Modulation of Macrophage Phenotype by
Soluble Product(s) Released from Neutrophils
This information is current as
of July 31, 2017.
Jean M. Daley, Jonathan S. Reichner, Eric J. Mahoney,
Laura Manfield, William L. Henry, Jr., Balduino
Mastrofrancesco and Jorge E. Albina
J Immunol 2005; 174:2265-2272; ;
doi: 10.4049/jimmunol.174.4.2265
http://www.jimmunol.org/content/174/4/2265
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References
The Journal of Immunology
Modulation of Macrophage Phenotype by Soluble Product(s)
Released from Neutrophils1
Jean M. Daley,2 Jonathan S. Reichner, Eric J. Mahoney, Laura Manfield, William L. Henry, Jr.,
Balduino Mastrofrancesco, and Jorge E. Albina
N
eutrophils are the first blood-borne nucleated cells to
infiltrate sites of injury or infection, where they produce
multiple effector molecules, including reactive oxygen
and nitrogen intermediates, proteolytic enzymes, and cytokines.
The anti-infectious capacity of neutrophils provides obvious functional advantage to their recruitment into infected tissue. Neutrophils, however, also accumulate in large numbers at sites of sterile
inflammation, such as uninfected wounds, myocardial and cerebral
infarctions, and fractures where, in the absence of infectious challenge, their role in the inflammatory response is less readily
apparent.
Experiments reported in this study were initially designed to
define the contribution of neutrophils to the cytokine profile of an
acute sterile inflammatory site, such as that provided by the polyvinyl alcohol (PVA)3 sponge wound model. The findings demonstrated that neutrophil depletion results in increased proinflammatory cytokine concentrations in early tissue inflammation. This
finding suggested that neutrophils produce a factor(s) that inhibits
the release of proinflammatory cytokines by macrophages. The
Department of Surgery, Division of Surgical Research, Rhode Island Hospital and
Brown Medical School, Providence, RI 02903
Received for publication April 27, 2004. Accepted for publication December 1, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was funded by National Institutes of Health Grants GM42859 (to J.E.A.)
and GM66194 (to J.S.R.) and by funds allocated to the Department of Surgery by
Rhode Island Hospital, a Lifespan partner. J.M.D. was supported by a National Institutes of Health Supplement to Promote Reentry into Biomedical and Behavioral
Research Careers. E.J.M. was supported by the Carter Family Charitable Trust
(Armand D. Versaci Research Scholar in Surgical Sciences Award).
2
Address correspondence and reprint requests to Dr. J. M. Daley, Division of Surgical Research, Rhode Island Hospital, NAB 214, 593 Eddy Street, Providence, RI
02903. E-mail address: [email protected]
3
Abbreviations used in this paper: PVA, polyvinyl alcohol; CM, complete medium.
Copyright © 2005 by The American Association of Immunologists, Inc.
results supported and extended the predictions of this hypothesis
by demonstrating that wound neutrophils produce a soluble factor(s) that suppresses LPS-mediated activation of a macrophage
cell line and primary peritoneal macrophages. The present results
thus reveal an additional capacity of neutrophils in inflammation,
namely, the ability of these cells to modulate macrophage inflammatory phenotype.
Materials and Methods
Animals
B6D2F1 male mice (Taconic Farms), 8 –12 wk of age, were housed at the
Central Research Facilities of Rhode Island Hospital and fed mouse chow
and water ad libitum. Mice were certified free of common pathogens by the
supplier and were monitored by Brown University/Rhode Island Hospital
veterinary personnel. Animal protocols were approved by the animal care
committee at Rhode Island Hospital.
PVA sponge wound model
Mice were anesthetized with pentobarbital (50 mg/kg i.p.; Abbott Laboratories). Five PVA sponges (PVA Unlimited), measuring 1 ⫻ 1 ⫻ 0.6 cm,
were inserted into individual s.c. pockets through a midline dorsal incision
under sterile conditions, and the skin was closed with clips (1). Mice were
euthanized by CO2 asphyxiation, and the sponges were removed under
sterile conditions. Culture of randomly selected PVA sponges at the time
of removal confirmed sterility in this experimental model. Wound cells
were isolated by rapid repeated compression of sponges in a Stomacher
(Tekmar). When necessary, cells were subjected to hypotonic lysis of
erythrocytes, followed by reconstitution in PBS (Invitrogen Life Technologies). Viable cells were identified by trypan blue exclusion and were
counted using a hemocytometer; viability was ⬎95%. Differential cell
counts were performed on Hema-3 (Biochemical Sciences)-stained Cytospins (Shandon), and cell phenotype was confirmed by flow cytometry, as
described below. Extracellular fluid from the PVA sponges (wound fluid)
was obtained by centrifugation (400 ⫻ g for 5 min) of two or three sponges
in the barrel of a syringe that was seated in a sterile tube. Wound fluid was
stored at ⫺80°C until analysis.
0022-1767/05/$02.00
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The regulation of macrophage phenotype by neutrophils was studied in the s.c. polyvinyl alcohol sponge wound model in mice
made neutropenic by anti-Gr-1 Ab, as well as in cell culture. Wounds in neutropenic mice contained 100-fold fewer neutrophils
than those in nonneutropenic controls 1 day after sponge implantation. Wound fluids from neutropenic mice contained 68% more
TNF-␣, 168% more IL-6, and 61% less TGF-␤1 than those from controls. Wound fluid IL-10 was not different between the two
groups, and IL-4 was not detected. Intracellular TNF-␣ staining was greater in cells isolated from neutropenic wounds than in
those from control wounds. The hypothesis that wound neutrophil products modulate macrophage phenotype was tested in
Transwell cocultures of LPS-stimulated J774A.1 macrophages and day 1 wound cells (84% neutrophils/15% macrophages).
