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Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, macrophage inflammatory
proteins-1 and -2, and KC in specific tissues. Collectively, these
results indicate novel regulatory activities of MDC in innate immunity
during sepsis and suggest that MDC may aid in an adjunct therapy in
sepsis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Macrophage-derived chemokine (MDC),3 a recently identified member of the CC chemokine family, has been shown to be a potent chemoattractant for dendritic cells, NK cells, and the Th2 subset of T cells (8). MDC binds to CCR4, which is expressed on Th2 cells (9, 10). T cells attracted by MDC generated cell lines predominantly producing Th2-type cytokines such as IL-4 and IL-5 (10). Recently, MDC has been shown to be involved in T cell-mediated allergic airway inflammation and atopic dermatitis (11, 12). In addition, it has been reported that MDC is highly expressed by epithelial cells of normal thymus and attracts CD3- and CD4-positive T cells in the thymus (13). Thus, most of the studies to date have focused on the involvement of MDC in the setting of chronic inflammation and dendritic cell and lymphocyte homing. However, MDC is also known to attract monocytes both in vitro and in vivo (11, 14). The fact that the recruitment and subsequent activation of monocytes into infectious foci have a pivotal role to clear bacteria (4, 15) suggests that MDC may play a crucial role in innate immunity during sepsis induced by bacterial infection. Furthermore, mRNA and protein expressions for MDC from cultured monocytes were induced by IL-4 and IL-13 (16). IL-4 and IL-13 are known to exert beneficial effects in experimental models of sepsis via modulating the production of inflammatory cytokine/chemokines (17, 18). Thus, it is interesting to speculate that IL-4- and IL-13-inducible CC-chemokine MDC may play a beneficial role in innate immunity during sepsis.
In the present study, we explored the role of MDC during sepsis induced by a well-established murine septic peritonitis model, cecal ligation and puncture (CLP) (19). The studies have demonstrated for the first time that MDC induces both a respiratory burst in macrophages and the release of lysozomal enzyme, enhances phagocytosis, increases the bactericidal activity of macrophages, and protects mice from the lethality induced by CLP. In addition, MDC treatment ameliorated systemic tissue inflammation and tissue dysfunction induced by CLP via modulation of the levels of inflammatory cytokines and chemokines in specific tissues. These data indicate that MDC exerts novel immune-regulatory activities during the innate immune response associated with acute septic peritonitis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Specific pathogen-free female CD-1 mice (6–8 wk of age) were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed in the animal care facility unit (University Laboratory of Animal Medicine, University of Michigan Medical School). The Animal Use Committee at the University of Michigan approved all studies.
Cecal ligation and puncture
The mice underwent CLP surgery, as described previously (19). In brief, the mice were anesthetized with i.p. injection of ketamine HCl (Vetamine; Mallinckrodt, Mundelein, IL) and then inhaled methoxyflurane (Metafane; Mallinckrodt). Under sterile conditions, the cecum was exposed through a 1- to 2-cm incision of the lower left abdomen, ligated with a 3-0 silk suture below the ileocecal valve, and punctured through and through once with a 21-gauge needle. The cecum was replaced in the peritoneum, and the abdomen was closed with surgical staples. The mice were injected with 1 ml of saline s.c. for fluid resuscitation and placed on a heating pad until they recovered from anesthesia.
Experimental protocol
Immediately after CLP, the mice were treated with either i.p or i.v. injection of recombinant murine MDC (1 µg/mouse; R&D Systems, Minneapolis, MN; endotoxin content < 0.1 ng/µg). Equivalent volume of saline was used as a control. In the first set of experiments, the mice were monitored for 7 days after CLP to determine the survival rate induced by CLP. In the next set of experiments, the CLP mice were anesthetized, bled, and euthanized at appropriate intervals after CLP. The peritoneal cavities were washed with 2 ml of sterile saline, and the lavage fluids were harvested. After taking a 10-µl aliquot of lavage fluids for the assessment of bacteria CFU, the fluids were centrifuged at 6000 x g for 1 min at 4°C. Cell-free peritoneal fluids were stored at -20°C, the cell pellets were resuspended in saline, and the cell numbers were counted in a hemocytometer. Differential cell analyses were performed by Diff-Quik-stained cytospin preparations (Dade, Düdingen, Switzerland). The liver, lung, and kidney were excised, weighed, frozen in liquid nitrogen, and stored at -20°C for subsequent analyses.
