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*
Department of Immunology and
Minimally Invasive Surgery Center, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH 44195
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, KC, macrophage-inflammatory protein
(MIP)-2, and monocyte chemoattractant protein-1 were the predominant
cytokines and chemokines produced; TNF- was not detected. Chemokine
mRNA expression and protein secretion occurred in two temporal stages.
The first, which reached a maximum at 6 h, was associated with
high levels of IL-1 and KC and low levels of MIP-2. This stage could
be reproduced by intradermal injection of IL-1 or IL-1 and was
partially blocked by injection of neutralizing Ab against IL-1 but
not IL-1. In animals depleted of circulating neutrophils, chemokine
expression was reduced by nearly 70% during this stage. In the second
stage, which peaked at 24 h after injury, modest but significant
levels of IL-1 were detected in association with low levels of KC
and high levels of MIP-2. This pattern of chemokine expression could
not be mimicked by injection of IL-1 or IL-1 (even with prolonged
exposure), although MIP-2 expression could be partially inhibited by
intradermal injection of neutralizing Ab against IL-1. Surprisingly,
neutrophil depletion before injury resulted in sustained high levels of
both KC and MIP-2 expression. These observations demonstrate that these
two closely related chemokines are under distinct regulatory controls
in vivo that are likely to reflect the temporally ordered participation
of different cell types and/or extracellular stimuli and
inhibitors. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The chemoattractant cytokine or chemokine gene superfamily contains >40 members that can be subdivided into at least four families based upon the position of the first two cysteine residues in the amino acid sequence (6, 7, 8). The CC and CXC families include the majority of chemokines. Within the CXC family, there are at least two subgroups that are distinguished by the presence of a 3-aa sequence motif (ELR) that immediately precedes the CXC motif. There are at least four members of the ELR CXC chemokine family in the mouse, two of which (KC and macrophage-inflammatory protein (MIP)3-2), are believed to participate in neutrophil recruitment to sites of inflammation in many tissue locations (10, 11, 12). CXC chemokines are among the earliest new proteins produced at sites of inflammation, consistent with their role in regulating the early infiltration of neutrophils (13, 14). In contrast to the CXC family, the CC chemokine family exhibits broader target cell specificity including lymphocytes, eosinophils, basophils, and monocyte/macrophages (6, 7, 8).
The regulation of chemokine gene expression in vivo may be important in
two respects. Certainly, the expression of gene products with
inflammatory activities must be closely controlled to minimize
unnecessary tissue damage. In addition, many chemokines exhibit
functional redundancy but are expressed in distinct temporal and/or
spatial patterns indicating differential regulation (6, 7, 8, 14). Furthermore, unlike many proinflammatory cytokines whose
abundant expression is largely restricted to inflammatory leukocytes,
almost all cells exhibit the potential to produce substantial amounts
of proinflammatory chemokines in response to a broad spectrum of
stimuli (6, 7). The prototypic proinflammatory cytokines
IL-1 and TNF- are well recognized as among the most potent
inducers of chemokine expression both in vitro and in vivo and have
been demonstrated in many inflammatory sites within minutes to hours
following injury (1, 2). Following their synthesis and/or
release, these agents could stimulate chemokine expression in multiple
surrounding cell types. The observed temporal patterns of chemokine
expression may then reflect differential participation of distinct cell
types and stimuli.
In the present study we examined the temporal expression patterns of
the two major neutrophil-directed chemokines in the mouse (MIP-2 and
KC) following an incisional skin wound. The results define two distinct
stages during the first 24 h after injury; KC expression
predominates during the first 12 h and declines by 24 h while
MIP-2 expression is modest during the first phase, with highest
expression at the 24-h time point. The two stages of chemokine
expression depend in part on the release and de novo synthesis of
IL-1 and IL-1, respectively. Furthermore, the differential
patterns of expression are dependent upon and/or regulated by the
infiltration of inflammatory neutrophils in opposing fashion.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cesium chloride, guanidine thiocyanate, agarose, SDS, Tris,
proteinase K, RNase-free DNase, and random priming kits were purchased
from Boehringer Mannheim (Indianapolis, IN). Formamide was obtained
from U.S. Biochemical (Cleveland, OH). Dextran sulfate was obtained
from Pharmacia Biotech (Uppsala, Sweden). Nylon transfer membrane was
purchased from Micron Separations (Westborough, MA). DuPont NEN
Research Products (Boston, MA) was the source of
[-32P]dCTP and Renaissance ECL reagent.