Overnight cocultures accumulated 60% less TNF-␣ and IL-6 than cultures of J774A.1 alone. The suppression of cytokine release
was mediated by a soluble factor(s), because culture supernatants from wound cells inhibited TNF-␣ and IL-6 release from
LPS-stimulated J774A.1 cells. Culture supernatants from purified wound neutrophils equally suppressed TNF-␣ release from
LPS-stimulated J774A.1 cells. Wound cell supernatants also suppressed TNF-␣ and superoxide release from murine peritoneal
macrophages. The TNF-␣ inhibitory factor has a molecular mass <3000 Da and is neither PGE2 nor adenosine. The present
findings confirm a role for neutrophils in the regulation of innate immune responses through modulation of macrophage
phenotype. The Journal of Immunology, 2005, 174: 2265–2272.
2266
NEUTROPHIL PRODUCT(S) MODULATE MACROPHAGE PHENOTYPE
Induction of neutropenia
Anti-mouse Gr-1 mAb (RB6.8C5 hybridoma originally produced by R. L.
Coffman, DNAX Research Institute) was produced under serum-free conditions using a bioreactor (Cell Pharm Micro Mouse; UniSyn Technologies). Mice were rendered neutropenic by a single i.p. injection of 0.5 mg
of anti-Gr-1 Ab 3 days before PVA sponge insertion. Control animals were
injected i.p. with an equal volume of normal saline or with 0.5 mg of rat
IgG (Rockland Immunochemicals).
Cells were removed by centrifugation, and the supernatants were used as
described above.
Macrophage viability was assessed after 24 h of incubation by trypan
blue exclusion, reduction of MTT (Sigma-Aldrich) (2), and release of lactate dehydrogenase (3). Protein synthesis was determined by measuring the
24-h incorporation of L-[ring-2,6-N-3H]phenylalanine into TCA-precipitable protein. When indicated, cells were lysed in buffer containing 0.01 M
Tris, 0.15 M NaCl, and 0.5% Igepal CA 630 (Sigma-Aldrich) with Complete (Roche) protease inhibitor.
Circulating leukocyte counts
Heparinized blood was obtained from the lateral tail vein or the inferior
vena cava. Leukocyte count was determined using a hemocytometer after
dilution and erythrocyte lysis with 0.01% crystal violet in 3% acetic acid.
Differential cell counts were performed on Hema-3 stained blood smears.
Purification of neutrophils from the wound cell suspension
Cell culture
All cell cultures were performed in complete medium (CM; RPMI 1640
(Invitrogen Life Technologies) supplemented with 10 mM MOPS, 5 ⫻
10⫺5 M 2-ME, 100 U/ml penicillin-streptomycin, and 1% FBS (HyClone))
unless noted otherwise. The murine macrophage cell line J774A.1 was
obtained from American Type Culture Collection. Murine resident peritoneal macrophages were obtained by peritoneal lavage with HBSS and 1%
FBS. Thioglycolate-elicited peritoneal macrophages were harvested 4 days
after i.p. injection of 3 ml of 3% Brewer modified thioglycolate medium
(BD Biosciences). J774A.1 macrophages or peritoneal macrophages were
allowed to adhere to tissue culture-treated plastic for 2 h, and nonadherent
cells were removed before initiation of experiments.
Three culture formats were used for coculture experiments: 1) J774A.1
cells, 5 ⫻ 105 in 1 ml of CM; 2) wound cells, 2.5 ⫻ 106 in 1 ml of CM;
and 3) cocultures in Transwell plates (0.4-␮m pore size; Costar) containing
5 ⫻ 105 J774A.1 cells in the lower chamber and 2.5 ⫻ 106 wound cells in
the upper chamber in a total volume of 1 ml of CM. LPS (Escherichia coli
serotype 055:B5, Sigma-Aldrich; 0.1 ␮g/ml) was added 1 h after the initiation of all cultures. Where noted, indomethacin (Sigma-Aldrich) or NS398 (Cayman Chemicals) was added at the start of cell incubation.
J774A.1 cells or peritoneal macrophages were also cultured with supernatants conditioned by day 1 wound cells. The wound cell-conditioned
supernatant was generated by isolating wound cells from PVA sponges 1
day after insertion and culturing these cells in CM at 2.5 ⫻ 106/ml for 24 h.
Cells were then removed from the medium by centrifugation, and the supernatant was stored at ⫺80°C. Supernatants conditioned by purified
wound neutrophils were generated in similar manner. J774A.1 cells (1 ⫻
105) or primary peritoneal macrophages (3 ⫻ 105) were incubated in 200
␮l of CM or a 1/1 (v/v) dilution of wound cell-conditioned supernatant in
CM for 1 h before addition of LPS, then cultured for an additional 24 h.
Wound cell-conditioned supernatant was fractionated using Amicon Centriplus YM-3 centrifugal filter devices (Millipore). When indicated, wound
cell-conditioned supernatant or an equal volume of CM was treated with
adenosine deaminase (type X from bovine spleen; Sigma-Aldrich) at 0.02
U/ml for 20 min at 25°C, and adenosine deaminase was removed, or not,
using a Centricon-10 concentrator (Millipore) before addition of the treated
supernatant to cell cultures. Indomethacin, NS-398, AH6809 (Cayman
Chemicals), L161982 (gift from Dr. R. Young, Merck Frosst Canada), or
8 phenyl-theophylline (Sigma-Aldrich) was added to cell cultures where
noted.