Passive neutralization of MDC was conducted in some CLP mice by i.p. injection of 0.5 ml of anti-murine MDC antiserum 2 h before CLP. Anti-MDC antiserum was raised by immunizing New Zealand White rabbits with murine MDC (R&D Systems). The polyclonal Abs were titered by direct ELISA, and the Abs recognized MDC even at a dilution of 1 x 10-6. The Abs completely blocked MDC-induced T cell chemotactic activity in vitro (data not shown). The Abs did not cross-react with a number of other murine cytokines, including CXC and CC chemokines, as seen in the fact that MDC ELISA established with the Abs did not detect any murine cytokines at a concentration as high as 100 ng/ml. As a control, 0.5 ml of pre-immune serum was used. The endotoxin content in both anti-MDC antiserum and control serum was below detection level (<0.05 EU/ml; PYROGENT; BioWhittaker, Walkersville, MD).
In other studies, mice received an i.p. inoculation of live Escherichia coli (3 x 108 CFU) that were previously recovered from the peritoneum of CLP mice. Immediately after the injection, each mouse received an i.p. injection of either MDC (1 µg/mouse) or saline. Twenty-four hours later, the mice were anesthetized, bled, and euthanized, and the peritoneal cavities were washed with 2 ml of sterile saline. Ten-microliter aliquots of lavage fluids were used for the assessment of bacteria CFU in the peritoneum.
Determination of CFU
A total of 10 µl of peritoneal lavage fluids and peripheral blood from each mouse was placed on ice and serially diluted with sterile saline. Ten microliters of each dilution was plated on thymic shared Ag (TSA) blood agar plates (Difco, Detroit, MI) and incubated overnight at 37°C, after which the number of colonies was counted. Data were expressed as CFU/10 µl.
Phagocytic and killing activities of macrophages in vitro
Peritoneal cells (1 x 106 cells) harvested from normal mice were suspended in antibiotic-free RPMI 1640 containing 5% FCS and were incubated at 37°C in two 24-well culture dishes. Two hours later, nonadherent cells were removed, the medium was replaced, and the adherent cells were preincubated with various concentrations of MDC (0, 20, or 100 ng/ml) for 1 h. The cells were then infected with 1 x 106 CFU of E. coli recovered from the peritoneum after CLP. After 1-h incubation, the wells were extensively washed out to remove unphagocytosed bacteria, and the wells in one plate were lysed with sterile 0.5% Triton X-100 for bacterial phagocytosis assay. Wells in the other plate were replaced with prewarmed fresh medium and incubated for an additional 2 h, after which the cells were lysed with 0.5% Triton X-100 for bacterial killing assay. The serially diluted cell lysates were plated on TSA blood agar plates (Difco) and incubated overnight at 37°C, and the numbers of colonies were counted.
Superoxide production
The production of superoxide from cultured macrophages was
measured using the reduction of ferricytochrome c (20, 21). In brief, peritoneal macrophages (1 x
106 cells) harvested from normal mice were
cultured in phenol red-free RPMI 1640 in 24-well culture dishes for
2 h at 37°C. Nonadherent cells were removed and the medium was
replaced with fresh medium containing cytochrome c (1.3
mg/ml; Sigma, St. Louis, MO). The adherent cells were stimulated with
various concentrations of MDC (0, 1, 10, or 100 ng/ml) at 37°C. After
30-min incubation, the plate was shaken gently, and the supernatants
were harvested. Superoxide production in the supernatants was measured
spectrophotometrically at 550 nm as a function of ferricytochrome
c reduction. To exclude the presence of other reductants, 15
µl of superoxide dismutase (SOD; 4.5 mg/ml; Sigma) was added as a
control for each sample. The amount of superoxide was calculated and
expressed as pmol SOD-inhibitable cytochrome c
reduction/min/106 cells according to the
following formula (21): 
A/min x reaction
volume x 106 nmol/mmol x
1000/(Cy/mmol/L) x 1000 ml/L = pmol cytochrome c,
where A/min = change in absorbance per minute and Cy =
extinction coefficient of cytochrome c (21.1 mM/cm).
