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1
*
Department of Trauma Surgery, University of Freiburg Medical School, Freiburg/Breisgau, Germany; and
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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, IL-1), complement components, and
chemotactic cytokines (chemokines). In the current studies, we
determined the requirements for TNF- and the complement activation
product C5a in chemokine production in vitro and in vivo. Two rat CXC
chemokines (macrophage inflammatory protein (MIP)-2 and
cytokine-induced neutrophil chemoattractant (CINC)) as well as three
rat CC chemokines (MIP-1, MIP-1ß, and monocyte chemoattractant
protein (MCP)-1) were investigated. Chemokine generation in vitro was
studied in rat alveolar macrophages stimulated with IgG immune
complexes in the absence or presence of Abs to TNF- or C5a. The rat
lung injury model induced by IgG immune complex deposition was employed
for in vivo studies. Abs to TNF- or C5a were administered
intratracheally or i.v., and effects on chemokine levels in
bronchoalveolar lavage fluids were quantitated by ELISA. Both in vitro
and in vivo studies demonstrated the requirements for TNF- and C5a
for full generation of CXC and CC chemokines. In vitro and in vivo
blockade of TNF- or C5a resulted in significantly reduced production
of chemokines. Supernatant fluids from in vitro-stimulated macrophages
revealed by Western blot analysis the presence of
C5a/C5adesArg, indicating intrinsic generation of
C5a/C5adesArg by alveolar macrophages and explaining the
higher efficiency of intratracheal vs i.v. blockade of C5a in reducing
chemokine production. These results underscore the central role of both
TNF- and C5a, which appear to function as autocrine activators to
promote CXC and CC chemokine generation by alveolar
macrophages. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and IL-1ß) and chemokines by activated
macrophages 3, 4 . Subsequently, due to vascular adhesion molecules
and the presence of chemotactic activities, leukocytes are recruited to
the inflammatory site 5, 6, 7 . The ensuing release of cytokines,
enzymes, and oxygen- and nitrogen-derived radicals 8, 9, 10, 11, 12 ultimately
causes tissue damage, increased vascular permeability, and hemorrhage.
The main source in lung for proinflammatory cytokines and chemokines
appears to be lung macrophages 3, 4, 13 . It is known that macrophages
are capable of producing and secreting a variety of different
complement components 14, 15 . Furthermore, cleavage of the complement
component C5 by phagocytic cell-derived proteases can cause generation
of C5a 16 , indicating that this powerful chemoattractant can be
generated both from complement convertases as well as from cell- and
tissue-derived enzymes. As demonstrated recently, C5a and TNF- are
crucial requirements for the full development of inflammatory injury in
the IgG immune complex-induced alveolitis model 3, 17, 18 . Blockade
of either C5a or TNF- in rats results in a substantial reduction in
lung injury 6, 19 . Mice genetically deficient in C5aR demonstrate
nearly complete protection from the response to an intrapulmonary IgG
immune complex formation 20 . In the immune complex-induced
inflammatory models of the reverse passive Arthus reaction in skin or
the peritoneal cavity, C5aR-deficient mice were substantially protected
against injury, exhibiting lower TNF- and IL-6 levels and local
neutrophil accumulation 21 . The requirements for C5a and early
response cytokines (TNF- and IL-1) for expression of vascular
adhesion molecules such as ICAM-1 and E-selectin have been demonstrated
5, 6, 17 . It has also been shown that C5a can directly stimulate
endothelial cells, resulting in expression of P-selectin 22 .
Requirements for CXC chemokines (macrophage inflammatory protein
(MIP)3-2 and cytokine-induced
neutrophil chemoattractant (CINC)) and the CC chemokine MIP-1 for
full development of lung injury following IgG immune complex deposition
have been recently assessed 13, 23 . MIP-2 and CINC are involved in
neutrophil chemotaxis and activation and are required for recruitment
of neutrophils into lung and the subsequent increase in vascular
permeability. Although chemotactic activity for rat macrophages and
neutrophils has been demonstrated for MIP-1, a main in vivo function
of this CC chemokine may be autocrine stimulation of macrophages
resulting in enhanced production of TNF- 23 . In vivo blockade of
MIP-1 results in clearly attenuated lung injury observed as reduced
vascular permeability and reduced neutrophil recruitment, presumably
related to the decrease of TNF- levels in BAL fluids.