Conditioned culture supernatants were also prepared from splenic lymphocytes. Splenocytes were obtained by mechanical dissociation of spleens
and elimination of fibrous tissue using wire mesh. Erythrocytes were removed by hypotonic lysis; macrophages and dendritic cells were depleted
by adherence to plastic for 2 h. Supernatants were prepared from the resulting cell suspension (90% lymphocytes, 4% monocytes, and 4% neutrophils) by culture at 2.5 ⫻ 106/ml for 24 h. Conditioned supernatants
were also prepared from murine L929 fibroblasts and from fibroblasts obtained from a murine wound by culture at a density of 5 ⫻ 105/ml for 24 h.
Wound cells were isolated from neutropenic or control animals 1 day after
sponge insertion and pooled. Abs used for cell staining and flow cytometry
were obtained from BD Biosciences unless noted otherwise.
Surface Ag staining. Erythrocytes were removed from the wound cell
suspension by hypotonic lysis. Cells were incubated with FcR-blocking
agent (Accurate Chemical & Scientific), washed in PBS with 1% FBS, and
incubated with predetermined optimal concentrations of fluorochromeconjugated Abs to B220 and IgM (for B cells), CD3⑀ (for T cells), or the
appropriate isotype control Abs.
Intracellular Ag staining. Erythrocytes were removed from the wound
cell suspension by hypotonic lysis. Cells were incubated with FcR-blocking agent (Accurate Chemical & Scientific), washed in PBS with 1% FBS,
fixed in paraformaldehyde with saponin (Cytofix/Cytoperm; BD Biosciences) according to the manufacturer’s recommendations, washed in
Permwash (BD Biosciences), and incubated with a predetermined optimal
concentration of fluorochrome-conjugated Ab directed against the intracellular macrophage Ag macrosialin (anti-CD68; Serotec) or the appropriate
isotype control Ab.
Intracellular cytokine staining. Cells were incubated (106 cells/ml) in
CM for 6 h with brefeldin A (Golgiplug; BD Biosciences) at 1 ␮l/106 cells
and stained for intracellular Ag as described above, using anti-CD68, antiTNF-␣, and/or the appropriate isotype control Abs.
Cells were analyzed and sorted using a FACSort and CellQuest software
(BD Biosciences). Cells were stained with propidium iodide after sorting,
and nuclear morphology was determined by light microscopy.
Assays
TNF-␣ (BioSource), IL-6 (BD Biosciences), TGF-␤1 (R&D Systems),
IL-4, and IL-10 (4) were assayed by ELISA. PGE2 was quantified by enzyme immunoassay (Cayman Chemical). ATP, ADP, AMP, and adenosine
were assayed by HPLC (5) with a limit of detection of 1 ␮M. TNF-␣
bioactivity was determined by lysis of actinomycin D (Sigma-Aldrich)treated L929 murine fibroblasts (NCTC clone 929; American Type Culture
Collection) (6), using a recombinant murine TNF-␣ standard (R&D Systems). Superoxide release was measured from the reduction of ferricytochrome c (Sigma-Aldrich) after cell stimulation with 200 nM PMA (LC
Services) over 90 min (7).
Data presentation and analysis
Data reported are the mean ⫾ 1 SD unless otherwise noted. Results from
cell culture experiments are from triplicate wells in a representative experiment. Statistical analysis was performed using Student’s unpaired t
test, Mann-Whitney U test, or ANOVA with Newman-Keuls test, as
appropriate.
Results
Characterization of the cellular infiltrate in the PVA sponge
wound model
The leukocytic infiltration of the wound increased over the first 7
days after PVA sponge insertion (Fig. 1A). Day 1 wound cells
were 84 ⫾ 4% neutrophils, 15 ⫾ 4% macrophages, ⬍1% lymphocytes, and ⬍1% eosinophils in six experiments with at least
five animals per experiment (mean ⫾ SEM). Neutrophils remained
the most abundant cell type through at least 3 days after wounding
(Fig. 1B). Macrophages constituted 52% of the cells isolated from
the wound 7 days after wounding. All subsequent experiments
using wound cells or wound fluids were performed using cells or
fluids isolated 1 day after PVA sponge insertion.
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Wound cells were isolated 1 day after PVA sponge insertion, suspended in
biotin-free FcR-blocking agent (Accurate Chemical & Scientific) at 0.3
ml/106 cells, washed, and incubated with biotin-conjugated Ab directed
against the macrophage cell surface Ag F4/80 (1 ␮g/106 cells; Caltag Laboratories). Cells were washed in PBS and incubated with MACS AntiBiotin MicroBeads (Miltenyi Biotec) according to the manufacturer’s recommendations, and macrophages were depleted using the MiniMACS
magnetic cell separator (Miltenyi Biotec). The resulting cell suspension
was 96% neutrophils by light microscopy.
Cell staining and flow cytometry
The Journal of Immunology
2267
Table I. Cytokine concentration in wound fluids: neutrophil depletion
selectively increases proinflammatory cytokinesa
TNF-␣ (pg/ml)
IL-6 (ng/ml)
TGF-␤1 (pg/ml)
IL-10 (pg/ml)
IL-4
Effect of anti-Gr-1 Ab on circulating and wound leukocyte
counts
Intraperitoneal injection of 0.5 mg of mAb directed against the
neutrophil surface Ag Gr-1 reduced neutrophils from 13.3 ⫾ 1.5 to
1.4 ⫾ 1.0% of the total circulating leukocytes. Neutropenia persisted throughout the time course of these experiments (4 days).
Differential blood lymphocyte and monocyte counts were not altered by Ab treatment (data not shown).
The effect of anti-Gr-1 Ab on leukocyte infiltration of the
wounds was quite marked: wounds from neutropenic animals contained 67-fold fewer total cells than controls, with 100-fold less
neutrophils and 10-fold less macrophages (Fig. 2).