ELISA kits for mouse cytokines and chemokines including TNF-,
IL-1, IL-1, KC, MIP-2, and monocyte chemoattractant protein
(MCP)-1(JE), neutralizing polyclonal Abs against mouse IL-1 and
IL-1, and rIL-1 and rIL-1 were obtained from R&D Systems
(Minneapolis, MN). A total of 0.3 ng of anti-IL-1 and 1 ng of
anti-IL-1 Ab could neutralize as much as 25 pg of IL-1 or
IL-1, respectively. Rat anti-neutrophil mAb (no. MCA771G) was
obtained from Serotec (Raleigh, NC) and the RB6-C85 mAb
(15) was purified by protein G-Sepharose chromatography
from cell culture supernatants. Biotinylated anti-rat Ig was
obtained from Vector Laboratories (Burlingame, CA) and HRP-conjugated
goat anti-rat IgG was obtained from Jackson ImmunoResearch
Laboratories (West Grove, PA).
Surgical wound
Six- to 8-wk-old female C57BL/6 mice (Charles River Breeding Laboratories, Wilmington, MA) were anesthetized with i.p. injection of sodium pentobarbital (75 mg/kg) and subjected to 20-mm full thickness abdominal incisions. The incisions were closed with Ethibond 5/0 braided nylon suture (Ethicon, Somerville, NJ). At specific times after wounding, mice were sacrificed by CO2 asphyxiation and the tissue surrounding the wound site (4-mm border on both sides) was harvested and used for preparation of total RNA or total protein extract, or fixed for immunohistology. Each experimental group contained 6–10 animals.
RNA preparation, Northern hybridization, and RNase protection assays
Mouse tissue was homogenized for total RNA extraction by the guanidinium isothiocyanate-CsCl method as described previously (16, 17). The levels of cytokine and chemokine mRNA were determined either by Northern hybridization using radiolabeled cDNA fragments as described previously (16, 17) or by use of the RiboQuant RPA system (BD PharMingen, San Diego, CA) according to the manufacturer’s instructions. Plasmids containing KC, MIP-2, and GAPDH cDNAs were prepared as described previously (18, 19).
Preparation of tissue extracts and ELISA
Tissue sections for measurement of cytokine protein levels were
homogenized by mechanical disruption with a PowerGen tissue homogenizer
(Fisher Scientific, Pittsburgh, PA) in 150 mM NaCl and 50 mm Tris-HCl
(pH 7.4) with Minimix protease inhibitors (Roche Pharmaceuticals,
Indianapolis, IN). Tissue homogenates were centrifuged at 18,000
x g for 10 min and the supernatants were collected,
aliquoted, and stored at -20°C. Protein concentrations were measured
by Bio-Rad Protein Assay (Bio-Rad, Hercules, CA). Quantification of
IL-1, IL-1, KC, MIP-2, MCP-1, MIP-1, RANTES, and eotaxin was
done using Quantitative Colorimetric Sandwich ELISA according to the
manufacturer’s instructions.
Immunohistochemistry
Tissue specimens were fixed in 10% buffered formalin for 18 h, dehydrated, embedded in paraffin, and cut in 5-µm sections. The slides were stained for neutrophils using a rat IgG anti-mouse neutrophil mAb (MCA771G). The secondary biotinylated anti-rat Ab was diluted 1/100 in PBS for use and subsequently followed by incubation with avidin-biotin peroxidase. Color development was performed with 3',3'-diaminobenzidine and the slides were counterstained with hematoxylin.