Measurement of lysozomal enzyme release
The lysozomal enzyme release from macrophages was determined by lysozyme activity in the culture supernatants. Peritoneal macrophages (1 x 106 cells) harvested from normal mice were cultured in phenol red-free RPMI 1640 containing 5% FCS and antibiotics in 24-well culture dishes for 2 h at 37°C. Nonadherent cells were removed, the medium was replaced, and the adherent cells were stimulated with various concentrations of MDC (0, 1, 10, or 100 ng/ml) for 48 h. Lysozyme activity was assayed by a turbidometric method (22). In brief, 50 µl of culture supernatant was added to 200 µl of the assay mixture, which consisted of 30 mg/ml Micrococcus lysodeikticus (Sigma) in 50 mM sodium acetate buffer (pH 6.0) and 0.05% Triton X-100. Chicken egg white lysozyme (Sigma) was used as a standard. The rate of substrate lysis was measured at 540 nm after 30 min. The activity was expressed as micrograms of lysozyme activity/106 cells.
Clinical chemistry
Serum levels of aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine were measured by Clinical Pathology at the University of Michigan Medical School using standardized techniques.
Measurement of cytokines and myeloperoxidase (MPO)
Murine MDC was quantitated using a standard method of sandwich
ELISA. In brief, microtiter plates were coated with 50 µl of
affinity-purified anti-murine MDC IgG (R&D Systems; 1 µg/ml) in
coating buffer (0.6 M NaCl + 0.26 M
H3BO4 + 0.08 M NaOH (pH
9.6)). Detection and processing were made by using biotinylated rabbit
anti-MDC IgG (3.5 µg/ml), streptavidin-peroxidase conjugate
(Bio-Rad, Richmond, CA), and chromogen substrate (Bio-Rad). TNF-,
macrophage inflammatory protein (MIP)-1, MIP-2, KC, and monocyte
chemoattractant protein (MCP)-1 were quantitated by specific sandwich
ELISA, as described elsewhere (23, 24). The ELISAs
employed in this study did not cross-react with other murine cytokines
available and consistently detected murine cytokine concentrations
above 25 pg/ml. MPO in tissue extracts was measured using an ELISA kit
(Calbiochem-Novabiochem, San Diego, CA) according to the
manufacturer’s instruction. The lower detection limit was 1.6
ng/ml.
Preparation of tissue extracts
The excised tissues (0.1 g) were placed in 1 ml of homogenization buffer (500 mM NaCl + 50 mM HEPES (pH 7.4) containing 0.1% Triton X-100, 0.5 µg/ml leupeptin, 1 mM PMSF, and 0.02% NaN3) and were homogenized with a Tissue Tearor (model 985-370; Biospec Products, Racine, WI). The homogenates were then subjected to one freeze/thaw extraction before ELISA or three freeze/thaw extractions before MPO assay. The homogenates were centrifuged at 6000 x g for 20 min at 4°C, and the cell-free supernatants were used for measurement of cytokines or MPO.
Statistics
Statistical significance was evaluated by ANOVA. In case of survival curve and CFU count, the data were analyzed by the log-rank test and Mann-Whitney test, respectively. A p value <0.05 was regarded as statistically significant. All data were expressed as mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To determine whether the CC chemokine MDC is capable of exerting
beneficial effects on the lethality induced by CLP, either MDC or
vehicle was injected i.p. or i.v. immediately after CLP surgery, and
survival rates were monitored. The data in Fig. 1
show the protective effects of MDC on
the survival of mice after CLP. When MDC was administered into the
peritoneum, the protective effects were seen after 48 h post-CLP.
At 48 h after CLP, the mortality rate in control mice was 73%
(11/15 mice), whereas only a 20% mortality rate (3/15 mice) was found
in mice treated with MDC (Fig. 1). On day 7, 13% of control mice (2/15
mice) survived compared with 40% in MDC-treated mice (6/15 mice).