In the current report, we demonstrate the requirements for C5a and
TNF- in chemokine (MIP-2, CINC, MIP-1, MIP-1ß, and MCP-1)
production in vitro and in vivo. The data obtained suggest that,
besides the ability of alveolar macrophages to release TNF-, there
is generation of C5a by an unknown C5-cleaving enzyme, these mediators
presumably functioning as autocrine stimulators. These studies suggest
that both C5a and TNF- are involved directly or indirectly in
neutrophil recruitment, not only through up-regulation of vascular
adhesion molecules but also through generation of chemokines.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages were isolated by repeatedly lavaging lungs
of anesthetized Long-Evans rats (Charles River Breeding Laboratories,
Portage, MI). After centrifugation of lavage fluids, cells were
resuspended in medium (DMEM; Whittaker Bioproducts, Wakersville, MA)
supplemented with 10% heat-inactivated (56°C for 30 min) FBS
(HyClone Laboratories, Logan, UT), plated in 48 microtiter well plates
(Corning, New York, NY) at a concentration of 1 x 106
cells/well and allowed to settle for at least 1 h. Wells were then
washed with medium to remove nonadherent cells. Immune complexes were
formed by incubation of BSA with goat anti-BSA (in molar ratio of
1:4). The centrifuged precipitate containing IgG immune complexes was
resuspended in DMEM to a final concentration of 100 µg/ml. This
concentration was established based on dose responses of alveolar
macrophages using increasing amounts of immune complexes and resulted
in maximal stimulation of cell cultures. All studies involved at least
quadruplicate replicates. Nonspecific goat IgG, goat anti-rat C5a,
or goat anti-rat TNF- was added to wells as indicated at a
concentration of 100 µg/ml. After an incubation period of 4 h,
supernatant fluids were collected and used for chemokine detection.
Animal model of lung injury
Male Long-Evans specific pathogen-free rats (275–300 mg) were
used for all studies. Anesthesia was induced by i.p. ketamine (15
mg/100 mg body weight). Lung injury was produced by intrapulmonary
deposition of IgG immune complexes as described elsewhere 24, 6 .
Briefly, 10 mg BSA was given i.v. following intratracheal
administration of 2.5 mg polyclonal goat anti-BSA in a total volume
of 300 µl. Negative control animals received anti-BSA
intratracheally with omission of the BSA injection. Animals were
sacrificed after 4 h, and BAL fluids were collected by
instillation of 5 ml PBS, flushing and withdrawing three times via an
intratracheal cannula. After adding a protease inhibitor mixture
(leupeptin 1 µg/ml, aprotinin 1 µg/ml, trypsin inhibitor 10
µg/ml, pepstatin 1 µg/ml), samples were centrifuged at 3,000
x g for 10 min, and supernatant fluids were used for
chemokine quantitation (for all studies, n 
5 per
group).
Reagents
Unless otherwise specified, all reagents were purchased from
Sigma (St. Louis, MO). Affinity-purified anti-BSA Ab was obtained
by column purification (AminoLink Immobilization Kit; Pierce, Rockford,
IL) of serum from an immunized goat. Goat anti-BSA, rather than
rabbit anti-BSA, was used to facilitate ELISA technology in which
rabbit Abs to chemokines were used. Purification of Abs (and their
blocking activities) to rat MIP-2 and CINC as well as to TNF- and
C5a has been described elsewhere 13, 17, 18, 25 . IgG Ab to rat MIP-2
was obtained from rabbit serum. Rabbit serum rich in anti-CINC was
kindly provided by Dr. Arthur Whittwer (Monsanto, St. Louis, MO). Abs
to rat CINC and MIP-2 were subjected to double affinity methods to
resume any cross-reactivity 13 . The Ab and reference protein used for
MIP-1 ELISA quantitation were purchased from Peprotech (Rocky Hill,
NJ). Components for the MIP-1ß ELISA were obtained from R&D Systems
(Minneapolis, MN). Anti-MCP-1 Ab was acquired from PharMingen (San
Diego, CA). Recombinant rat MCP-1 protein was purchased from BioSource
(Camarillo, CA). Biotinylation steps were performed with EZ-Link
NHS-LC-Biotinylation Kit (Pierce) according to the manufacturer’s
instructions. The doses and administration techniques of Abs to C5a or
TNF- were determined on the basis of earlier studies 3, 17 . For in
vivo blocking experiments, 400 µg goat anti-rat TNF- or
goat anti-rat C5a was administered i.v. or intratracheally, as
indicated. Positive reference control animals received 400 µg
nonspecific goat IgG instead. All Ab preparations were found to contain
less than 10 ng endotoxin/ml, using the Limulus Amebocyte
Test Kit QCL-1000 (Whittaker).
Quantitation of chemokines
Chemokine detection was performed using Ab-sandwich ELISA
as described recently for MIP-2 and CINC 13 . The same ELISA technique
and reagents were used for developing immunoassays to measure levels of
MIP-1, MIP-1ß, and MCP-1. For MIP-1, 60 µl/well of 5 µg/ml
Ab were used for coating, and samples were diluted 1:2. The
biotinylated secondary Ab was used at a concentration of 25 µg/ml.
