsinki, Finland).
Chemotaxis assay
Cells (5 x 106/mL) suspended in Hanks balanced
salt solution (HBSS; Sigma) and 0.2 % BSA were incubated with 5 µM
calcein AM (Molecular Probes Inc, Eugene, OR) for 15 min at 37°C in
5% CO2 in air, washed twice with HBSS, and resuspended at
0.5 x 106/mL in RPMI 1640 and 0.2% BSA. Assays were
performed using Neuro Probe MBA 96-well chambers fitted with
polycarbonate membranes (Neuro Probe, Inc., Bethesda, MD) with pore
sizes of 5 µm for neutrophils and monocytes, or 8 µm for THP-1
cells. Native A12 or rA12 (10-13 to 10-8 M)
diluted in RPMI 1640 and 0.2% BSA (410 µL) were placed in the lower
compartment, and cells (300 µL) in the upper compartment. The chamber
was incubated at 37°C in 5% CO2 in air for 90 min for
neutrophil chemotaxis or for 2 h for monocytes or lymphocytes. The
number of migrated cells collected in the lower chamber was measured by
fluorescence at 
ex = 485 nm and
em = 530 nm using a CytoFluor® multiwell plate
reader system (PerSeptive Biosystems, Framingham, MA). Cell numbers
were extrapolated from a standard curve calibrated based on a
fluorescence reading of a known number of cells. C5a (Sigma) or
N-formyl-methionyl-leucyl-phenylalanine (fMLP) (Sigma;
10-9 M) was used as a positive control in all experiments.
Data from at least three experiments were analyzed using the Student’s
t-test.
Actin polymerization
Cells (0.5 x 106/mL) suspended in HBSS and 0.2
% BSA were prewarmed at 37°C in 5% CO2 before addition
of A12 preparations diluted in the same buffer at 37°C. The reaction
was performed in 96-well plates and initiated by mixing 2.5 x
104 cells with 50 µL of A12 preparations to give final
concentrations between 10-12 to 10-9 M. At
various times, fixative (3.7% formaldehyde) was added, the plate was
immediately placed on ice, and cells were permeablized and stained for
F-actin for 60 min with a mixture of 0.05% (w/v) digitonin
(Calbiochem, San Diego, CA) and 0.5 µM fluorescein isothiocyanate
(FITC)-phalloidin (Sigma). Cells were then pelleted and washed twice in
HBSS. Fluorescence emission was assayed at ex = 485
nm and em = 530 nm. fMLP (10-9 M) was
used as positive control in all experiments.
Measurement of cytosolic Ca2+ flux
Changes in [Ca2+]i were monitored
using the fluorescent probe Fluo-3 (Molecular Probes Inc.). Cells
(5 x 106/mL) suspended in HBSS containing 20 mM
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES), 10 mM glucose, and 0.2% BSA were incubated for 30 min at
37°C with 10 µM Fluo-3 added from a 10 mM dimethyl sulfoxide stock
solution. The cell suspension was then diluted with an equal volume of
HBSS and 0.2% BSA, incubated for 30 min at 37°C, centrifuged, washed
in HBSS, and resuspended in HBSS plus 20 mM HEPES, 10 mM glucose, and
0.2% BSA (2 x 106/mL). Prior to the assay, 5 µL of
anti-fluorescein–rabbit IgG (A-889; Molecular Probes Inc.) per mL of
cell suspension was added, and 50 µL of cell suspension was then
loaded to a 96-well plate containing 50 µL of various dilutions of
A12. Fluorescence emission was assayed immediately for 10–20 cycles
(10 s/cycle) at ex = 480 nm and
em = 530 nm. C5a or fMLP (10-8 M) was
used as positive controls in all experiments.
Leukocyte recruitment in vivo
Specific pathogen-free-BALB/c mice (8–10 weeks old) were
injected intraperitoneally with 1.0 mL of HBSS containing 10 µg of
rA12 or native A12 (total endotoxin level < 2 pg). Cells were
harvested by peritoneal lavage using 6–8 mL of HBSS plus 3.8% (w/v)
sodium citrate, and cell numbers were counted. Viability of the washed
cell suspensions was >98% as determined by trypan blue exclusion.