Although the survival rate on day 7 after i.v. injection of MDC was
similar to that after i.p. injection (42%; 8/19 mice), the i.v.
administration did not affect the survival of mice at 48 h after
CLP, causing no statistical significant difference compared with
control (mortality rates in control vs MDC-treated mice at 48 h
after CLP, 58% (11/19 mice) vs 53% (10/19 mice), respectively). The
results clearly indicate a beneficial role of MDC in CLP-induced
lethality and suggest that MDC needs to act locally in the peritoneal
cavity for mouse survival.
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CLP permits the leakage of polymicrobial flora in the peritoneal
cavity, which leads to a local inflammatory response directed at
eliminating bacteria from the peritoneum (19). Experiments
were conducted to assess the local effects of MDC in this model of
sepsis. Intraperitoneal injection of MDC increased the recruitment of
macrophages, but not of neutrophils, in the peritoneum at 24 h
after CLP, compared with control (Fig. 2A). The amount of viable
bacteria recovered from the peritoneum at 24 h after CLP was
markedly decreased in mice treated with MDC compared with control (Fig. 2B). The mean CFU in MDC-treated mice (6.3 x
105/10 µl) was 40-fold lower than that in
control (2.7 x 107/10 µl;
p < 0.01). At the same time point (24 h), bacteremia
was found in 10 of 11 control mice (mean CFU = 3.3 x
104/10 µl blood), whereas 5 of 14 mice treated
with MDC were bacteremic and the mean CFU was 12-fold lower than that
in control (2.7 x 103/10 µl blood;
p < 0.05; Fig. 2C). MDC treatment resulted
in significant decreases in the peritoneal levels of TNF-, MIP-2,
KC, MIP-1, and MCP-1 at 24 h after CLP (Table I). Thus, MDC appears to elicit the
recruitment of macrophages into the peritoneum independently of other
cytokines/chemokines that may attract the cells and also seems to
increase bacterial clearance.
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To confirm the in vivo effects of MDC in bacterial clearance,
either MDC (1 µg) or vehicle was given i.p. immediately after i.p.
inoculation of E. coli (3 x 108
CFU/mouse). Twenty-four hours later, the bacterial load in the mice was
assessed. Fig. 3 shows that MDC treatment
lessened the viable bacteria recovered from the peritoneum and blood
compared with that from control. The mean CFU in the peritoneum in
MDC-treated mice (1.6 x 104/10 µl) was
16-fold lower than that in control (2.6 x
105/10 µl; p < 0.01; Fig. 3A). Bacteremia was found in 70% of control mice (7/10
mice), whereas 20% of mice treated with MDC were bacteremic (2/10
mice) (Fig. 3B).
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, MDC dose dependently increased the
CFU counts inside the cells after 1-h incubation. When the infected
cells were cultured for an additional 2 h, the killing rate of
bacteria was markedly increased by the treatment with MDC in a
dose-dependent fashion. Bacterial killing rate by macrophages treated
with 100 ng/ml MDC was five times higher than that with control (Table II). It appeared that MDC cleared bacteria through enhancing the
phagocytic and bactericidal activities of macrophages.
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Above-described findings clearly show that MDC treatment was
effective in bacterial clearance. We next examined whether
endo- genous MDC was capable of exerting the bactericidal activity
in this model of sepsis. As shown in Fig. 4A, the level of MDC in the
peritoneal fluids significantly increased with time and remained
elevated at 48 h after CLP. MDC blockade with anti-MDC Abs
decreased the number of infiltrating macrophages, but not neutrophils,
in the peritoneum at 8 h after CLP, compared with the number in
control (Fig. 4B). At this time point, no differences were
found in the peritoneal levels of TNF-, MIP-2, KC, MIP-1, and
MCP-1 (data not shown). Although the numbers of leukocyte populations
at 24 h were similar between the groups, the recovery of viable
bacteria from the peritoneum at this time point markedly increased in
mice treated with anti-MDC Abs, compared with that of control (Fig. 4C). The peritoneal levels of TNF-, MIP-2, KC, MIP-1,
and MCP-1 at 24 h after CLP were significantly increased by 2- to
3-fold by anti-MDC treatment (data not shown). At the same time
point (24 h), bacteremia was found in four of seven mice treated with
control Abs, whereas all mice that received anti-MDC Abs were
bacteremic (nine of nine mice). The mean CFU in anti-MDC-treated
mice was 103-fold higher than that in the control
(Fig. 4D). Furthermore, neutralization of endogenous MDC
significantly deteriorated the survival of mice after CLP compared with
that of the control (Fig. 5). The data
support a host defense role of MDC during experimental sepsis because
neutralization of MDC resulted in a reduced ability to clear bacteria
from the host.