Anti-MIP-1ß was coated at 10 µg/ml with 75 µl/well. Biotinylated
anti-MIP-1ß was used at 2.5 µg/ml. For MCP-1 detection, the
concentrations used were 10 µg/ml for coating Ab (100 µl/well) and
0.5 µg/ml for secondary Ab. For this ELISA, all incubations were
conducted at room temperature. Incubation times were 2–4 h. All other
procedures were performed as described earlier.
Western blot analysis of macrophage-derived C5a
Ab to rat C5a was immobilized on the surface of beads (AminoLink; Pierce) according to the manufacturer’s instructions. Supernatant fluids of untreated and stimulated macrophages (1 x 107) were incubated at 4°C overnight with 50 µl bead slurry (50%) per condition. Beads were then centrifuged at 1300 rpm for 5 min at 4°C, washed once with PBS, and centrifuged again. Proteins were dissociated from beads with 100 µl Laemmli’s sample buffer and boiled for 10 min. Fifteen percent SDS PAGE was performed, and proteins were transferred to a polyvinylidene difluoride membrane (Schleicher & Schuell, Keene, NH) using a semidry electrophoresis apparatus (LKB Multiphor II; Pharmacia Biotech, Uppsala, Sweden). The blot was incubated with 5% milk for 1 h and then probed with goat anti-C5a as primary Ab overnight, followed by incubation with an alkaline phospatase-conjugated donkey anti-goat Ab (Cappel-Organon Teknika, Durham, NC) (both at a 1:1000 dilution) and alkaline phosphatase substrate color development (Bio-Rad Laboratories, Hercules, CA).
Statistical analysis
In groups with equal variances, data sets were analyzed using one-way ANOVA, and individual group means were then compared with the Student-Newman-Keuls multiple comparison test. In groups containing unequal variances, Kruskal-Wallis ANOVA was performed followed by Dunnett’s method for multiple comparison. All values were expressed as mean ± SEM. Significance was assigned where p < 0.05. For percentage change between groups, values obtained from negative controls were subtracted from each data point.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on in vitro production of
chemokines
In experiments involving in vitro stimulation with IgG immune
complexes, a concentration of 100 µg immune complexes per milliliter
was used. For each condition, negative controls consisted of no
treatment or addition of 20 µg BSA/ml, 100 µg anti-C5a or
anti-TNF-/ml, or 100 µg anti-BSA/ml (in the absence of
BSA). The results are shown in Fig. 1
.
The presence of BSA, anti C5a, anti-TNF-, and anti-BSA (IgG)
alone resulted in no increase in basal level of MIP-2 (34.2 ±
1.94 ng/ml). As expected, in vitro stimulation of alveolar macrophage
monolayers with IgG immune complexes in presence of nonspecific IgG led
to increased MIP-2 production, with levels rising to 90.8 ± 5.33
ng/ml (p < 0.05). The addition of anti-C5a
reduced IgG immune complex-induced expression of MIP-2 by 60.9%
(p < 0.05), falling to 54.1 ± 0.83
ng/ml. Anti-TNF- had a similar effect, reducing MIP-2 levels by
38.5% (p < 0.05), to 69.0 ± 2.56 ng/ml
(Fig. 1A). In supernatant fluids from alveolar macrophages,
CINC levels rose from 76.6 ± 7.2 ng/ml in negative controls
(untreated) to 703 ± 51.8 ng/ml after addition of IgG immune
complexes and nonspecific IgG (Fig. 1B). The presence of
anti-C5a led to a 32.8% (p < 0.05)
decrease, to 497 ± 27.1 ng CINC/ml, while the blockade of TNF-
was similarly effective, causing a 30.6% reduction
(p < 0.05), to 511 ± 26.1 ng/ml).
Nonstimulated macrophage monolayers generated MCP-1 levels of 9.65
± 0.71 ng/ml (Fig. 1C). Similar low levels of MCP-1 were
found when BSA, anti-C5a, anti-TNF-, or anti-BSA (IgG)
was added to macrophages. After 4 h of stimulation with IgG immune
complexes together with nonspecific IgG, MCP-1 levels rose to 19.4
± 0.32 ng/ml (p < 0.05). These levels dropped
by 42.0% (p < 0.05) in the presence of
anti-C5a (to 15.3 ± 0.77 ng/ml) and by 43.6%
(p < 0.05) in the presence of Ab to TNF-
(to 15.17 ± 0.41 ng/ml). Supernatant fluids of untreated
macrophages contained no measurable MIP-1 (<1 ng/ml), but MIP-1
levels rose to 392 ± 38.5 ng/ml in the presence of IgG immune
complexes and nonspecific IgG (Fig. 1D). Again, the presence
of anti-C5a reduced production of MIP-1 by 40.2%
(p < 0.05), to 234 ± 19.0 ng/ml, while
the presence of anti-TNF- resulted in a 39.7%
(p < 0.05) drop, to 236 ± 16.9 ng/ml.
