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From the Department of Surgery, Division of Surgical Research, Brown University and Rhode Island Hospital, Providence, Rhode Island
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Introduction |
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The relevance of the product of iNOS, NO, and its putative derivatives to inflammatory processes in general and more specifically to wound healing has recently been highlighted.5 It has been proposed, in this regard, that the sustained expression of NOS in healing wounds is critical to the accumulation of collagen and the acquisition of mechanical strength in wounds.5 These results were, interestingly, obtained in part using the same wound model used in previous reports from this laboratory. Because they differed so substantially from observations contrary to the significant expression of NOS in the wound past the initial 24 to 72 hours after wounding, experiments were performed to better define the temporal pattern of iNOS expression and identify the cells expressing this enzyme in healing wounds. Results presented herein further support a redundant system of NOS regulation in healing wounds that includes, along with the degradation of extracellular L-arginine by arginase in late wounds,1,2 the restricted expression of iNOS to macrophages in the early phases of repair and the suppression of iNOS induction in macrophages from late wounds by factors present in the wound's extracellular fluid.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male Fischer rats (150 to 200 g; VAF-Plus, Charles River Breeding Laboratories, Wilmington, MA) were used in all wounding experiments. VAF-Plus animals are certified free of common rat pathogens by the supplier and housed in an isolation environment on their arrival at the laboratory. The animals were monitored by Brown University/Rhode Island Hospital veterinary personnel. Sterile circular polyvinyl alcohol sponges (Unipoint Industries, High Point, NC) measuring approximately 1 cm in diameter and 0.4 cm in thickness were implanted subcutaneously through a 7-cm midline incision in the dorsum of each animal (10 sponges per animal) under anesthesia (Pentobarbital, Abbott Laboratories, North Chicago, IL; 5 mg/100 g body weight).1,2
At designated times after sponge implantation, the animals were sacrificed with CO2, and cells contained in the sponges were harvested exactly as described previously.1,2 That cellular recovery from the sponges is virtually complete was demonstrated experimentally by the lack of detectable DNA in the sponges after cell extraction (not shown). Total cell yields were used for differential counts and for the immunoblot detection of iNOS. Wound-derived macrophages were isolated from the wound cell preparation by adherence to plastic and recovered with ice-cold Ca- and Mg-free Hanks' balanced salt solution. Purity was greater than 90% as determined by Wright-Giemsa staining and by immunofluorescence using an anti-rat macrophage antibody (Ab).6 Viability at the time of harvest was >95% by trypan blue exclusion. Viability after various periods of incubation in culture medium (RPMI 1640 (GIBCO, Grand Island, NY) supplemented with 1% bovine serum albumin, 10 mmol/L 3-(N-morpholino)-propanesulfonic acid, and antibiotics) was determined by lactic dehydrogenase release into supernatants and was >85% at 24 hours. Cell-free wound fluid was obtained by centrifugation of whole sponges in the cold as described previously.2
Peritoneal exudate cells were obtained by peritoneal lavage 6 hours after the intraperitoneal injection of oyster glycogen (Sigma Chemical Co., St. Louis, MO). These cells were >90% PMNs by staining with a specific mouse anti-rat PMN monoclonal (M)Ab.7
Immunoblot Analysis of iNOS Expression
Postnuclear supernatants of cell lysates obtained from freshly harvested or cultured cells were size fractionated in 7.5% SDS-polyacrylamide gel electrophoresis loaded with 15 µg of cell protein per lane. Proteins were transferred to a nitrocellulose membrane and incubated overnight in blocking buffer containing 10 mmol/L Tris/HCl, pH 7.5, 1% bovine serum albumin, 100 mmol/L NaCl, and 0.1% Tween-20. Blots were probed with a mouse MAb against macrophage iNOS (Transduction Laboratories, Lexington, KY) diluted 1:500 in blocking buffer for 1 hour at room temperature followed by washing for 30 minutes in blocking buffer. Antibody was detected using alkaline-phosphatase-conjugated goat anti-mouse immunoglobulin (Sigma) and visualized with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Bio-Rad Laboratories, Hercules, CA). A contour plot of each band was generated, and optical density of the area was determined using a PDI desktop densitometer and Quantity One one-dimensional gel analysis software (PDI, Huntington Station, NY).