Neutrophil depletion increases proinflammatory cytokines in
wound fluids
Table I shows the cytokine content of wound fluids from control
and neutropenic animals. Despite the decrease in the inflammatory
cell infiltrate, immunoreactive TNF-␣ and IL-6 concentrations in
wound fluid from neutropenic animals were 68 and 168% higher,
respectively, than those from controls, whereas TGF-␤1 concen-
FIGURE 2. Anti-Gr-1 Ab depletes leukocyte populations in the PVA
sponge wound. Mice were injected i.p. with 0.5 mg of anti-Gr-1 Ab or an
equal volume of saline, and PVA sponges were inserted into control (n ⫽
6) and neutropenic (n ⫽ 7) mice 3 days later. Wound cells were isolated 1
day after sponge insertion. The graph shows cell counts per animal (E) and
group means (⫺). Neutropenic wounds had lower total cell, neutrophil, and
macrophage counts than control wounds (p ⬍ 0.05, by Mann-Whitney U
test).
Neutropenic
322 ⫾ 99
22 ⫾ 14
1331 ⫾ 429
116 ⫾ 38
ND
542 ⫾ 125ⴱ
59 ⫾ 19ⴱ
520 ⫾ 176ⴱ
108 ⫾ 32
ND
a
Mice were injected i.p. with 0.5 mg of anti-Gr-1 Ab or saline, and PVA sponges
were inserted into neutropenic and control mice 3 days later. Wound fluids obtained
1 day after sponge insertion were assayed for cytokines as described in Materials and
Methods. The concentrations of TNF-␣ and IL-6 in wound fluids from animals that
had been injected i.p. with 0.5 mg of rat IgG were not different from those in salineinjected controls (data not shown). A minimum of six animals was tested in each
group. The table shows means ⫾ one SD.
ⴱ, p ⬍ 0.05, Mann-Whitney U test.
trations were 61% lower. Wound fluid IL-10 concentrations were
not different between the two groups. IL-4 was below the limit of
detection of the assay (10 pg/ml) in wound fluids from either
group.
The increases in proinflammatory cytokines in wound fluids
from neutropenic animals were independent of plasma cytokines.
Plasma TNF-␣ concentrations at the time of sponge removal were
below the limit of detection of the assay (30 pg/ml) in both groups.
There was no difference in the plasma concentrations of IL-6 (control, 59 ⫾ 21 pg/ml; neutropenic, 48 ⫾ 14 pg/ml; p ⬎ 0.05, by
Mann-Whitney U test). Neutropenic animals did not exhibit
obvious differences in the appearance of the surgical site or in
postoperative behavior compared with wounded control animals.
Cells from neutropenic wounds contain more TNF-␣ than those
from control wounds
To determine the source of increased TNF-␣ in the wound fluids of
neutropenic animals, cells were isolated from control and neutropenic wounds, cultured with brefeldin to inhibit intracellular cytokine transport, then stained for intracellular TNF-␣ and the intracellular macrophage Ag CD68. The validity of CD68 staining
for the identification of wound macrophages was confirmed by
microscopic examination of the cells after FACS. In control animals, CD68high cells were 98.5% macrophages/mononuclear cells
and 1.5% polymorphonuclear leukocytes; CD68⫺/low cells were
95.0% polymorphonuclear leukocytes (including bands) and 4.0%
macrophages/mononuclear cells. As will be discussed below, in
FIGURE 3. Intracellular TNF-␣ staining in cells from control and neutropenic wounds. Mice were injected i.p. with 0.5 mg of anti-Gr-1 Ab or
saline, and PVA sponges were inserted 3 days later. Wound cells isolated
1 day after sponge insertion were cultured with brefeldin A and stained for
intracellular TNF-␣ and macrophage intracellular Ag CD68 as described in
Materials and Methods. MCF, mean channel fluorescence of the quadrant.
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FIGURE 1. Characterization of the cellular infiltrate in the PVA sponge
wound. PVA sponges were inserted as described in Materials and Methods, and six animals each were euthanized at 1, 3, and 7 days after sponge
insertion. Wound cells were isolated, identified, and counted as described
in Materials and Methods. A, Wound cell count per animal. Counts differed
at all time points (p ⬍ 0.05, by ANOVA and Newman-Keuls test). B,
Differential cell count. f, neutrophils; _, macrophages.
Control
2268
NEUTROPHIL PRODUCT(S) MODULATE MACROPHAGE PHENOTYPE
FIGURE 5. Kinetics of TNF-␣ release in J774A.1/wound cell cocultures stimulated with LPS. Transwell cocultures of J774A.1 cells and day
1 wound cells were performed as described in Fig. 4. TNF-␣ concentrations were lower in cocultures than in J774A.1 cells cultured alone at 20
and 24 h (p ⬍ 0.001, by two-factor ANOVA).
Wound cells inhibit TNF-␣ and IL-6 release by LPS-stimulated
J774A.1 macrophages
FIGURE 4. Wound cells inhibit TNF-␣ and IL-6 release by LPS-stimulated J774A.1 macrophages. Wound cells were isolated from nonneutropenic mice 1 day after sponge insertion. Wound cells (2.5 ⫻ 106) or
J774A.1 cells (J774; 5 ⫻ 105) were cultured in 1 ml of CM. Transwell
cocultures (J774 and wound cells) contained 5 ⫻ 105 J774A.1 cells in the
lower chamber and 2.5 ⫻ 106 wound cells in the upper chamber in a total
volume of 1 ml. Cells were cultured with LPS (0.1 ␮g/ml) for 24 h. Culture
supernatants were assayed for TNF-␣ by ELISA (A), for TNF-␣ bioactivity
by lysis of actinomycin-treated L929 cells (B), or for IL-6 by ELISA (C).
TNF-␣ and IL-6 concentrations were lower in cocultures than in J774A.1
cells cultured alone (p ⬍ 0.05, by Student’s unpaired t test).
neutropenic animals the CD68⫺/low cells included a greater proportion of mononuclear cells with morphologic characteristics of
monocytes, macrophages, and lymphocytes than those in controls.