Finally, for MIP-1ß, the baseline level of 15.7 ± 3.19 ng/ml in
negative control culture fluids rose to 280 ± 8.22 ng/ml in the
presence of IgG immune complexes in the copresence of nonspecific IgG
(Fig. 1E). Levels of MIP-1ß were attenuated by 46.3%
(p < 0.05) in the presence of anti-C5a
(157 ± 9.58 ng/ml). Anti-TNF- had a similar protective effect,
causing a 41.3% reduction (p < 0.05) of
MIP-1ß generation (to 171 ± 9.06 ng/ml).
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To account for the inhibitory effects of anti-C5a Ab in vitro,
we hypothesized that alveolar macrophages not only produce the
complement component C5 but also contain an enzyme capable of cleaving
C5 into its active C5a fragment. Western blot analysis of supernatant
fluids of stimulated macrophages was performed using extraction of C5a
by immunoprecipitation with anti-C5a immobilized on beads,
revealing a band at the 14-kDa position, which aligned with a product
obtained from complement-activated rat serum. Since serum
carboxypeptidase was not blocked in the activated serum, the more
intense band likely represents C5adesArg. The faint, more
slowly migrating band likely represents C5a consistent with the
electrophoretic mobilities of rat C5a and C5adesArg 26 .
Immunoprecipitation using supernatant fluids from nonstimulated cells
failed to produce a similar band. Furthermore, this Ab did not react
with fresh rat serum that had not been complement activated (Fig. 2). Thus, stimulated alveolar macrophages
release a product that is defined as C5a/C5adesArg by
immunochemical analysis.
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on in vivo lung
expression of chemokines
The finding that in vitro production of chemokines by IgG immune
complex-stimulated alveolar macrophages was suppressible by
anti-C5a and anti-TNF- led to companion in vivo experiments.
For these experiments, animals were treated intratracheally or i.v.
with anti-C5a or anti-TNF-. The amount of blocking Ab
(present as IgG) employed (400 µg) was based on earlier reports 2, 11 and preliminary studies (data not shown). The results are shown in
Fig. 3. Negative controls (untreated)
consisted of animals receiving anti-BSA intratracheally in the
absence of i.v. infused BSA. In BAL fluids from negative control
animals only traces of MIP-2 were detectable (0.55 ± 0.63 ng/ml)
(Fig. 3A). After intrapulmonary IgG immune complex
deposition in the presence of intratracheally instilled nonspecific
goat IgG, MIP-2 levels rose 80-fold, to 42.4 ± 2.19 ng/ml. In
animals that had been treated intratracheally with anti-C5a, MIP-2
levels in BAL fluids decreased by 43.6% (p <
0.05), to 24.1 ± 2.48 ng/ml. Intratracheal instillation of
anti-TNF- had a similar effect, with a 31.1%
(p < 0.05) reduction in levels of MIP-2, to
29.41 ± 3.71 ng/ml. i.v. blockade of C5a or TNF- proved to be
less effective, but nevertheless statistically significant, with
reductions of 28.8% (p < 0.05) in BAL MIP-2
for anti-C5a, and 25.9% (p < 0.05) for
anti-TNF-, respectively. CINC was undetectable (<1 ng/ml) in
BAL fluids from negative untreated control animals (Fig. 3B). Four hours after injury, BAL levels of CINC rose to
1291 ± 44.9 ng/ml in positive control animals receiving 400 µg
nonspecific goat IgG intratracheally. Intratracheal administration of
anti-C5a caused a reduction of 48.6% (p <
0.05), to 663 ± 50.6 ng/ml, whereas the treatment with
anti-TNF- led to a 27.8% (p < 0.05)
decrease in BAL levels of CINC (to 931 ± 118 ng/ml). The impact
of i.v. treatment with anti-C5a or anti-TNF- was less
effective. The i.v. blockade of C5a caused a 28% drop
(p < 0.05) of CINC levels in BAL fluids, while
anti-TNF- administered i.v. led to a 24.8% decline
(p < 0.05). No measurable MCP-1 was found in
BAL fluids of untreated negative control animals (<1 ng/ml) (Fig. 3C). After lung injury, MCP-1 levels rose to 14.4 ±
0.46 ng/ml. In the presence of anti-C5a administered
intratracheally, MCP-1 levels in BAL fluids dropped by 17.4%
(p < 0.05), to 11.9 ± 0.41 ng/ml. While
the intratracheal blockade of TNF- led to a 26.3% decrease
(p < 0.05) in MCP-1 to 10.6 ± 0.21
ng/ml, there was no significant effect when anti-TNF- was
administered i.v. For reasons that are not understood, the blocking Ab
to C5a proved to be more effective when given i.v. with a 39.7% drop
(p < 0.05) of MCP-1 levels in BAL fluids. No
constitutive expression of lung MIP-1 was detected in untreated
negative control animals (<1 ng/ml) (Fig. 3D). In BAL
fluids of positive controls MIP-1 receiving nonspecific goat IgG