Immunofluorescence Analysis
Cells were either cytospun onto glass slides when immunostaining was to be performed on freshly isolated preparations or adhered in Permanox eight-well chambered slides (Nunc, Naperville, IL) in experiments warranting an incubation period. Fixation was accomplished in acetone at -20°C for 5 minutes. Cells were blocked in PBS containing 1% normal goat serum and incubated with primary Ab in PBS plus 1% normal goat serum for 45 minutes at room temperature. Primary antibodies included rabbit anti-iNOS (1:50; Transduction Laboratories) and ascitic fluids containing either mouse anti-rat macrophage IgG (KU-1) at 1:200 dilution6 or mouse anti-rat neutrophil IgG (RP3) at 1:200 dilution.7 Slides were washed and incubated with fluorescein-conjugated goat anti-mouse IgG (1:100; Sigma) and Texas-Red-conjugated sheep anti-rabbit IgG (1:500; Biosource, Camarillo, CA) for 30 minutes at room temperature. Cells were observed using a Nikon Microphot-FXA, and images were captured using a SenSys CCD fitted with PVCAM acquisition software (Photometrics, Tucson, AZ) and analyzed using IPLab Spectrum 3.1 (Signal Analytics Co., Vienna, VA).
L-Arginine Metabolism by Wound-Derived Cells
Cells freshly harvested from 1-day-old wounds were enriched for macrophages or PMNs using NIM.2 density gradients (Cardinal Associates, Santa Fe, NM). Enriched populations were cultured in medium containing 1 mmol/L L-arginine and 0.1 µCi of [guanido-14C]L-arginine (DuPont NEN, Cambridge, MA) for 1, 3, or 5 hours at a density of 106 cells/ml. At each time point the appearance of radiolabeled L-citrulline in culture supernatant was determined using a Bio-LC amino acid analyzer (Dionex, Sunnyvale, CA) and on-line peak detection. Flux of L-arginine through iNOS is reported as cpm in its product, [ureido-14C]L-citrulline. Similar metabolic experiments were performed using freshly harvested or overnight cultured 10 day wound-derived macrophages. In this case, cultures with radiolabeled L-arginine were conducted for 2 hours. Flux of L-arginine through iNOS was calculated from the radioactivity in L-citrulline and the initial L-arginine-specific radioactivity in the medium at the beginning of culture.
NADPH Diaphorase Activity
Wound-derived cells were fixed for 5 minutes with 4% paraformaldehyde and rinsed with PBS. NADPH diaphorase activity was demonstrated by incubating cells at 37°C for 45 minutes with 0.5 ml of NADPH (1.25 mg/ml), nitroblue tetrazolium (100 µg/ml), and 0.2% Triton X-100 in 0.1 mol/L phosphate buffer (pH 8.0) and an additional 1 ml of PBS. The cells were washed with PBS and stained with nuclear fast red (1%) for 3 minutes to chelate ammonium ions.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Figure 1
displays immunoblots for
iNOS in lysates of unfractionated cell preparations harvested from
wounds 1 through 10 days after injury. As can be seen, iNOS
immunoreactivity was prominently present in lysates from freshly
harvested wound-derived cells on days 1 and 3 and declined thereafter.
Rat blood monocytes lacked immunodetectable iNOS protein (not shown).
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. PMNs constitute the
bulk of the inflammatory infiltrate during the initial 48 hours after
wounding and are progressively replaced by macrophages. By 10 days
after injury, practically all cells isolated from the wounds are
macrophages. Because of the high proportion of PMNs contained in early
wounds and their reported ability to express iNOS,8
it was
proposed from this laboratory that it was these cells that contained
and expressed iNOS in the early wounds.2
Adding support to
this hypothesis, Miles, et al reported that lysates of peritoneal cells
isolated shortly after the intraperitoneal injection of oyster
glycogen, a technique for the enrichment of peritoneal exudate for
PMNs, strongly immunoreacted to an Ab against iNOS.9
Immunofluorescent examination of cells isolated from 1-day-old wounds
(Figure 2)
, however, demonstrated that
PMNs provide only low-level signal for iNOS antigen and indicated that
iNOS antigen present in wound cells at that time is predominantly
associated with the macrophages found in the preparation. Evidence in
this regard includes the coincident staining of macrophages with an
anti-rat macrophage-specific Ab and the anti-iNOS Ab (Figure 2C)
as
well as the mutual exclusion of iNOS staining and that obtained with an
Ab specific for rat PMNs (Figure 2G)
. Almost identical findings
resulted from the examination of wounds harvested 6 hours after
wounding, with a predominance of iNOS-negative PMNs in the preparations
and the presence of occasional macrophages that were, for the most
part, iNOS positive (not shown).
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. Applying this
criterion to results from six independent experiments, the majority of
day 1 wound macrophages were positive for iNOS, whereas approximately
one-half of PMNs in these wounds contained detectable iNOS antigen.
Although there were, indeed, iNOS-positive PMNs to be found in these
preparations, there was an order of magnitude difference in the
intensity of iNOS-specific staining between macrophages and PMNs (Table 2)
. Moreover, as will be shown later, PMNs found by immunofluorescent
staining to contain iNOS antigen may not actually express iNOS
activity.