Western slot-blot analysis
One microgram of each tissue extract was applied to nitrobind/nitrocellulose membrane (Osmonics, Westborough, MA) in a Bio-Dot apparatus (Bio-Rad) and subsequently rinsed with PBS. The slot-blot membrane was preincubated 1 h at 20°C in 5% nonfat dry milk in TBS plus Tween 20 (TBST). Rat anti-mouse neutrophil Ab (MCA771G) was used at a dilution of 1/5000 and incubated with filters for 1 h at 20°C. Primary Ab was removed, filters were washed, and secondary Ab (HRP-conjugated goat anti-rat IgG diluted 1/3000 in 5% milk/TBST) was added for an additional 1-h incubation at 20°C. Blots were developed using ECL reagent and quantified by densitometry using IMAGE software (National Institutes of Health, Bethesda, MD). The specificity of the anti-neutrophil Ab was assessed by analysis of cross-reactivity between elicited peritoneal neutrophils and macrophages obtained 6 and 48 h, respectively, after i.p injection of 1 ml of 3% Brewer’s thioglycolate broth.
Intradermal injection
Recombinant cytokines or neutralizing polyclonal Abs were injected intradermally at three separate sites along a 20-mm segment of the abdominal midline using a 26-gauge needle and a tuberculin syringe. Cytokines and Abs were injected in a total volume of 100 µl. For animals undergoing surgery, injections were made 30 min before the procedure. For Ab neutralization studies, control mice were injected with comparable levels of nonimmune goat IgG. Tissue samples were harvested and used for analysis of cytokine and chemokine protein expression as described above.
Statistical analysis
Comparisons of treatment arms over the course of the study were performed with ANOVA F tests. If significant differences were found, then pairwise Student’s t tests were used to compare groups. Standard errors were calculated using least-squares means techniques based on the two-way ANOVA. All calculations were performed with SAS version 8.1 software (SAS Institute, Cary, NC).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Injury induced by an incisional wound resulted in the production
of multiple cytokines and chemokines within the first 24 h as
measured by ELISA of tissue homogenates (Fig. 1
). The most prevalent cytokines detected
were IL-1 and IL-1; no TNF-, IL-6, IFN-
, or TGF was
observed in the initial 24 h following injury. The most highly
expressed chemokines were KC, MIP-2, and MCP-1, while little or no
MIP-1, MIP-1, eotaxin, or RANTES protein was measurable during
this early time period. IL-1 was present in the tissue before
injury, most likely sequestered within keratinocytes (20, 21). In addition, the levels of IL-1 increased rapidly to a
peak at 4 h postsurgery, presumably due to de novo synthesis of
IL-1 mRNA (data not shown). IL-1 protein declined to preinjury
levels within 12 h and remained at this level throughout the
experimental period. IL-1 mRNA and protein production commenced soon
after wounding, but the amounts produced were markedly lower than
IL-1. This response was biphasic, reaching an initial peak around
4–8 h, followed by a modestly higher peak at the 24-h time point (Fig. 1A). Expression of the CXC chemokine KC exhibited a biphasic
pattern in which early levels of KC production generally exceeded
levels observed at the late peak (Fig. 1B). In contrast,
though MIP-2 expression was also biphasic, the later component of the
response was three to four times greater than the early component (Fig. 1C). Thus KC levels exceeded MIP-2 during the early phase
while MIP-2 levels predominated during the later phase. The CC
chemokine MCP-1 exhibited roughly equivalent expression at 8 and
24 h (Fig. 1D).
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). In contrast, MIP-2 mRNA was first
detected at 4 h after injury and increased to maximal levels
between 12 and 16 h after injury. These data demonstrate that the
distinct pattern of KC and MIP-2 protein secretion results from
alterations at the level of gene expression and not from differences in
use or sequestration of secreted chemokine protein.