intratracheally, levels rose to 1764 ± 111 ng/ml. The
intratracheal treatment with anti-C5a resulted in a 56.2%
attenuation, p < 0.05, to 771 ± 31.3 ng/ml,
compared with a 43% (p < 0.05) decline when
administered i.v. The intratracheal administration of anti-TNF-
led to a 37.3% (p < 0.05) decrease of
MIP-1 (to 1104 ± 33.2 ng/ml), while i.v. blockade of TNF-
reduced MIP-1 levels by 33.8%, p < 0.05. The
regulation of MIP-1 and MIP-1ß appeared to be similar for in vitro
(Fig. 1
) and in vivo (Fig. 3) conditions. In BAL fluids of untreated
negative control animals, a baseline expression for MIP-1ß of
8.91 ± 3.2 ng/ml was found (Fig. 3E). Lung injury
caused by IgG immune complex deposition in the presence of nonspecific
IgG instilled intratracheally led to strong up-regulation of MIP-1ß,
rising to 448 ± 13.8 ng/ml, p < 0.05. When
treated with anti-C5a intratracheally, the MIP-1ß levels in BAL
fluids dropped by 50.7% (p < 0.05), to
225 ± 46 ng/ml. The intratracheal presence of anti-TNF- at
onset of injury decreased MIP-1ß by 36.5% (p
< 0.05), to 287 ± 9.18 ng/ml. The i.v. blockade of C5a was less
effective when compared with the intratracheal intervention,
resulting in a drop of MIP-1ß levels by 37.1%, p <
0.05. Anti-TNF- given i.v. had no significant effect on BAL content
of MIP-1ß.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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and a profound
reduction in the up-regulation of lung vascular ICAM-1 17 . Using
genetically altered mice with an induced deficiency of C5aR or the
receptor for substance P (NK-1R), complete protection against lung
injury in the model of IgG immune complex-induced alveolitis was
demonstrated 20 . In mice with C5aR deficiency, intrapulmonary
challenge with immune complexes resulted in lower, but statistically
not significant, concentrations of BAL TNF- with wide variances. In
contrast, a substantial decrease of levels of TNF- in peritoneal
fluids was found in mice deficient in C5aR when the inflammatory
reaction was induced by i.p. challenge with immune complexes 21 . It
is unclear whether the discrepancies in intrapulmonary TNF- levels
in rats and C5aR-deficient mice are due to differences of species, or
if, with smaller error bars, the decrease in TNF- levels might be
statistically significant in C5aR-deficient mice. The finding that anti-C5a inhibits rat alveolar macrophage production of chemokines after stimulation suggests that these cells are generating C5a, which binds with membrane C5aR, and that this process enhances generation of chemokines. In data described above, we have shown that supernatant fluids of alveolar macrophages stimulated in vitro contain immunoreactive C5a, which was not detected in supernatant fluids of nonstimulated macrophages. Synthesis of C5, among other complement components, by monocytes and macrophages has been described 14, 15 , and we speculate that, under conditions of cell activation, locally produced C5 is cleaved to generate C5a, rendering alveolar macrophages autonomous from plasma-derived complement. As noted above, serine proteases from granulocytes have the ability to cleave C5 at neutral pH, generating C5a. Since macrophages are known to produce much more matrix metalloproteases than serine proteases, these matrix metalloproteases seem more likely to be readily available for the cleavage of locally generated C5. Alternatively, C5 cleavage could occur by complement-specific convertases, the components for which are also produced by macrophages 29, 30 . Against the hypothesis of locally assembled complement-specific convertases are reports of a normal inflammatory reaction of mice deficient in C3 or C4 in a skin model of IgG immune complex-mediated injury 31 . Assuming local generation and cleavage of C5, it seems unlikely in these deficient mice that complement convertases could cause C5 cleavage, implying the involvement of some other enzyme. Thus, it is possible in C3- or C4-deficient mice that the inflammatory response is C5 dependent, with a noncomplement-derived C5 convertase coming into play.
Intrapulmonary blockade of TNF- by Ab causes a substantial decrease
in lung injury, with a 60% reduction of vascular permeability and 67%
drop of neutrophil influx as assessed by myeloperoxidase analysis of
lung tissue 3, 18 . Lung injury in this model requires intrapulmonary
recruitment of neutrophils. The reduced tissue accumulation of
neutrophils in the presence of blocking Ab to TNF- has been shown to
be at least partly due to significantly decreased intrapulmonary
vascular ICAM-1 6 .