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Previous findings from this laboratory demonstrated that overnight
culture of wound-derived macrophages obtained 10 days after injury
resulted in increased cellular content of iNOS antigen.3
In agreement with these findings, immunofluorescent staining
demonstrated that 5% to 7% of the wound day 10 cells expressed iNOS
immediately on harvest, increasing to 29% to 49% iNOS-positive cells
after overnight culture (Figure 3)
. This variability represents the
range found in data collected from six separate experiments. iNOS was
found to be present in a subset of macrophages rather than being
uniformly distributed among the entire population both in freshly
harvested and overnight cultured cells. No quantitative differences
were detected in the intensity of iNOS immunofluorescent staining
between iNOS-positive macrophages stained immediately on harvest and
those examined after overnight culture. A redistribution of the antigen
recognized by the anti-rat macrophage Ab KU-1 may be noted after
overnight culture (Figure 3, A and D)
. This is a reproducible, albeit
incidental, finding in the context of this report as the Ab is used
here solely as a phenotypic marker. The nature of the epitope
recognized by this Ab remains uncharacterized although preliminary data
suggest it might be the rat equivalent of the human CD68 antigen
(unpublished observations).
More closely examining the acquisition of iNOS by macrophages harvested
from 10-day-old wounds in culture, results shown in Figure 4
illustrate the time course of the
appearance of iNOS antigen in these cells during an overnight
incubation as well as evidence that iNOS mRNA accumulates in the cells
before the appearance of iNOS protein. That the expression of iNOS in
cultured wound macrophages did not result from cell adherence in
culture was demonstrated in experiments where cells were incubated in
Teflon bags. Cells so cultured also acquired iNOS protein during
overnight incubation (not shown). Differentiation into wound
macrophages is apparently necessary for the induction of iNOS in
culture as blood monocytes did not express iNOS after overnight culture
(not shown).
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, removal of macrophages from the wound and
their culture results in increased expression of iNOS in the cells.
Although this phenomenon could result from the effects of any of
multiple media components, it could also be explained by the presence
of inhibitors or suppressors of iNOS induction in the extracellular
space of the wound. To explore this possibility, wound macrophages
harvested 10 days after wounding were cultured overnight in the
presence of varying concentrations of cell-free wound fluid obtained
from the same animals (1% to 10% v/v) or normal rat serum up to 20%
by volume. Figure 5
illustrates results
from these experiments. Data shown there demonstrate, first, that the
addition of 1 mmol/L L-arginine to culture media suppresses
the induction of iNOS relative to results obtained when
L-arginine was added at 6 µmol/L, a concentration closely
resembling that actually found in the extracellular space of wounds 10
days after wounding.2
This finding supports the conclusion
made previously, on the basis of experiments where NO production by
wound macrophages was modulated through the regulation of
O2 availability, that NO down-regulates iNOS
expression.3
In regard to the potential presence of an
inhibitor of iNOS expression in wound fluids, addition of such fluids
to 6 µmol/L L-arginine medium resulted in a dose-related
suppression of iNOS appearance. Rat serum was without effect and is not
shown.
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To further define the activity of iNOS in freshly harvested day 1
and day 10 wound cells, these cells were analyzed for their ability to
metabolize L-arginine to L-citrulline in
culture and for NADPH diaphorase activity.10
Figure 6
illustrates results of experiments
where wound cells isolated 1 day after wounding were partially enriched
for PMNs or macrophages and cultured for up to 5 hours in the presence
of [guanido-14C]L-arginine.
Results in Figure 6
indicate that most L-arginine
catabolism through NOS to
[ureido-14C]L-citrulline was
conducted by the macrophage-enriched population, with little flux
through this enzyme in the PMN-enriched fraction. Moreover, Figure 7A
demonstrates that only macrophages in
day 1 wound-cell preparation expressed NADPH diaphorase activity.
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, demonstrating that freshly harvested
macrophages produced little radiolabeled L-citrulline
from radiolabeled L-arginine. Results shown in Figure 7C
,
in addition, demonstrate that no NADPH diaphorase activity was
detectable in these cells. Overnight cultured day 10 macrophages
produced substantially more labeled L-citrulline from
L-arginine (Table 3)
and stained strongly for NAPDH
diaphorase activity (Figure 7D)
. These results suggest that iNOS
present in freshly harvested day 10 wound-derived macrophages, in
contrast to that found in day 1 cells and in overnight cultured day 10
macrophages, has limited to nil functional activity.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Additional results identified the cellular population expressing iNOS in early wounds as macrophages and not PMNs. Although multiple cell types, including endothelial cells and platelets, could contribute to the production of NO in the early phases of inflammation, it was of interest to note that blood monocytes lacked iNOS and that wound macrophages expressed this enzyme as early as 6 hours after wounding. PMNs present in wounds harvested at that time, although theoretically capable of expressing iNOS, were mostly silent for immunodetectable iNOS, consumed little L-arginine in culture, and showed no histochemical evidence for NADPH diaphorase activity.