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differentially regulate the temporal expression of KC
and MIP-2
The presence of IL-1 and IL-1 in wound tissue in the absence
of TNF- suggested that IL-1 may be an important stimulus of CXC
chemokine expression during the response to injury. As a first test of
this possibility, mice were treated intradermally with neutralizing Abs
against IL-1 or IL-1 30 min before surgery. Injection of 50 µg
of anti-IL-1 or anti-IL-1 reduced the detectable levels
of the cognate cytokine by >95% (Fig. 3, A and B). Though
these Abs exhibit <1% cross-reactivity, neutralizing IL-1 also
reduced the level of IL-1 measured 6 h following surgery.
Neutralizing IL-1 with 50 µg of Ab blocked the production of KC
and MIP-2 by 
50% at the 6-h time point, while neutralization of
IL-1 reduced MIP-2 levels but had no effect on KC levels (Fig. 3, C and D). Consistent with the finding that
TNF- was not expressed, no effect was observed following injection
of neutralizing Ab directed against this cytokine (data not shown).
When chemokine expression was measured 24 h following Ab injection
and surgery, neutralization of IL-1 had little effect on either
chemokine, whereas neutralization of IL-1 caused a marked reduction
in expression of both KC and MIP-2 (Fig. 3, C and
D). The neutralizing efficacy of the Ab treatment at 24
h after injury may be diminished due to the diffusion of Ab from the
tissues. Nevertheless, these findings indicate that IL-1 and IL-1
contribute differentially to the temporally distinct waves of KC and
MIP-2 expression.
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in the induction of IL-1, KC, and MIP-2 was
further tested by injection of 100 ng of recombinant mouse (rm)IL-1
intradermally at three different sites along a 20-mm section of the
abdomen. Injection of saline vehicle caused no change in cytokine or
chemokine expression. Injection of rmIL-1 had a modest effect on the
expression of IL-1 but did not induce TNF- (Fig. 4A). KC and MIP-2 were both
strongly induced by IL-1, exhibiting a sharp peak at 4 h which
declined to near basal levels by 12 h postinjection (Fig. 4B). Both chemokines were expressed at levels higher than
observed following surgery, but the quantitative relationship between
the two was similar to that seen during the early postsurgical phase
(e.g., KC 
MIP-2). Notably, no secondary wave of chemokine
expression was observed. KC expression was also more sensitive than
MIP-2 to IL-1, as indicated by an 5-fold difference in the
threshold dose required to elicit expression of either chemokine (Fig. 4C). Both IL-1 and IL-1 induced comparable patterns of
expression following a single injection (Fig. 4D). Hence the
differential roles for IL-1 and IL-1 depend upon their temporal
expression patterns rather than their inherent capacity to stimulate
chemokine expression. The lack of prolonged chemokine expression seen
with a single bolus injection of IL-1 might reflect a requirement for
continued exposure to the stimulus. Indeed, in mice injected three
times at 8-h intervals, KC expression remained high. However, even
under these conditions MIP-2 expression was only modest (Fig. 4D). These findings demonstrate that IL-1 functions as a
relatively selective stimulus for KC expression in the skin.
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To test the role of IL-1 in neutrophil recruitment to the
wound site, tissue from operated animals or animals injected with
rmIL-1 was harvested at 6 or 24 h postinjury or postinjection
for analysis of neutrophil content. The magnitude of neutrophil
infiltration was assessed by measuring the amount of
neutrophil-specific Ag present in tissue extracts using a Western
immunoblot strategy, while the distribution of infiltrating neutrophils
was assessed by immunohistology. One-microgram aliquots of tissue
extract were blotted onto filters using a slot-blot apparatus and
probed with a neutrophil-specific mAb. This method was relatively
specific for neutrophils as indicated by a lack of reactivity with
extracts from untreated skin. Furthermore, there was <3%
cross-reactivity using extracts obtained from purified populations of
peritoneal neutrophils and macrophages. A time course of representative
samples from individual operated mice over a period of hours to days
illustrates that neutrophils are first detected at the wound site
within 4 h, reach maximum levels by 24 h, and disappear
gradually over the next 2–3 days (Fig. 5). This finding is confirmed by the
immunohistology results, which illustrate that the neutrophil
infiltrate is much greater at 18–24 h after surgery than at 6 h
and that the primary site of infiltration is at the wound edge (Fig. 6, A–C). When neutrophil
infiltration was evaluated in tissues from animals injected with
rmIL-1 or , the response was quantitatively less than that
observed in operated animals. While the leukocyte content in operated
and IL-1-injected mice was comparable through the first 12 h,
it increased through 48 h in operated animals but declined in
injected animals between 12 and 48 h (Figs. 5B and 6,
D–E). The distribution of neutrophils in IL-1 injection
sites was diffuse compared with the pattern seen in operated animals.