In the current study, using IgG immune complex stimulation in vitro and
in vivo, it seems clear that endogenously generated TNF- and C5a
each promote chemokine production, which appears in vivo to be
essential for full recruitment of neutrophils. When CINC, MIP-2, or
MIP-1 were blocked after IgG immune complex deposition in vivo,
reductions of injury in terms of permeability (approximately 40%) and
neutrophil accumulation (approximately 70%) were noted 13, 23 . It is
clear that intratracheal blockade of C5a or TNF- is, in general,
more effective than i.v. intervention in reducing generation of
chemokines. This supports the concept of locally produced complement
activation products and cytokines by lung macrophages. It remains to be
determined whether C5a and TNF- exert their regulatory function on
chemokine generation by similar or different mechanisms. Experiments
combining the blockade of C5a and TNF- did not result in a further
decrease in protection or in chemokine levels compared with either
intervention alone (data not shown). Since the Ab preparations used
were whole IgG fractions, the combination of anti-C5a and
anti-TNF- resulted in administration of a large amount of IgG,
representing a proinflammatory stimulus (data not shown). To overcome
this technical problem, affinity-purified Ab preparations will be used
to reduce the total mass of IgG instilled into the airways. Because
MIP-1 appears to act as an autocrine stimulator for TNF-
production 23 , it is obvious that reducing expression of this
chemokine attenuates the positive feedback mechanism for TNF-,
thereby enhancing the ability of anti-C5a to suppress generation of
this and other chemokines. It can now be speculated that, like
MIP-1, C5a and TNF-, both of which appear to be produced by
activated macrophages, function as autocrine activators resulting in
enhanced chemokine generation. It is to be noted that blockade of C5a
or TNF- does not completely inhibit chemokine production in vitro
and in vivo. This might indicate other involved pathways or mediators
upstream of complement and early response cytokines, for example direct
activation of macrophages by Fc receptor engagement, as has been
demonstrated for IL-8 production in human monocytes 32 .
In conclusion, our observations indicate that blocking TNF- or C5a
not only causes reduced expression of vascular adhesion molecules in
vivo but also decreased chemokine expression. The knowledge of
stimulatory patterns for chemokine expression not only provides a basis
for a better understanding of the early onset of the acute inflammatory
response but also suggests new therapeutic approaches for suppressing
chemokines that are thought to play important roles in a variety of
inflammatory responses, such as in shock/sepsis, adult respiratory
distress syndrome, physical trauma, and burn trauma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Boris J. Czermak, Department of Pathology, University of Michigan Medical School, Room 7520 MSRB I, 1301 Catherine Road, Ann Arbor, MI 48109-0602. E-mail address:
3 Abbreviations used in this paper: MIP, macrophage inflammatory protein; CINC, cytokine-induced neutrophil chemoattractant; MCP, monocyte chemoattractant protein; IC, immune complexes; BAL, bronchoalveolar lavage.
Received for publication May 14, 1998. Accepted for publication November 2, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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regulates in vivo intrapulmonary expression of ICAM-1. Am. J. Pathol. 142:1739.[Abstract]
and IL-1. J. Immunol. 149:331.[Abstract]
in acute lung injury in rats. J. Immunol. 154:4793.[Abstract]
receptor cross-linking induces IL-8 production. J. Immunol. 155:3161.[Abstract]
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A. Mullick, Z. Leon, G. Min-Oo, J. Berghout, R. Lo, E. Daniels, and P. Gros Cardiac Failure in C5-Deficient A/J Mice after Candida albicans Infection. Infect. Immun., August 1, 2006; 74(8): 4439 - 4451. [Abstract] [Full Text] [PDF]
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R.-F. Guo, N. C. Riedemann, L. Sun, H. Gao, K. X. Shi, J. S. Reuben, V. J. Sarma, F. S. Zetoune, and P. A. Ward Divergent Signaling Pathways in Phagocytic Cells during Sepsis J. Immunol., July 15, 2006; 177(2): 1306 - 1313. [Abstract] [Full Text] [PDF]