As mentioned previously, a report by Miles et al9 concluded that PMNs present in the peritoneal cavity of rats injected with oyster glycogen expressed iNOS. This conclusion was based on findings in immunoblots performed on lysates of unfractionated peritoneal exudate cells. It appeared unlikely that two methods for PMN elicitation, wounding or glycogen injection, would give discordant results in terms of iNOS detection. Immunodetection experiments were performed, therefore, using glycogen-elicited peritoneal PMNs. Just as described by Miles et al, immunoblotting of lysates prepared from these cells provided a strong signal for iNOS (not shown). However, immunofluorescent staining of the cells indicated that, just as found in wound cells, it is the macrophages present in the preparation that account for the positive identification of iNOS (not shown).
Macrophages proved to be, on a cell-by-cell basis, the predominant cell in the expression of iNOS in early wounds. It must be kept in mind, however, that over 85% of all cells isolated from those wounds are PMNs, so that low-level iNOS expression by these cells could result in substantial aggregate NO production. This does not seem to be the case because these cells were not capable of metabolizing significant amounts of L-arginine to L-citrulline in culture and because NADPH diaphorase staining of early wound cells demonstrated no enzyme activity in the PMNs. It may be that iNOS protein that is immunodetected in these cells is functionally inert.
iNOS expression in wound cells declined as the wound aged. The markedly reduced immunodetectable iNOS in macrophages freshly harvested from day 10 wounds, their limited capacity to metabolize L-arginine to L-citrulline when assayed immediately after harvesting, and results of NADPH diaphorase staining experiments are in line with previous observations indicating the inability of late wound explants to process L-arginine to L-citrulline in culture.2
As demonstrated in this communication, freshly harvested day 10 wound macrophages contain little immunoreactive iNOS and iNOS mRNA. iNOS-specific mRNA appears in the cells within 3 hours of culture, and iNOS protein follows at approximately 12 hours. The suppression of iNOS appearance in the cells by culture with wound fluids and not with normal rat serum suggests the presence of a fluid-phase inhibitor of iNOS induction in late wounds. Preliminary results argue against transforming growth factor (TGF)-ß as the cytokine responsible for the suppressive effects of wound fluids because the inclusion of a neutralizing anti-TGF-ß antibody in cultures of wound macrophages with wound fluid failed to allow the appearance of iNOS protein in the cells. The potential roles of other compounds known to be able to inhibit the induction of iNOS in macrophages (interleukin-10, interleukin-4, or corticosterone) are under current investigation. Incidentally, the appearance of iNOS protein and activity in day 10 wound-derived macrophages during culture explains the finding of high concentrations of NO2-, a degradation product of NO, in supernatants of overnight incubations of these cells that was reported by others.11
The temporal restriction in the expression of iNOS in wounds shown here may not precisely apply to other species or wound types. A recent report describes the delayed expression of iNOS, with peak message found in the wound 4 days after injury, in cutaneous wounds in mice.12 Moreover, that report indicates that wound closure proceeds slower in iNOS knock-out mice than in wild-type animals and that delayed repair in iNOS-deficient animals could be reversed by the local application of an adenoviral vector containing human iNOS cDNA at the time of injury. It must be kept in mind that knock-out animals did, after all, close their wounds. Thus, although potentially relevant in determining the rate of repair, early iNOS expression does not appear to be essential to wound healing.
It can be proposed that the suppression of iNOS in late wounds may be important to the process of repair. In this connection, evidence reported here and elsewhere indicates that the late wound environment is particularly adverse to the expression of iNOS. First because, as shown here, wound fluids suppress the expression of iNOS in wound macrophages. Second, because wound fluids harvested from late wounds contain a remarkably high arginase activity, which results in the almost complete disappearance of L-arginine from the free amino acid pool of the wound extracellular space.1,2 The wound environment, then, both suppresses iNOS expression and decreases substrate availability to this enzyme. As judged from the amino acid composition of wound fluid1,2 and the lack of L-citrulline production by cultured whole wounds previously reported from this laboratory,2 this double hit effectively suppresses iNOS activity in late wounds. The antiproliferative effects of NO or its derivatives,13,14 their ability to suppress protein synthesis,15 and their capacity to inhibit prolyl hydroxylase,16 an enzyme that is rate limiting for the processing of nascent collagen, may explain the need to suppress the expression of iNOS in wounds after the phase of early inflammation by multiple and overlapping mechanisms.
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Acknowledgements |
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Footnotes |
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Supported in part by National Institutes of Health grants GM-42859 (J.E. Albina) and GM-51493 (J.S. Reichner). A.J. Meszaros and C.A. Louis are supported in part by NIEHS Pathobiology Graduate Program training grant T32 ES07272. Additional funding was provided by the Anita Allard Memorial Fund and by Rhode Island Hospital.
Accepted for publication December 15, 1998.
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