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The ability of MDC to activate macrophages was evaluated next.
During activation of macrophages, reactive oxygen products are
generated and lysozomal enzymes are released from the granules
(25, 26). In the present study, we measured superoxide
anion (O2-) generation (a
NADPH-dependent oxidase enzyme) and lysozyme release (a lysozomal
enzyme) from macrophages after the stimulation with MDC. The data in
Fig. 6 show that MDC elicited the
generation of O2- and lysozyme
release from macrophages in a dose-dependent manner. Heat-inactivated
MDC (100°C for 3 min) did not induce these observations (data not
shown). The data clearly indicate that MDC can activate macrophages and
likely can enhance intracellular bacterial killing.
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Sepsis frequently causes severe systemic inflammation
(27). Next, experiments were conducted to assess the
systemic effects of MDC in CLP mice by measuring the tissue level of
MPO, an indirect marker of the number of neutrophils. Intraperitoneal
injection of MDC significantly decreased CLP-induced MPO level in
liver, lung, and kidney at 24 h post-CLP compared with that of the
control (Fig. 7A). The
treatment dramatically decreased serum levels of AST, BUN, and
creatinine, compared with those in the control (Fig. 7B). To
identify the mechanism whereby MDC treatment ameliorated the tissue
inflammation induced by CLP, the levels of cytokine/chemokines known to
attract neutrophils were examined. Administration of MDC resulted in
significant decreases in the levels of TNF-, MIP-2, KC, and MIP-1
in the liver and lung compared with those in the control (Fig. 8). KC level in kidney was also decreased
by MDC treatment (Fig. 8). Conversely, neutralization of MDC
dramatically increased CLP-induced MPO level in tissues and serum
levels of AST, BUN, and creatinine compared with those in the control,
which were accompanied by increased levels of TNF-, MIP-2, KC, and
MIP-1 in specific tissues (data not shown). Thus, it appears that
MDC ameliorated systemic tissue inflammation as well as tissue injury
in part via modulation of the production of inflammatory
cytokines/chemokines.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The mechanism whereby MDC exerted a protective role in CLP mice appears
to be due to its novel activities on phagocytic macrophages.
Administration of MDC not only induced the recruitment of macrophages
but also served to clear bacteria from CLP mice. Conversely,
neutralization of endogenous MDC decreased the recruitment of
macrophages and increased bacterial load in CLP mice.
Cytokines/chemokines known to attract monocytes directly or indirectly,
which include TNF-, MIP-2, KC, MIP-1, and MCP-1, were inhibited
by MDC treatment. Furthermore, the levels of these mediators were
unchanged after anti-MDC treatment by the time when the recruitment
of macrophages was inhibited. Thus, MDC appears to elicit macrophages
directly in this model of septic peritonitis. The changes of the
peritoneal levels of cytokine/chemokines by MDC or anti-MDC appear
to be due to the change in the pathogen load that alters cytokine
production. In addition to its bactericidal activity in CLP mice,
bacterial load in the peritoneum and peripheral blood after inoculation
of live E. coli was decreased by MDC treatment. In vitro,
MDC enhanced bacterial phagocytic and killing activities of macrophages
in a dose-dependent fashion. Like MCP-1 (31, 32), MDC was
found to induce a respiratory burst in macrophages
(O2- generation) and to induce
lysozomal enzyme release from macrophages (lysozyme enzyme release),
both of which are crucial molecules for bacterial clearance (3, 4). Altogether, the data suggest that the accumulation and
activation of macrophages by MDC are the main mechanisms underlying the
beneficial effects of MDC in our model of sepsis.