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 A. G. Kostyk, K. M. Dahl, M. W. Wynes, L. A. Whittaker, D. J. Weiss, R. Loi, and D. W.H. Riches Regulation of Chemokine Expression by NaCl Occurs Independently of Cystic Fibrosis Transmembrane Conductance Regulator in Macrophages Am. J. Pathol., July 1, 2006; 169(1): 12 - 20. [Abstract] [Full Text] [PDF]
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 M. Nozaki, B. J. Raisler, E. Sakurai, J. V. Sarma, S. R. Barnum, J. D. Lambris, Y. Chen, K. Zhang, B. K. Ambati, J. Z. Baffi, et al. Drusen complement components C3a and C5a promote choroidal neovascularization PNAS, February 14, 2006; 103(7): 2328 - 2333. [Abstract] [Full Text] [PDF]
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 T. Iizuka, Y. Ishii, K. Itoh, T. Kiwamoto, T. Kimura, Y. Matsuno, Y. Morishima, A. E. Hegab, S. Homma, A. Nomura, et al. Nrf2-deficient mice are highly susceptible to cigarette smoke-induced emphysema Genes Cells, December 1, 2005; 10(12): 1113 - 1125. [Abstract] [Full Text] [PDF]
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 M. R. Wilson, S. Choudhury, and M. Takata Pulmonary inflammation induced by high-stretch ventilation is mediated by tumor necrosis factor signaling in mice Am J Physiol Lung Cell Mol Physiol, April 1, 2005; 288(4): L599 - L607. [Abstract] [Full Text] [PDF]
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H. Cho, T. M. Lasco, S. S. Allen, T. Yoshimura, and D. N. McMurray Recombinant Guinea Pig Tumor Necrosis Factor Alpha Stimulates the Expression of Interleukin-12 and the Inhibition of Mycobacterium tuberculosis Growth in Macrophages Infect. Immun., March 1, 2005; 73(3): 1367 - 1376. [Abstract] [Full Text] [PDF]
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A. Mullick, M. Elias, S. Picard, L. Bourget, O. Jovcevski, S. Gauthier, A. Tuite, P. Harakidas, C. Bihun, B. Massie, et al. Dysregulated Inflammatory Response to Candida albicans in a C5-Deficient Mouse Strain Infect. Immun., October 1, 2004; 72(10): 5868 - 5876. [Abstract] [Full Text] [PDF]
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J. Godau, T. Heller, H. Hawlisch, M. Trappe, E. Howells, J. Best, J. Zwirner, J. S. Verbeek, P. M. Hogarth, C. Gerard, et al. C5a Initiates the Inflammatory Cascade in Immune Complex Peritonitis J. Immunol., September 1, 2004; 173(5): 3437 - 3445. [Abstract] [Full Text] [PDF]
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A. H. J. Kim, I. D. Dimitriou, M. C. H. Holland, D. Mastellos, Y. M. Mueller, J. D. Altman, J. D. Lambris, and P. D. Katsikis Complement C5a Receptor Is Essential for the Optimal Generation of Antiviral CD8+ T Cell Responses J. Immunol., August 15, 2004; 173(4): 2524 - 2529. [Abstract] [Full Text] [PDF]
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G. C. Hildebrandt, K. M. Olkiewicz, L. A. Corrion, Y. Chang, S. G. Clouthier, C. Liu, and K. R. Cooke Donor-derived TNF-{alpha} regulates pulmonary chemokine expression and the development of idiopathic pneumonia syndrome after allogeneic bone marrow transplantation Blood, July 15, 2004; 104(2): 586 - 593. [Abstract] [Full Text] [PDF]
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H. Gao, R.-F. Guo, C. L. Speyer, J. Reuben, T. A. Neff, L. M. Hoesel, N. C. Riedemann, S. D. McClintock, J. V. Sarma, N. Van Rooijen, et al. Stat3 Activation in Acute Lung Injury J. Immunol., June 15, 2004; 172(12): 7703 - 7712. [Abstract] [Full Text] [PDF]
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 J.-W. Park, C. Taube, A. Joetham, K. Takeda, T. Kodama, A. Dakhama, G. McConville, C. B. Allen, G. Sfyroera, L. D. Shultz, et al. Complement Activation Is Critical to Airway Hyperresponsiveness after Acute Ozone Exposure Am. J. Respir. Crit. Care Med., March 15, 2004; 169(6): 726 - 732. [Abstract] [Full Text] [PDF]
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T. W. Wright, G. S. Pryhuber, P. R. Chess, Z. Wang, R. H. Notter, and F. Gigliotti TNF Receptor Signaling Contributes to Chemokine Secretion, Inflammation, and Respiratory Deficits during Pneumocystis Pneumonia J. Immunol., February 15, 2004; 172(4): 2511 - 2521. [Abstract] [Full Text] [PDF]
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 M. R. Wilson, S. Choudhury, M. E. Goddard, K. P. O'Dea, A. G. Nicholson, and M. Takata High tidal volume upregulates intrapulmonary cytokines in an in vivo mouse model of ventilator-induced lung injury J Appl Physiol, October 1, 2003; 95(4): 1385 - 1393. [Abstract] [Full Text] [PDF]