The intensity of staining for TNF-␣ was greater in CD68high
cells than in CD68⫺/low cells in both neutropenic and control
animals (Fig. 3). Macrophages from neutropenic wounds stained
more intensely for TNF-␣ than those from controls. The blood
The hypothesis that wound cells, specifically wound neutrophils,
suppress cytokine release by macrophages in vivo was modeled
and tested in vitro. The murine macrophage cell line J774A.1 used
in these experiments was confirmed in the laboratory to exhibit
minimal release of TNF-␣ in unstimulated culture and submaximal
cytokine release when stimulated with 0.1 ␮g/ml LPS. J774A.1
macrophages were cultured in Transwells with or without day 1
wound cells (86% neutrophils) from nonneutropenic animals. Fig.
4A shows that supernatants from J774A.1/wound cell cocultures
contained 60% less immunoreactive TNF-␣ than those from cultures of J774A.1 cells alone. Results obtained by ELISA for
TNF-␣ were confirmed in a bioassay (Fig. 4B). Similarly, supernatants from J774A.1/wound cell cocultures contained 60% less
IL-6 than cultures of J774A.1 cells (Fig. 4C). Comparison of the
kinetics of TNF-␣ release in cultures of LPS-stimulated J774A.1
cells vs those in J774A.1/wound cell cocultures showed that suppression of TNF-␣ release in cocultures was not evident until after
8 h of incubation (Fig. 5).
Culture supernatants conditioned by day 1 wound cells inhibit
TNF-␣ and IL-6 release by LPS-stimulated J774A.1
macrophages
LPS-stimulated J774A.1 macrophages were also cultured in supernatants previously conditioned by day 1 wound cells. As shown in
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leukocytes of control and neutropenic animals did not differ in
intracellular TNF-␣ staining (data not shown).
The CD68⫺/low cells from neutropenic wounds contained more
TNF-␣ than those from control wounds. These CD68⫺/lowTNF-␣⫹
cells comprised 30% of the total cells from neutropenic animals.
Morphologic examination of these cells after FACS showed 59%
polymorphonuclear leukocytes and 41% mononuclear cells. In
control animals, the CD68⫺/lowTNF-␣⫹ population of cells constituted only 6.7% of the wound cell suspension and was comprised almost entirely of polymorphonuclear leukocytes (96%),
with the remaining 4% being mononuclear cells. The absolute
number of these CD68⫺/lowTNF-␣⫹ mononuclear cells, as calculated from the respective total cellularity of control vs neutropenic
wounds, was actually higher in controls (4800/animal) than in neutropenic wounds (2100/animal). These cells probably represent extravasated monocytes, found in the laboratory to be CD68⫺/low
(data not shown). In this regard it has been shown that CD68 Ag
expression is enhanced during macrophage recruitment (8).
The Journal of Immunology
FIGURE 7. Kinetics of TNF-␣ release in LPS-stimulated J774A.1 macrophages incubated with wound cell-conditioned supernatants. J774A.1
cells were cultured with LPS (0.1 ␮g/ml), with or without wound cellconditioned supernatants, as described in Fig. 6. J774A.1 cultures with
wound cell-conditioned supernatants contained less TNF-␣ than cultures in
CM at all time points (p ⬍ 0.05, by two-factor ANOVA).
The neutrophil supernatant and the wound cell supernatant equally
suppressed TNF-␣ release by J774A.1 macrophages (Fig. 6A).
Suppression of TNF-␣ release from LPS-stimulated J774A.1
macrophages was not observed when supernatants were conditioned by splenic lymphocytes or L929 murine fibroblasts.
(J774A.1 in CM, 94.2 ⫾ 14.1 ng/ml; J774A.1 in splenic lymphocyte-conditioned supernatant, 83.5 ⫾ 15.6 ng/ml; J774A.1 in
L929-conditioned supernatant, 114.4 ⫾ 13.8 ng/ml; p ⬎ 0.05, by
ANOVA and Newman-Keuls test). Supernatants conditioned by
fibroblasts isolated from a murine wound suppressed TNF-␣ release from LPS-stimulated J774A.1 cells (J774A.1 in mouse
wound-derived fibroblast-conditioned supernatant, 18.0 ⫾ 0.8 ng/
ml; p ⬍ 0.05 vs J774A.1 in CM (above), by ANOVA and
Newman-Keuls test).
The suppression of proinflammatory cytokine release by wound
cell-conditioned supernatants did not result from cytotoxicity or
from nonspecific inhibition of protein synthesis. There was no difference between cells cultured in CM and those cultured in wound
cell supernatant with respect to cell viability at the end of culture,
as assessed by trypan blue exclusion, lactate dehydrogenase release, or reduction of MTT (Table II). Protein synthesis, as measured by incorporation of radiolabeled phenylalanine into protein,
was identical in both culture conditions.
Wound cell-conditioned supernatants inhibit release of TNF-␣
and production of superoxide by murine peritoneal macrophages
FIGURE 6. Culture supernatants from wound cells inhibit TNF-␣ and
IL-6 release by LPS-stimulated J774A.1 macrophages. A, Wound cells
were isolated 1 day after PVA sponge insertion, and macrophages were
depleted from the wound cell suspension as described in Materials and
Methods. The unfractionated wound cells (86% neutrophils) or the purified
wound neutrophils (96% neutrophils) were cultured for 24 h at 2.5 ⫻
106/ml. Supernatants from these cultures were diluted 1/1 with CM and
added to J774A.1 cells (5 ⫻ 105/ml) along with LPS (0.1 ␮g/ml). The
TNF-␣ concentration of the culture supernatant was assayed by ELISA
after 24 h. J774A.1 cultures with either wound cell supernatant or wound
neutrophil supernatant contained less TNF-␣ than cultures in CM (p ⬍
0.05, by ANOVA and Newman-Keuls test). B, J774A.1 cells were cultured
with LPS (0.1 ␮g/ml), with or without wound cell-conditioned supernatants. IL-6 was assayed by ELISA after 24-h culture. J774A.1 cultures with
wound cell-conditioned supernatants contained less IL-6 than cultures in
CM (p ⬍ 0.01, by Student’s unpaired t test).