Differential staining was performed using a modified Wright-Giemsa
stain (Diff-Quik Set; Lab-Aids, Sydney, Australia) on cytospin
preparations. An average of 300 cells/slide were counted, and mean
numbers of total cells, monocytes, lymphocytes, and neutrophils from at
least three animals were recorded.
Isolation of S100 proteins from SF
SF obtained from RA patients was depleted of cells by
centrifugation at 600 g for 5 min at 4°C, and 3 mL of the
fluid was mixed with an equal volume of ice-cold PBS and applied to
rabbit anti-A9 or anti-A12 IgG coupled to CNBr-Sepharose (Pharmacia).
Samples were mixed for 16 h at 4°C on a rotating wheel, and
unbound protein was removed by washing twice in 10 mL PBS plus
10 mL of 1.0 M NaCl and twice with 10 mL of PBS. Bound protein was
eluted with two 2-mL washes of 0.1 M glycine, pH 3.0, and separated on
a Vydac C4 RP-HPLC column as described above. Eluted proteins were
concentrated by lyophilization and analyzed by SDS-10% PAGE followed
by silver staining and Western blot analysis.
Immunohistochemical staining of rheumatoid synovium
Formalin-fixed, paraffin-embedded, inflamed synovial tissue
(n = 4) from patients with RA was serially sectioned (4
µm) and processed immunohistochemically to determine the expression
and cellular source of A12. In brief, tissue sections were blocked with
goat serum [1:5 (v/v)] diluted with 2% BSA plus 0.05 M Tris-buffered
saline, pH 7.6, for 30 min at room temperature, followed by an
overnight incubation at 4°C with 2 to 10 µg/mL of antibody to A12,
CD68 (Dako, Sydney, Australia) or isotype control IgG (Dako). Sections
were extensively washed in Tris-buffered saline, after which
biotin-conjugated antibodies (Dako) were added (goat anti-rabbit for
A12 and goat anti-mouse for CD68) for 30 min. Sections were rinsed and
incubated with horseradish peroxidase-conjugated streptavidin (Dako),
and immunoreactivity was revealed by adding 3-amino-9-ethylcarbazole
(Sigma), followed by counterstaining with hematoxylin. Other control
reactions included sections incubated in the absence of primary
antibody.
Statistical analysis
Data are presented as the means ± SE of the
specified number of experiments. Statistical comparisons were performed
using Student’s t-test for unpaired data.
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Figure 1. Chemotactic response of THP-1 cells (A) and monocytes (B) to increasing
doses of hA12 (A), rA12, or (B) native A12 (•). Responses
were compared to the basal migration in RPMI 1640-0.2% BSA ( ) and
10-9 M C5a ( ). The ability of A12 to induce directional
versus random migration was confirmed by adding equivalent
concentrations of A12 in the upper and lower chambers ( ). Data,
expressed as mean numbers of migrated cells in six wells ±
SD of a single experiment, are given and are representative
of three experiments.
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Figure 2. Chemotactic response of THP-1 cells, monocytes, neutrophils and
lymphocytes towards C5a (10-9 M) (hatched bars) or
recombinant A12 (10-10 M) (filled bars). Data shown are
means ± SD of results of three experiments of six
wells, in which the ratio of migration compared with control is
given.
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30 s) in polymerization provoked by fMLP
and rA12 compared with unstimulated cells. Increases in actin
polymerization were followed by characteristic oscillations of
depolymerization and polymerization. The initial response to rA12 at
between 10 and 30 s was followed by stronger polymerization
between 45 and 60 s.
