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Jean M. Daley,2 Jonathan S. Reichner, Eric J. Mahoney, Laura Manfield, William L. Henry, Jr.,
Balduino Mastrofrancesco, and Jorge E. Albina
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).
N eutrophils are the first blood-borne nucleated cells to results supported and extended the predictions of this hypothesis
infiltrate sites of injury or infection, where they produce by demonstrating that wound neutrophils produce a soluble fac-
multiple effector molecules, including reactive oxygen tor(s) that suppresses LPS-mediated activation of a macrophage
and nitrogen intermediates, proteolytic enzymes, and cytokines. cell line and primary peritoneal macrophages. The present results
The anti-infectious capacity of neutrophils provides obvious func- thus reveal an additional capacity of neutrophils in inflammation,
tional advantage to their recruitment into infected tissue. Neutro- namely, the ability of these cells to modulate macrophage inflam-
phils, however, also accumulate in large numbers at sites of sterile matory phenotype.
inflammation, such as uninfected wounds, myocardial and cerebral
infarctions, and fractures where, in the absence of infectious chal-
lenge, their role in the inflammatory response is less readily Materials and Methods
apparent. Animals
Experiments reported in this study were initially designed to
B6D2F1 male mice (Taconic Farms), 8 12 wk of age, were housed at the
define the contribution of neutrophils to the cytokine profile of an
Central Research Facilities of Rhode Island Hospital and fed mouse chow
acute sterile inflammatory site, such as that provided by the poly- and water ad libitum. Mice were certified free of common pathogens by the
vinyl alcohol (PVA)3 sponge wound model. The findings demon- supplier and were monitored by Brown University/Rhode Island Hospital
strated that neutrophil depletion results in increased proinflamma- veterinary personnel. Animal protocols were approved by the animal care
tory cytokine concentrations in early tissue inflammation. This committee at Rhode Island Hospital.
finding suggested that neutrophils produce a factor(s) that inhibits
the release of proinflammatory cytokines by macrophages. The PVA sponge wound model
Mice were anesthetized with pentobarbital (50 mg/kg i.p.; Abbott Labo-
ratories). Five PVA sponges (PVA Unlimited), measuring 1 1 0.6 cm,
Department of Surgery, Division of Surgical Research, Rhode Island Hospital and
Brown Medical School, Providence, RI 02903
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
Received for publication April 27, 2004. Accepted for publication December 1, 2004. euthanized by CO2 asphyxiation, and the sponges were removed under
The costs of publication of this article were defrayed in part by the payment of page sterile conditions. Culture of randomly selected PVA sponges at the time
charges. This article must therefore be hereby marked advertisement in accordance of removal confirmed sterility in this experimental model. Wound cells
with 18 U.S.C. Section 1734 solely to indicate this fact. were isolated by rapid repeated compression of sponges in a Stomacher
1
This work was funded by National Institutes of Health Grants GM42859 (to J.E.A.) (Tekmar). When necessary, cells were subjected to hypotonic lysis of
and GM66194 (to J.S.R.) and by funds allocated to the Department of Surgery by erythrocytes, followed by reconstitution in PBS (Invitrogen Life Technol-
Rhode Island Hospital, a Lifespan partner. J.M.D. was supported by a National In- ogies). Viable cells were identified by trypan blue exclusion and were
stitutes of Health Supplement to Promote Reentry into Biomedical and Behavioral counted using a hemocytometer; viability was 95%. Differential cell
Research Careers. E.J.M. was supported by the Carter Family Charitable Trust counts were performed on Hema-3 (Biochemical Sciences)-stained Cyto-
(Armand D. Versaci Research Scholar in Surgical Sciences Award).
spins (Shandon), and cell phenotype was confirmed by flow cytometry, as
2
Address correspondence and reprint requests to Dr. J. M. Daley, Division of Sur- described below. Extracellular fluid from the PVA sponges (wound fluid)
gical Research, Rhode Island Hospital, NAB 214, 593 Eddy Street, Providence, RI was obtained by centrifugation (400 g for 5 min) of two or three sponges
02903. E-mail address: jdaley@lifespan.org in the barrel of a syringe that was seated in a sterile tube. Wound fluid was
3
Abbreviations used in this paper: PVA, polyvinyl alcohol; CM, complete medium. stored at 80C until analysis.
Induction of neutropenia Cells were removed by centrifugation, and the supernatants were used as
described above.
Anti-mouse Gr-1 mAb (RB6.8C5 hybridoma originally produced by R. L. Macrophage viability was assessed after 24 h of incubation by trypan
Coffman, DNAX Research Institute) was produced under serum-free con- blue exclusion, reduction of MTT (Sigma-Aldrich) (2), and release of lac-
ditions using a bioreactor (Cell Pharm Micro Mouse; UniSyn Technolo- tate dehydrogenase (3). Protein synthesis was determined by measuring the
gies). Mice were rendered neutropenic by a single i.p. injection of 0.5 mg 24-h incorporation of L-[ring-2,6-N-3H]phenylalanine into TCA-precipita-
of anti-Gr-1 Ab 3 days before PVA sponge insertion. Control animals were ble protein. When indicated, cells were lysed in buffer containing 0.01 M
injected i.p. with an equal volume of normal saline or with 0.5 mg of rat Tris, 0.15 M NaCl, and 0.5% Igepal CA 630 (Sigma-Aldrich) with Com-
IgG (Rockland Immunochemicals). plete (Roche) protease inhibitor.