Wound cell-conditioned supernatants also inhibited LPS-stimulated TNF-␣ release from thioglycolate-elicited murine peritoneal
macrophages. As shown in Fig. 8A, macrophages incubated in
Table II. Wound cell conditioned supernatants are not toxic to J774A.1
macrophagesa
Wound Cell
Conditioned
Complete Medium Supernatant
Trypan blue exclusion (%)
LDH in media (U/ml)
MTT reduction (OD)
[3H]Phenylalanine 3 protein (cpm)
⬎95%
2.95 ⫾ 1.32
0.9 ⫾ 0.1
3021 ⫾ 94
⬎95%
2.91 ⫾ 0.4
1.0 ⫾ 0.1
3432 ⫾ 298
a
J774A.1 cells were cultured in CM or with wound cell supernatant as described
in the legend to Fig. 6. LDH, lactate dehydrogenase; [3H]phenylalanine 3 protein,
incorporation of [3H]phenylalanine into protein.
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Fig. 6, J774A.1 cultures containing wound cell-conditioned supernatants accumulated 56% less TNF-␣ and 60% less IL-6 at 24 h
than those in CM. The kinetics of TNF-␣ release differed from
those in J774A.1/wound cell cocultures, because the inhibition of
TNF-␣ release by wound cell supernatants was evident as early as
1 h after stimulation with LPS (Fig. 7). The suppression of TNF-␣
release was not due to cellular accumulation of the cytokine during
culture, because lysates from J774A.1 cells incubated with wound
cell-conditioned supernatants contained less TNF-␣ than those in
CM (J774A.1 in supernatants, 133 ⫾ 6 pg/106 cells; J774A.1 in
CM, 147 ⫾ 6 pg/106 cells; p ⬍ 0.05, by Student’s unpaired t test.).
IL-6 was not assayed in these samples because there is no cellassociated form of the cytokine. The suppression of macrophage
TNF-␣ release by wound cell supernatants was also evident in the
absence of LPS (J774A.1 cells in CM, 2.1 ⫾ 0.2 ng/ml; J774A.1
in wound cell supernatants, 1.6 ⫾ 0.1 ng/ml; p ⬍ 0.05, by Student’s unpaired t test.).
To specifically assess the contribution of neutrophils to the
TNF-␣-suppressive capacity of wound cells, LPS-stimulated
J774A.1 macrophages were cultured in supernatants conditioned
by neutrophils purified from day 1 wound cells (96% neutrophils).
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NEUTROPHIL PRODUCT(S) MODULATE MACROPHAGE PHENOTYPE
FIGURE 8. Wound cell-conditioned supernatants inhibit TNF-␣ release
and superoxide production by LPS-stimulated primary murine peritoneal
macrophages. A, Thioglycolate-elicited peritoneal macrophages (3 ⫻ 105
in 200 ␮l) were cultured with LPS (0.1 ␮g/ml) with or without wound
cell-conditioned supernatants, as described in Fig. 6. TNF-␣ was assayed
by ELISA after 24-h culture. Macrophages cultured with wound cell supernatants released less TNF-␣ than those cultured in CM (p ⬍ 0.01, by
Student’s unpaired t test). B, Resident peritoneal macrophages (3 ⫻ 105 in
200 ␮l) purified by overnight adherence were cultured with or without
wound cell-conditioned supernatants at varying doses of LPS. Macrophage
superoxide production was assayed after 24-h culture. Macrophages cultured with wound cell-conditioned supernatants (f) produced less superoxide than those in CM (䡺; p ⬍ 0.05, by two-factor ANOVA).
indomethacin (10 ␮M) reversed the inhibition of TNF-␣ release in
the cocultures (Fig. 10A). The effectiveness of cyclooxygenase
inhibitors was confirmed by measurement of PGE2 in the culture
medium (Fig. 10B).
wound cell supernatants released 71% less TNF-␣ than those cultured in CM. Resident peritoneal macrophages cultured in wound
cell supernatants also produced less superoxide after PMA stimulation than those cultured in CM at all tested LPS concentrations
(Fig. 8B).
Partial characterization of the neutrophil-derived factor(s) that
inhibits proinflammatory cytokine release from J774A.1
macrophages
The neutrophil-derived factor(s) that inhibits proinflammatory cytokine release by LPS-stimulated J774A.1 macrophages is resistant
to freezing and thawing and has a molecular mass ⬍3000 Da. Fig.
9 shows that the ⬍3000-Da filtrates of wound cell-conditioned
supernatants inhibited TNF-␣ release by LPS-stimulated J774A.1
macrophages similar to unfractionated supernatants. IL-6 release
was also inhibited by the ⬍3000-Da fraction of wound cell-conditioned supernatants (J774A.1 in CM, 19.5 ⫾ 1.4 ng/ml; J774A.1
in supernatants, 11.4 ⫾ 0.7 ng/ml; p ⬍ 0.01, by Student’s unpaired
t test).