 View larger version (12K): [in a new window] |
Figure 3. (A) Relative F-actin content in THP-1 cells stimulated with fMLP
(10-9 M) () or rA12 (10-11 M) (•) over
120 s. Basal F-actin content of cells in RPMI 1640/0.2% BSA is
shown as the dashed line. Samples were analyzed by fluorescence
emission of FITC-phalloidin-labeled cells as described in Materials and
Methods, and data are representative of three experiments. (B) Effect
of C5a (10-8 M) () or rA12 (10-11 M) (•)
on [Ca2+]i in THP-1 cells. Basal
[Ca2+]i of cells in RPMI 1640/0.2% BSA is
shown as the dashed line. Samples were analyzed by fluorescence
emission of Fluo-3-labeled cells as described in Materials and Methods.
The data are representative of three experiments.
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A12 recruits leukocytes in vivo
To determine whether A12 activities in vitro reflect its ability
to recruit leukocytes in vivo (Fig. 4
), A12 (n = 9) was injected intraperitoneally into
mice and was found to increase peritoneal leukocyte numbers from
1.8 ± 1.0 x 106 in animals (n =
12) injected with vehicle control to 4.2 ± 1.9 x
106 after 8 h (P < 0.01). Neutrophils
increased from 1.59 ± 0.718 x 105 in control
mice to 18.27 ± 7.42 x 105 cells in mice
injected with A12 (P < 0.05) and declined to normal
levels by 24 h (P < 0.05 compared with 8 h).
At 8 h, macrophage numbers increased above those of control mice
[from 13.86 ± 0.42 x 105 to 18.97 ±
1.29 x 105 cells (P < 0.01)] and
were even higher at 24 h [23.1 ± 1.852 x
105 (P < 0.05) compared with levels at 8
h]. In contrast, lymphocyte numbers at 8 and 24 h remained
relatively unchanged compared with numbers in control-injected mice.
Total leukocyte numbers in A12-injected mice declined by 24 h
although levels were still somewhat higher compared with controls, but
differences were not significant [3 ± 2.1 x
106 (n = 6; P > 0.05)].
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Figure 4. Accumulation of leukocytes elicited by intraperitoneal injection of 10
µg A12. Insert table represents the total number of peritoneal
leukocytes accumulated 8 and 24 h after injection with A12 or
vehicle control. Monocytes, neutrophils, and lymphocytes recruited at 8
(gray bars) and 24 h (black bars) postinjection with A12 or with
vehicle control (white bars) were determined by differential staining.
Results are the means ± SD from at least three
animals.
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Figure 5. S100 proteins in SF from RA patients. RP-HPLC elution profiles of
S100 proteins extracted by anti-A12 IgG Sepharose (—) and anti-A9 IgG
Sepharose (----) from 2 mL of fluid. Insert shows silver-stained
image of the SDS-PAGE; lane 1, A12 bound by anti-A12 IgG Sepharose
(retention time 17.7–17.9 min), lane 2, A8 (18.7–19.2 min), and lane
3, A9 (19.5–19.9 min) bound by anti-A9 IgG Sepharose. Samples from
three patients were analyzed with similar results.
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Figure 6. Localization of A12 in RA synovium. (A) Positive staining of
perivascular neutrophils (x60); (B) almost total absence of A12 in the
lining layer (L; x20); (C) A12-positive macrophages (asterisk) at the
lining layer (x60); (D) sequential section to panel C, stained with
macrophage marker CD68 (x60). Digital images were on the same
synovium, representative of three patients.
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Figure 7. LPS induces A12 mRNA expression in monocytes. Dose-dependent induction
of A12 mRNA in Mono Mac 6 cells by LPS for 24 h (A). Time course
of A12 mRNA induction by LPS (100 ng/mL) in Mono Mac 6 cells (B) and
peripheral blood monocytes (C). mRNA was analyzed by semiquantitative
RT-PCR as described in Materials and Methods; quantitation densitometry
of A12 and GADPH mRNA is given. Results are representative of two
experiments.