Control Neutropenic
wound cell supernatants released 71% less TNF- than those cul-
tured in CM. Resident peritoneal macrophages cultured in wound
cell supernatants also produced less superoxide after PMA stimu-
lation than those cultured in CM at all tested LPS concentrations
(Fig. 8B).
The use of PG receptor antagonists confirmed that PGE2 is not wound cells, the most likely source for the soluble mediator(s) that
the inhibitory factor under study. Neither the EP4 PG receptor inhibited macrophage cytokine release in cocultures, because cell
antagonist L161982 (0.510 M) nor the EP1/EP2/DP PG receptor culture supernatants from purified wound neutrophils suppressed
antagonist AH6809 (0.110 M) reversed the inhibition of TNF- TNF- release by LPS-stimulated J774A.1 macrophages to the
release by LPS-stimulated J774A.1 macrophages cultured with same extent as supernatants from unfractionated wound cells.
wound cell-conditioned supernatants (data not shown). Moreover, the inhibition of proinflammatory cytokine release is
Stimulation of adenosine receptors has been reported to inhibit not due to a nonselective inhibition of protein synthesis, because
the production of TNF- by LPS-stimulated macrophages. No total protein synthesis was not altered by wound cell-conditioned
ATP, ADP, AMP, or adenosine was detected in wound cell-con- supernatants.
ditioned supernatant by HPLC. To rule out adenosine receptor ac- The suppressive effects of wound cell culture supernatants were
tivation by concentrations of adenosine lower than the limit of not limited to the J774A.1 macrophage cell line. Fig. 8A demon-
detection of the assay, wound cell-conditioned supernatants were strates that the supernatants also inhibited TNF- release from
treated with adenosine deaminase before addition to J774A.1 cells. primary peritoneal macrophages. Moreover, the suppression of
Adenosine deaminase did not reverse the inhibition of TNF- re- LPS-mediated macrophage activation was not restricted to the pro-
lease by LPS-stimulated J774A.1 macrophages. Furthermore, the duction of proinflammatory cytokines. The results shown in Fig.
adenosine receptor antagonist, 8-phenyl theophylline (120 M), 8B illustrate the reduction in macrophage superoxide release that
had no effect on TNF- release by LPS-stimulated J774A.1 mac- resulted from culture in wound cell-conditioned supernatant.
rophages (data not shown). Results from this study demonstrate that neutrophils mediate the
inhibition of proinflammatory cytokine and superoxide release
TNF- release. The findings of the present study suggest that 2. Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival:
application to proliferation and cytotoxicity assays. J. Immunol. Methods 65:55.
neutropenia in Gfi1-deficient animals resulted in up-regulation of 3. Korzeniewski, C., and D. M. Callewaert. 1983. An enzyme-release assay for
macrophage cytokine production. The latter conclusion is also natural cytotoxicity. J. Immunol. Methods 64:313.
supported by Steinshamns investigation (20) of the TNF- re- 4. Ayala, A., Z. K. Deol, D. L. Lehman, C. D. Herdon, and I. H. Chaudry. 1994.
Polymicrobial sepsis but not low-dose endotoxin infusion causes decreased
sponse to endotoxin in mice made neutropenic with cyclophosph- splenocyte IL-2/IFN- release while increasing IL-4/IL-10 production. J. Surg.
amide, showing increased lethality and increased serum TNF- Res. 56:579.
concentration compared with control animals after LPS stimula- 5. Micheli, V., H. A. Simmonds, M. Bari, and G. Pompucci. 1993. HPLC determi-
nation of oxidized and reduced pyridine coenzymes in human erythrocytes. Clin.
tion. A proinflammatory bias in neutropenia has thus been shown Chim. Acta 220:1.
to be independent of species, mechanism of neutropenia, and 6. Cui, S., J. S. Reichner, R. B. Mateo, and J. E. Albina. 1994. Activated murine
macrophages induce apoptosis in tumor cells through nitric oxide-dependent or
stressor imposed upon the animals. -independent mechanisms. Cancer Res. 54:2462.
The work reported in this study resulted in an unexpected and 7. Nessel, C. C., W. L. Henry, Jr., B. Mastrofrancesco, J. S. Reichner, and
paradoxical finding: that neutrophils, which are considered proto- J. E. Albina. 1999. Vestigial respiratory burst activity in wound macrophages.
Am. J. Physiol. 276:R1587.
typical inflammatory cells, could act as anti-inflammatory cells 8. Rabinowitz, S. S., and S. Gordon. 1991. Macrosialin, a macrophage-restricted
through suppression of the macrophage inflammatory phenotype. membrane sialoprotein differentially glycosylated in response to inflammatory
The present observations together with reports indicating that neu- stimuli. J. Exp. Med. 174:827.
9. Kunkel, S. L., M. Spengler, M. A. May, R. Spengler, J. Larrick, and D. Remick.
trophils modulate T cell subset selection in candidiasis (21) and 1988. Prostaglandin E2 regulates macrophage-derived tumor necrosis factor gene
dendritic cell activation in Toxoplasma gondii infection (22), shed expression. J. Biol. Chem. 263:5380.
10. Hasko, G., C. Szabo, Z. H. Nemeth, V. Kvetan, S. M. Pastores, and E. S. Vizi.
new light on the capacity of neutrophils to regulate multiple cel- 1996. Adenosine receptor agonists differentially regulate IL-10, TNF-, and ni-
lular immune responses. tric oxide production in RAW 264.7 macrophages and in endotoxemic mice.