PGE2 (9) and adenosine (10 –13) have been shown to suppress
macrophage TNF-␣ production and meet the criteria of stability to
freezing/thawing and molecular mass ⬍3000 Da. The results presented in Fig. 10 show that cyclooxygenase inhibitors did not alter
TNF-␣ release in cocultures of day 1 wound cells and J774A.1
macrophages. Neither the selective cyclooxygenase-2 inhibitor
NS398 (10 ␮M), nor the nonselective cyclooxygenase inhibitor
FIGURE 10. Effect of cyclooxygenase inhibitors on wound neutrophil inhibition of TNF-␣ release by LPS-stimulated J774A.1 macrophages. J774A.1
cells were cultured alone or with day 1 wound cells in Transwells as described
in Fig. 4. NS-398 (10 ␮M) or indomethacin (10 ␮M) was added to cultures 1 h
before stimulation with LPS (0.1 ␮g/ml). TNF-␣ (A) and PGE2 (B) concentrations in the J774A.1 culture medium were assayed after 24-h culture as
described in Materials and Methods. J774A.1/wound cell cocultures accumulated less TNF-␣ than J774A.1 cultures (p ⬍ 0.05, by two-factor ANOVA).
Neither cyclooxygenase inhibitor had an effect on TNF-␣ release.
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FIGURE 9. The ⬍3000-Da fraction of wound cell supernatant inhibits
TNF-␣ release by LPS-stimulated J774A.1 macrophages. J774A.1 cells
were cultured in CM (䡺) or wound cell supernatants (f) as described in
Fig. 6. The ⬍3000-Da fraction of supernatant or CM was also diluted 1/1
with CM and added to J774.1 cultures. All cultures were treated with LPS
(0.1 ␮g/ml). The TNF-␣ concentration of the J774A.1 culture supernatant
was assayed by ELISA after 24-h culture. Both the unfractionated and the
⬍3000-Da fraction of wound cell supernatants inhibited TNF-␣ release
compared with CM (p ⬍ 0.01, by two-factor ANOVA).
The Journal of Immunology
The use of PG receptor antagonists confirmed that PGE2 is not
the inhibitory factor under study. Neither the EP4 PG receptor
antagonist L161982 (0.5–10 ␮M) nor the EP1/EP2/DP PG receptor
antagonist AH6809 (0.1–10 ␮M) reversed the inhibition of TNF-␣
release by LPS-stimulated J774A.1 macrophages cultured with
wound cell-conditioned supernatants (data not shown).
Stimulation of adenosine receptors has been reported to inhibit
the production of TNF-␣ by LPS-stimulated macrophages. No
ATP, ADP, AMP, or adenosine was detected in wound cell-conditioned supernatant by HPLC. To rule out adenosine receptor activation by concentrations of adenosine lower than the limit of
detection of the assay, wound cell-conditioned supernatants were
treated with adenosine deaminase before addition to J774A.1 cells.
Adenosine deaminase did not reverse the inhibition of TNF-␣ release by LPS-stimulated J774A.1 macrophages. Furthermore, the
adenosine receptor antagonist, 8-phenyl theophylline (1–20 ␮M),
had no effect on TNF-␣ release by LPS-stimulated J774A.1 macrophages (data not shown).
The results reported in this study provide in vivo and in vitro
evidence that neutrophils modulate macrophage inflammatory phenotype through the release of a soluble mediator(s). In vivo findings in a model of acute sterile inflammation demonstrate that
neutrophil depletion results in selective alterations in local cytokine concentrations that include increases in TNF-␣ and IL-6 and
reduction in TGF-␤1. Intracellular staining evidenced increased
TNF-␣ staining in both neutrophils and macrophages from the
wounds of neutropenic animals compared with those from control
animals. The observed increases in TNF-␣ and IL-6 in the wound
fluid of neutropenic animals reflect a local, rather than a systemic,
effect, because plasma TNF-␣ and IL-6 were not different in neutropenic and control animals. Moreover, intracellular staining of
TNF-␣ in circulating leukocytes revealed no differences between
the groups.
The increases in TNF-␣ and IL-6 concentrations of wound fluids
from neutropenic animals were unexpected in view of the 67-fold
reduction in the number of total wound leukocytes. The reciprocal
changes in cell number vs proinflammatory cytokine content in the
wounds of control and neutropenic animals suggested that cell
density-dependent mechanisms regulate proinflammatory cytokine
production at sites of inflammation, with a higher cell number
correlating to decreased, rather than increased, cytokine levels.
One well-described mechanism that could account for the present
findings in vivo is the phagocytosis of apoptotic neutrophils and
the subsequent reduction in proinflammatory cytokine release by
macrophages at the inflammatory site (14, 15). Alternatively, the
present observations could be explained by a soluble factor(s) released from wound cells that down-regulates local proinflammatory cytokine release through an autocrine and/or paracrine mechanism. To test for both mechanisms simultaneously, a coculture
system that mimicked the cellularity of the early wound and prevented direct cell-to-cell contact was used.
The data in Fig. 4 show results from one such coculture experiment and demonstrate that day 1 wound cells from naive animals,
which are predominantly neutrophils, inhibited TNF-␣ and IL-6
release by LPS-stimulated J774A.1 macrophages by 60%. The results depicted in Fig. 5 suggest synthesis and release of the inhibitory factor(s) into the medium during coculture, because TNF-␣
accumulation in the medium was not suppressed until after 8 h in
coculture. Inhibition of TNF-␣ release was detectable by 1 h after
the addition of conditioned supernatant to J774A.1 cells (Fig. 7),
suggesting that those supernatants contain a preformed inhibitory
factor(s). Fig. 6 provides evidence that neutrophils are, among
wound cells, the most likely source for the soluble mediator(s) that
inhibited macrophage cytokine release in cocultures, because cell
culture supernatants from purified wound neutrophils suppressed
TNF-␣ release by LPS-stimulated J774A.1 macrophages to the
same extent as supernatants from unfractionated wound cells.
Moreover, the inhibition of proinflammatory cytokine release is
not due to a nonselective inhibition of protein synthesis, because
total protein synthesis was not altered by wound cell-conditioned
supernatants.