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and IL-1ß was investigated. Induction
by TNF in Mono Mac 6 cells and monocytes was transient, with optimal
expression of more than twofold above baseline levels after 12 and
6 h, respectively, and declined to control values by 24 h
(Fig. 8
). IL-1ß failed to induce A12 mRNA (data not shown).
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Figure 8. Kinetics of TNF-induced A12 mRNA expression in Mono Mac 6 cells (A)
and monocytes (B). Cells were stimulated with 100 U/mL of TNF for
the indicated times. mRNA was analyzed by semiquantitative RT-PCR and
is representative of two experiments.
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-stimulated cells
with this technique. |
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The suggested ability of hA12 to regulate monocyte migration was supported by its positive effects on actin polymerization; an event immediately following receptor binding and signal transduction and associated with cell shape change during migration [21 , 22 ]. Actin in monocytes underwent typical reversible polymerization within 20–30 s of incubation with A12, in a manner similar to that induced by fMLP (Fig. 3A) . The strong response provoked by 10-11 M A12 increased over 40 s and, unlike fMLP, was sustained over 120 s. A12-stimulated actin reorganization was accompanied by almost instantaneous increases in [Ca2+]i similar to that initiated by C5a (Fig. 3B) , fMLP, and IL-8 [23 ], in which directional cell motility is accompanied by activation. These in vitro results indicate differences in the chemotactic mechanisms initiated by A12 compared with mA8, which provokes myeloid cell migration and actin polymerization but does not alter [Ca2+]i [10 , 24 ]. On the other hand, S100B promotes changes in glial cell morphology, neurite extension, and possibly growth cone motility, and it also rapidly increases [Ca2+]i in glial and neuronal cells [25 ].
Intraperitoneal injection of A12 (1 µM) into mice elicited a mild but sustained recruitment of leukocytes that reached more than twofold control levels after 8 h. Although A12 only weakly induced neutrophil migration in vitro, neutrophil numbers increased >10-fold during this time and declined to normal levels by 24 h (Fig. 4) . Small but significant increases in monocyte numbers were also evident after 8 h but were greatest after 24 h and declined thereafter. The discrepancy between in vitro and in vivo studies may be due to the more complex cellular environment in which A12 ligation with RAGE on monocytes/macrophages induces TNF and IL-1ß [7 ] which, in turn, could induce chemokines responsible for the early recruitment of neutrophils. The kinetics of leukocyte recruitment elicited by hA12 described here were remarkably similar to responses provoked by mA8 in vivo [10 , 11 ]. It is interesting that the chemotactic activity of mA8 is not shared by its human counterpart [26 ]. mA8 and human A8 (hA8) have only 56% amino acid identity [9 , 27 ] which is atypical of the highly conserved interspecies similarities of other members of the S100 family [28 , 29 ]. Screening of cDNA and genomic DNA libraries for a human homologue of murine CP-10 (mA8) failed to yield isolates with greater sequence similarity than hA8, and the murine protein was designated mA8. Despite the relatively low structural homology between mA8 and hA12 (33% identity), similarities in target cell specificities [26 ] and kinetics of leukocyte recruitment in vivo [10 , 11 ] suggest that they may be functional homologues.
Elevated levels of hA8 and hA9 are associated with a number of inflammatory diseases, particularly RA, in which high levels in serum and SF have been reported [30 ], as well as expression in the synovial membrane [31 ], but their functions in this disease are unclear. Here we demonstrate the presence of A12, together with A8 and A9, in SF of RA patients (Fig. 5) and expression of A12 by inflammatory cells in RA synovium (Fig. 6) . Approximately 70 nM A12, within the effective chemotactic concentration range (10-13 to 10-8 M) in vitro, was extracted from SF using anti-A12 IgG affinity chromatography. A12 in RA synovium was located in neutrophils at the sublining and interstitial region, often within the perivascular region (Fig. 6A) . A12-positive neutrophils at this site might be strategically poised to facilitate leukocyte recruitment. In contrast to S100A8 and -A9 [32 ], A12-positive cells were rare within the synovial-lining layer (Fig. 6B) although occasional macrophages in the sublining and lining layers were A12 positive (Fig. 6C) .