Sepsis frequently causes severe systemic inflammation, which leads to
multiple organ failure, a condition that is often fatal to the host
(27, 33). Interestingly, MDC treatment decreased the
intensity of CLP-induced tissue inflammation and also improved the
physiologic function of specific tissues. Neutralization of endogenous
MDC increased the severity of the tissue inflammation and worsened the
dysfunction. The beneficial effects of MDC appear to be dependent on
the reduced tissue production of TNF-, MIP-2, KC, and MIP-1
in that MDC treatment decreased and, in contrast, anti-MDC
treatment increased the levels of these cytokine/chemokines in specific
tissues. The above inflammatory cytokines/chemokines are regarded as
powerful mediators of inflammation and tissue damage (5, 34). Unlike IL-4, IL-10, and IL-13, all of which reduced
LPS-induced production of inflammatory cytokines by monocytes
(35, 36, 37), MDC failed to inhibit the production of TNF-,
MIP-2, KC, and MIP-1 from LPS-stimulated peritoneal macrophages in
vitro (data not shown). As discussed above, MDC played a pivotal role
to clear bacteria from the circulation. Thus, it is likely that the
modulation of systemic inflammation by MDC treatment is due to a
decrease in the pathogen load that alters tissue/organ damage.
Interestingly, the novel activities of MDC on macrophages are similar to those of MCP-1, which have both been shown to be associated with chronic inflammation. As shown in this study and elsewhere (31, 32), both of these CC chemokines are capable of activating macrophages. Administration of MCP-1 6 h before bacteria challenge protected mice in lethal Pseudomonas aeruginosa infection model by increasing the killing and clearance of bacteria (30); however, such beneficial effects were not evident when MCP-1 was given at the time of bacteria inoculation. Administration of MCP-1 (1 µg/mouse) failed to protect mice from CLP-induced lethality when treatment was initiated immediately after CLP (A. Matsukawa, unpublished data), although immune-neutralizing studies have demonstrated that MCP-1 played a protective role in the same model of sepsis (29). In this study, we have demonstrated that MDC can enhance bacterial clearance in two types of bacterial peritonitis models and that this chemokine can protect mice from CLP-induced lethality when administered after CLP surgery.
Investigations such as these are important. Sepsis remains a serious disorder, and the mortality due to sepsis and sepsis-mediated multiple organ failure has not significantly improved over the past three decades (38, 39), despite the development of powerful antibiotics and significant advances in the management of intensive care patients and intensive care unit technology. Experimental sepsis employed in the present study possesses a number of the hallmarks of clinical sepsis with peritonitis associated with postsurgical or accidental trauma (19). The novel immune-regulatory activities of MDC that enhance bacterial phagocytic and killing activity of macrophages, leading to the modulation of systemic inflammatory responses in this model, may pave the way for the development of novel therapeutic interventions in sepsis.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Steven L. Kunkel, Department of Pathology, University of Michigan Medical School, 1301 Catherine Road, Ann Arbor, MI 48109-0602.
3 Abbreviations used in this paper: MDC, macrophage-derived chemokine; CLP, cecal ligation and puncture; TSA, thymic shared Ag; AST, aspartate transaminase; BUN, blood urea nitrogen; MPO, myeloperoxidase; MIP, macrophage inflammatory protein; MCP, monocyte chemoattractant protein.
Received for publication December 21, 1999. Accepted for publication March 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T. L. Ness, C. M. Hogaboam, R. M. Strieter, and S. L. Kunkel Immunomodulatory Role of CXCR2 During Experimental Septic Peritonitis J. Immunol., October 1, 2003; 171(7): 3775 - 3784. [Abstract] [Full Text] [PDF]
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; 15 mice) immediately after CLP. The survival rates
were monitored for 7 days after CLP. Two different experiments were
conducted, and the data were pooled. The mortality rates were very
similar in individual experiments.
, Control
mice (11 mice). 
, MDC-treated mice (14 mice). B and
C, Ten microliters of peritoneal fluids
(B) and sera (C) were serially diluted
and plated on TSA blood agar plates, and the CFU was determined. Line
represents the median CFU count. 
,
p < 0.01; when compared with control.