The suppressive effects of wound cell culture supernatants were
not limited to the J774A.1 macrophage cell line. Fig. 8A demonstrates that the supernatants also inhibited TNF-␣ release from
primary peritoneal macrophages. Moreover, the suppression of
LPS-mediated macrophage activation was not restricted to the production of proinflammatory cytokines. The results shown in Fig.
8B illustrate the reduction in macrophage superoxide release that
resulted from culture in wound cell-conditioned supernatant.
Results from this study demonstrate that neutrophils mediate the
inhibition of proinflammatory cytokine and superoxide release
from macrophages via a soluble mediator(s) of ⬍3000 Da. Characterization of the inhibitory molecule(s) is not yet complete.
Known inhibitors of macrophage TNF-␣ release that are ⬍3000
Da include PGE2 and adenosine. The early accumulation of PGs at
inflammatory sites (16) and the suppressive effects of PGE2 on
macrophage TNF-␣ production (9) suggested that cyclooxygenase
products might mediate wound neutrophil suppression of macrophage TNF-␣ release in response to LPS. However, the data in Fig.
10 show that inhibition of PGE2 synthesis by NS 398 or indomethacin did not alter the neutrophil-mediated inhibition of TNF-␣ release by LPS-stimulated macrophages. Also, neither the EP4 receptor antagonist L161982 nor the EP2 receptor antagonist
AH6809 affected the capacity of wound cell-conditioned supernatants to inhibit TNF-␣ release by J774A.1 macrophages. In this
regard the receptor antagonists were selected because J774A.1
cells express only PG receptors of the EP4 and EP2 subtypes (17).
PGE2, therefore, is not the mediator of wound cell suppression of
TNF-␣ release by LPS-stimulated macrophages. Adenosine and
adenosine receptor agonists also inhibit macrophage inflammatory
responses, including the release of TNF-␣ (10 –13). Neither adenosine nor its metabolic precursors (ATP, ADP, and AMP) were
found in wound cell-conditioned supernatants. Also, the lack of
effect of adenosine deaminase or an adenosine receptor antagonist
argues against involvement of the adenosine receptor in the wound
cell-mediated inhibition of TNF-␣ release by LPS-stimulated
macrophages.
Supernatants from wound-derived fibroblasts, but not those
from splenic lymphocytes or from L929 immortalized murine fibroblasts, also suppressed LPS-stimulated TNF-␣ release from
J774A.1 cells. Although it is not known whether the suppressive
factor produced by wound fibroblasts is identical with that from
wound neutrophils, it is of interest that other cell types that come
into contact with macrophages within inflammatory sites are also
capable of inhibiting TNF-␣ release.
These results confirm a role for neutrophils in the regulation of
proinflammatory responses by macrophages. This conclusion finds
support and extension in published studies of neutrophil depletion.
Rats made neutropenic with vinblastine and subjected to hemorrhagic shock manifested decreased neutrophil sequestration and
increased expression of IL-6 mRNA and CD14 mRNA in the lung
compared with nonneutropenic controls (18). Mice lacking the
transcriptional repressor Gfi1 are severely neutropenic and have
increased lethality and higher plasma levels of TNF-␣, IL-10, and
IL-1␤ after LPS administration (19). Macrophages isolated from
neutropenic Gfi1-deficient mice exhibit increased LPS-stimulated
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Discussion
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Acknowledgments
We thank Jill Rose for assistance in preparing the manuscript.
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TNF-␣ release. The findings of the present study suggest that
neutropenia in Gfi1-deficient animals resulted in up-regulation of
macrophage cytokine production. The latter conclusion is also
supported by Steinshamn’s investigation (20) of the TNF-␣ response to endotoxin in mice made neutropenic with cyclophosphamide, showing increased lethality and increased serum TNF-␣
concentration compared with control animals after LPS stimulation. A proinflammatory bias in neutropenia has thus been shown
to be independent of species, mechanism of neutropenia, and
stressor imposed upon the animals.
The work reported in this study resulted in an unexpected and
paradoxical finding: that neutrophils, which are considered prototypical inflammatory cells, could act as anti-inflammatory cells
through suppression of the macrophage inflammatory phenotype.
The present observations together with reports indicating that neutrophils modulate T cell subset selection in candidiasis (21) and
dendritic cell activation in Toxoplasma gondii infection (22), shed
new light on the capacity of neutrophils to regulate multiple cellular immune responses.
As stated in the introduction, the role of neutrophils in sterile
inflammation has not been completely clarified. With specific regard to the role of neutrophils in the healing of wounds, the current
paradigm, which is based on the work of Simpson and Ross (23),
states that neutrophils are dispensable to the repair process, and
that their function in injured tissue is only to contain or eliminate
infection. Only recently has this paradigm been challenged. Dovi
et al. (24) demonstrated accelerated re-epithelialization of skin
wounds in transiently neutropenic mice, without effects on collagen content or tensile strength of the wound. These findings do not
appear to apply to all models of wound healing, because re-epithelialization of oral wounds in guinea pigs was neither accelerated
nor retarded in neutropenic animals (25). Thus, the role of neutrophils in repair remains undefined.
The present observations are unlikely to be limited to wounds.
The early inflammatory response in the PVA sponge wound model
is identical with that observed in other models of sterile tissue
injury, with the early development of a neutrophil-rich cellular
infiltrate that is later replaced by a macrophage-predominant infiltrate. The suppression of the macrophage proinflammatory phenotype by neutrophil products is probably common to all acute sterile
inflammatory processes. Characterization of a neutrophil-derived
soluble mediator(s) that suppresses macrophage proinflammatory
phenotype as well as their mechanism of action may provide
opportunities for therapeutic intervention to suppress macrophage inflammatory phenotype, especially during spontaneous
or therapy-induced neutropenia.