A12 is constitutive in neutrophils, but its expression in monocytes
remains in question. Small amounts of A12 were found in lysates of
monocytes [33
] and weak immunostaining in monocytes but
not in unstimulated monocytoid cell lines [34
]. However,
a recent report indicates no A12 in vitamin
D3-differentiated HL60 cells or monocytes
[35
]. We show that TNF, a proinflammatory mediator
that plays a pivotal role in RA [36
], induced A12 mRNA
in Mono Mac 6 cells and monocytes, with maximal expression after 6–12
h (Fig. 8)
. Although neutrophil-derived A12 may contribute
significantly to leukocyte recruitment in RA (Fig. 6)
, A12 from
TNF-stimulated monocytes/macrophages may be important in the early
phases of leukocyte recruitment, in particular when there are acute
inflammatory flares. Secretion of the protein, or the transient effect
of TNF may explain the low numbers of A12-positive macrophages
detected in the synovium, in which A12 expression may have subsided.
Induction of A12 by TNF is interesting, given that A12 induces
TNF and IL-1ß expression in macrophages [7
],
suggesting a positive feedback mechanism between TNF and A12,
perhaps in a manner similar to that between macrophage-inflammatory
protein- and TNF/IL-1 [37
, 38
].
Low basal levels of A12 mRNA were observed in monocytes, and stimulation with LPS increased transcript levels in blood monocytes and in Mono Mac 6 cells over a time course of 6–48 h (Fig. 7B) , similar to that observed for induction of mA8 in monocytoid cell lines and in elicited macrophages [39 ]. A12 gene induction was exquisitely sensitive to low levels of LPS (Fig. 7A) , typical of those found in the circulation during bacteremia. Increased A12 in plasma 2–24 h after infusion of mice with LPS [7 ] has been reported, clearly implicating A12 involvement in responses to gram-negative infection. It is interesting that Mono Mac 6 cells stimulated with high amounts of LPS (500 ng/mL) expressed less A12 mRNA than that induced by lower amounts of LPS (Fig. 7A) , suggesting production of monocyte-derived mediators that regulate A12 gene expression. Although cell-associated A12 was detected by ELISA, levels in supernatants of stimulated monocytes were below the level of detection by this method.
Despite the similar patterns of gene induction/expression [39 , 40 ] and similar chemotactic properties of mA8 and hA12, there are distinct structural differences between these proteins which may regulate other functions. mA8 and hA8 are exquisitely sensitive to oxidation by hypochlorite, the major oxidant generated by activated neutrophils [41 ], and hA8, injected as a complex with A9, suppresses adjuvant-induced arthritis [42 ]. Furthermore, A8 constitutes 20% of the cytosolic protein of neutrophils and potentially large amounts, released during phagocytosis [43 ] or as a result of cell necrosis [44 ] may play a protective role in inflammation. Moreover, oxidation of mA8 to the covalent homodimer negates chemotactic activity, possibly by sterically hindering and/or structurally altering exposure of the chemotactic hinge domain, restricting cellular target recognition. Taken together with hypochlorite’s ability to reduce the chemotactic activity of C5a and fMLP by formation of methionine sulfoxides [45 ], oxidative mechanisms may regulate excess leukocyte accumulation and contribute to terminating progression of acute inflammation. In marked contrast, hA12 is not prone to covalent dimerization or oxidation because it lacks Cys and Met residues, suggesting that its chemotactic properties may be more stable. Moreover, A12 up-regulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 in endothelial cells and TNF in monocytes, and blockade of A12/RAGE interactions inhibit delayed-type hypersensitivity responses and inflammatory colitis in murine models [7 ]. Together with the properties reported here, a proinflammatory role is indicated. The levels of this protein found in the SF of patients with RA may contribute to sustained neutrophil and monocyte recruitment during chronic inflammatory episodes.
Received December 13, 2000; revised April 1, 2001; accepted April 5, 2001.
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