The injection of saline vehicle did not produce detectable leukocytic
infiltration.
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Neutrophils have been shown to be important sources for chemokine
expression (13, 14, 22, 23, 24). In addition, both chemokine
expression and neutrophil infiltration differed dramatically between
operated and IL-1-injected animals. Hence we wished to assess the
importance of neutrophils in expression of KC and MIP-2 at sites of
surgical injury. Mice were depleted of circulating neutrophils by i.p.
injection of 100 µg of the anti-neutrophil mAb (RB6-C85) as
previously described (15, 25). This treatment led to a
95% reduction in the number of neutrophils elicited into the
peritoneal cavity 6 h following i.p. injection with thioglycolate
broth. One day after Ab treatment animals were either operated or
injected intradermally with rmIL-1 (single or multiple injections)
and the wound or injection sites were harvested 6, 24, or 48 h
later. Examination of wound or injection sites immunohistochemically or
by slot-blot analysis showed that neutrophil infiltration was
undetectable at 6 h and reduced to <20% of control values at
24 h after surgery (data not shown). This reduction in leukocyte
infiltration results in a marked diminution in expression of both KC
and MIP-2 at the wound site during the early phase of the response (6
h) (Fig. 7). Surprisingly, by 24 h,
levels of both KC and MIP-2 were markedly higher than those seen in
neutrophil-replete animals. By 48 h after injury, KC levels
returned to baseline in both normal and neutrophil-depleted animals,
while MIP-2 levels remained elevated in both cases. The effects of
neutrophil depletion in operated animals were most dramatic on KC
expression; in normal animals KC levels declined substantially by
24 h, while in neutrophil-depleted animals KC levels at 24 h
markedly exceeded even those seen in normal animals at the peak of
expression. In neutrophil-depleted animals injected intradermally with
rmIL-1, KC and MIP-2 levels were comparable or modestly reduced as
compared with controls at both 6 and 20 h (in animals receiving
three consecutive IL-1 injections). As in prior studies (see Fig. 4
), IL-1 injection was a potent stimulus of KC expression but had only
a modest effect on MIP-2 (Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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) and
chemokine (KC, MIP-2, and MCP-1) expression occurs in two distinct
temporal waves; 2) KC and MIP-2 are expressed differentially, as KC
exceeds MIP-2 early (6 h after injury) while MIP-2 predominates during
the later phase (24 h); 3) IL-1 is a potent stimulus of KC expression
but has only modest activity for MIP-2 (even prolonged exposure to IL-1
cannot reproduce the second, MIP-2-dominated stage of chemokine
expression); and 4) infiltrating neutrophils influence the two stages
in opposing fashion; in neutrophil depleted animals, chemokine
expression during the first stage is reduced while KC expression during
the second stage is markedly enhanced.
The temporal sequence of cytokine and chemokine expression at a wound
site during the first 24 h appears to be at least biphasic, and
the major components (IL-1, KC, MIP-2, and MCP-1) each exhibited
an early and late response pattern. Complex temporal patterns of
chemokine expression in vivo have been previously reported in a number
of settings, although the regulation of such expression has not been
explored in detail (13, 14, 26, 27, 28, 29, 30). The distinct patterns
of expression for the different CXC chemokine genes reported in this
study are consistent with studies of chemokine expression in excisional
wounds in human skin measured between 1 and 7 days after injury
(13, 14). However, the present study focuses upon patterns
observed within the first 24 h after injury. IL-1 and
appeared to be the predominant cytokines seen within minutes to hours
after injury. IL-1 was detected in uninjured skin, most likely in
preformed pools within keratinocytes (20, 21), and was
released into the tissue rapidly after surgery. In contrast, IL-1
protein secretion was induced at modest levels but exhibited two
distinct temporal phases with peaks at 4–6 h and at 24 h
following injury. Although these two isoforms of IL-1 appear to have
comparable stimulatory activity for promoting chemokine expression in
the skin, the use of neutralizing Abs suggests that they operate in a
temporally distinct fashion; IL-1 is present and active within the
first phase of response (up to 10 h after injury) while IL-1
contributes stimulatory activity in the second phase of response. The
very low induction of TNF- observed in this model is surprising and
in some conflict with previous literature (29, 31). The
differences in our results from those previously reported may reflect
differences in the methodologies used (RT-PCR vs ELISA) or use of
different mouse strains and/or wound models.
The patterns of KC and MIP-2 expression were markedly different from one another; while KC expression was substantially higher than MIP-2 during the first phase of response, MIP-2 levels significantly exceeded KC levels in the second phase of the response. A number of prior studies have observed differential expression both in terms of cell types and in terms of temporal pattern (32, 33, 34, 35, 36, 37). For example, in several models of bacterial infection in mouse lung, maximum KC expression occurs at day 1 while MIP-2 peaks on day 2 (34, 36). In models of ischemia/reperfusion injury and during peritoneal inflammation resulting from cecal ligation/puncture, the temporal pattern of KC and MIP-2 expression differs and appears to involve distinct cell populations (33, 35). The majority of these studies have studied expression at 24 h and later; whether the regulatory mechanisms are similar to those operative during the first 24 h following injury is not known. Interestingly, the differences between KC and MIP-2 expression in terms of time and magnitude suggest that these two functionally similar chemoattractants may have nonredundant roles in the inflammatory process. Indeed, because expression of MIP-2 reaches peak levels at or after the peak of neutrophil infiltration has occurred, MIP-2 may function in later aspects of the inflammatory response. In this regard, CXC chemokines have been reported to exhibit angiogenic activity (38, 39), and several studies have suggested important roles for CXC chemokines and CXCR2 in wound healing. Such an activity would be consistent with the pattern of MIP-2 expression observed in this study (40, 41).
The markedly different expression kinetics for KC and MIP-2
demonstrated in this work are most consistent with phase-specific
regulation of each gene by distinct stimuli and/or cell types.
Injection of rmIL-1 or rmIL-1 appears to reproduce the early
pattern of chemokine expression in which KC predominates over MIP-2.
Both IL-1 and IL-1 were capable of stimulating this early
response, but neither agent was able to stimulate a pattern of response
in which MIP-2 predominates even when tissues levels of IL-1 were
maintained over the 24-h time frame by multiple injections. This
suggests that the early wave of chemokine expression (in which KC
predominates) is dependent upon cells resident in the injured tissue
while the second phase may require additional cell types and/or stimuli
that are only found in operated animals.
The observation that neutrophil infiltration was modest in IL-1-injected as compared with operated animals suggested the possibility that neutrophil infiltration might be an important regulatory aspect of chemokine gene expression. Indeed, neutrophils exhibit substantial chemokine gene expression in vitro and have been shown to be an important source of chemokine production in vivo (13, 14, 22, 23, 24). The depletion of neutrophils had very dramatic but surprising impact on the pattern of chemokine expression. The marked reduction of chemokine levels at 6 h following surgery in neutrophil-depleted animals indicates that at least a portion of the early burst of KC production is derived from or dependent upon infiltrating neutrophils. The truly remarkable increase in KC expression at 24 h after surgery in neutrophil-depleted animals, though unexpected, is consistent with a recent report of MIP-2 and KC expression in CXCR2-/- mice with intracranial infections (37). This observation suggests that KC expression does not decline naturally but is turned off as a consequence of the heavy neutrophil infiltrate. Though possible, this effect is not likely to result from the sequestration of secreted chemokine by neutrophils, because the diminution in KC expression is also seen at the mRNA level. Furthermore, stimuli inducing expression of both KC and MIP-2 must be present at the site during the period from 16 to 24 h following injury, because expression of both genes is very high in the absence of neutrophil infiltration. Hence, in normal animals, neutrophils at the site 16–24 h after injury may act to 1) prevent the generation of stimulus, 2) sequester the stimulus, or 3) inhibit the subsequent response. Moreover, this effect acts in partially selective fashion as KC expression is more sensitive than MIP-2. Neutrophil production of stimulus antagonists such as IL-1RA or soluble TNFR could sequester the necessary stimuli as suggested (42). Alternatively, because neutrophils likely undergo apoptosis at the wound site and apoptotic cell bodies have been shown to be anti-inflammatory, this process may provide a source of selective chemokine regulation (43, 44).
Animals injected multiple times with rmIL-1 exhibited prolonged high
levels of KC expression with only modest neutrophil infiltration.
Hence, the inflammatory cell infiltration that follows the initial
surgical injury probably depends on chemoattractants other than KC and
MIP-2. Indeed, a broad range of chemotactic stimuli may be generated at
wound sites and provide partially redundant control of leukocyte
infiltration (45). For example, the activation of
complement and coagulation cascades associated with the traumatic wound
are likely contributors to this early phase. The chemotactic activity
of the C5a fragment of complement has long been recognized
(46). Numerous components of the coagulation system have
also been demonstrated to exhibit inflammatory properties
(47). Furthermore, the tissue necrosis certain to be part
of traumatic injury is believed to be a general stimulus of
proinflammatory cytokine expression, although the molecular details of
this effect are largely unknown (48, 49).
The findings presented in this study define, in detail, the patterns of cytokine and chemokine response which occur within the tissue microenvironment following surgical injury. The results illustrate a biphasic response that appears to be composed of distinct cell types and stimuli functioning in a temporally ordered fashion. The candidate cell types and stimuli that are likely participants have been identified both in vitro and in vivo in many previous studies. Furthermore, work with cell culture models has determined many of the molecular pathways that mediate response to external stimuli and translate such signals into altered patterns of gene expression. At present, however, the specific roles for individual cell types, extracellular stimuli, and intracellular regulatory mechanisms in producing the specific patterns of chemokine gene expression and their inflammatory sequellae in vivo remain to be fully appreciated.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Thomas A. Hamilton, Department of Immunology, Lerner Research Institute, Cleveland Clinic Foundation, NB30, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: hamiltt{at}ccff.org
3 Abbreviations used in this paper: MIP, macrophage-inflammatory protein; MCP, monocyte chemoattractant protein; rm, recombinant mouse.
Received for publication August 2, 2001. Accepted for publication January 18, 2002.
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 G. Sosne, P. L. Christopherson, R. P. Barrett, and R. Fridman Thymosin-{beta}4 Modulates Corneal Matrix Metalloproteinase Levels and Polymorphonuclear Cell Infiltration after Alkali Injury Invest. Ophthalmol. Vis. Sci., July 1, 2005; 46(7): 2388 - 2395. [Abstract] [Full Text] [PDF]
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 S. P. Matzer, F. Rodel, R. M. Strieter, M. Rollinghoff, and H. U. Beuscher Constitutive expression of CXCL2/MIP-2 is restricted to a Gr-1high, CD11b+, CD62Lhigh subset of bone marrow derived granulocytes Int. Immunol., November 1, 2004; 16(11): 1675 - 1683. [Abstract] [Full Text] [PDF]
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D. A. Armstrong, J. A. Major, A. Chudyk, and T. A. Hamilton Neutrophil chemoattractant genes KC and MIP-2 are expressed in different cell populations at sites of surgical injury J. Leukoc. Biol., April 1, 2004; 75(4): 641 - 648. [Abstract] [Full Text] [PDF]
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 Y. Dai, S. Datta, M. Novotny, and T. A. Hamilton TGF{beta} inhibits LPS-induced chemokine mRNA stabilization Blood, August 15, 2003; 102(4): 1178 - 1185. [Abstract] [Full Text] [PDF]
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