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E. S. Ong, X.-P. Gao, N. Xu, D. Predescu, A. Rahman, M. T. Broman, D. H. Jho, and A. B. Malik E. coli pneumonia induces CD18-independent airway neutrophil migration in the absence of increased lung vascular permeability Am J Physiol Lung Cell Mol Physiol, October 1, 2003; 285(4): L879 - L888. [Abstract] [Full Text] [PDF]
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 A. J. Chong, C. R. Hampton, and E. D. Verrier Microvascular Inflammatory Response in Cardiac Surgery Seminars in Cardiothoracic and Vascular Anesthesia, September 1, 2003; 7(3): 333 - 354. [Abstract] [PDF]
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B. de Vries, J. Kohl, W. K. G. Leclercq, T. G. A. M. Wolfs, A. A. J. H. M. van Bijnen, P. Heeringa, and W. A. Buurman Complement Factor C5a Mediates Renal Ischemia-Reperfusion Injury Independent from Neutrophils J. Immunol., April 1, 2003; 170(7): 3883 - 3889. [Abstract] [Full Text] [PDF]
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C. D. Kaplan, S. K. O'Neill, T. Koreny, M. Czipri, and A. Finnegan Development of Inflammation in Proteoglycan-Induced Arthritis Is Dependent on Fc{gamma}R Regulation of the Cytokine/Chemokine Environment J. Immunol., November 15, 2002; 169(10): 5851 - 5859. [Abstract] [Full Text] [PDF]
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M. Huber-Lang, E. M. Younkin, J. V. Sarma, N. Riedemann, S. R. McGuire, K. T. Lu, R. Kunkel, J. G. Younger, F. S. Zetoune, and P. A. Ward Generation of C5a by Phagocytic Cells Am. J. Pathol., November 1, 2002; 161(5): 1849 - 1859. [Abstract] [Full Text] [PDF]
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S. Nakashima, Z. Qian, S. Rahimi, B. A. Wasowska, and W. M. Baldwin III Membrane Attack Complex Contributes to Destruction of Vascular Integrity in Acute Lung Allograft Rejection J. Immunol., October 15, 2002; 169(8): 4620 - 4627. [Abstract] [Full Text] [PDF]
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N. Fernandez, M. Renedo, C. Garcia-Rodriguez, and M. Sanchez Crespo Activation of Monocytic Cells Through Fc{gamma} Receptors Induces the Expression of Macrophage-Inflammatory Protein (MIP)-1{alpha}, MIP-1{beta}, and RANTES J. Immunol., September 15, 2002; 169(6): 3321 - 3328. [Abstract] [Full Text] [PDF]
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H. Zhao, M. C. Montalto, K. J. Pfeiffer, L. Hao, and G. L. Stahl Murine model of gastrointestinal ischemia associated with complement-dependent injury J Appl Physiol, July 1, 2002; 93(1): 338 - 345. [Abstract] [Full Text] [PDF]
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D. R. Roach, A. G. D. Bean, C. Demangel, M. P. France, H. Briscoe, and W. J. Britton TNF Regulates Chemokine Induction Essential for Cell Recruitment, Granuloma Formation, and Clearance of Mycobacterial Infection J. Immunol., May 1, 2002; 168(9): 4620 - 4627. [Abstract] [Full Text] [PDF]
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X.-p. Gao, T. J. Standiford, A. Rahman, M. Newstead, S. M. Holland, M. C. Dinauer, Q.-h. Liu, and A. B. Malik Role of NADPH Oxidase in the Mechanism of Lung Neutrophil Sequestration and Microvessel Injury Induced by Gram-Negative Sepsis: Studies in p47phox-/- and gp91phox-/- Mice J. Immunol., April 15, 2002; 168(8): 3974 - 3982. [Abstract] [Full Text] [PDF]
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 C. M. Calkins, D. D. Bensard, B. D. Shames, E. J. Pulido, E. Abraham, N. Fernandez, Xianzhong Meng, C. A. Dinarello, and R. C. McIntyre JR IL-1 regulates in vivo C--X--C chemokine induction and neutrophil sequestration following endotoxemia Innate Immunity, February 1, 2002; 8(1): 59 - 67. [Abstract] [PDF]
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 M. J. Janatpour, S. Hudak, M. Sathe, J. D. Sedgwick, and L. M. McEvoy Tumor Necrosis Factor-dependent Segmental Control of MIG Expression by High Endothelial Venules in Inflamed Lymph Nodes Regulates Monocyte Recruitment J. Exp. Med., November 5, 2001; 194(9): 1375 - 1384. [Abstract] [Full Text] [PDF]
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Y. Song, L. Ao, C. D. Raeburn, C. M. Calkins, E. Abraham, A. H. Harken, and X. Meng A low level of TNF-{alpha} mediates hemorrhage-induced acute lung injury via p55 TNF receptor Am J Physiol Lung Cell Mol Physiol, September 1, 2001; 281(3): L677 - L684. [Abstract] [Full Text] [PDF]
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U. Baumann, N. Chouchakova, B. Gewecke, J. Kohl, M. C. Carroll, R. E. Schmidt, and J. E. Gessner Distinct Tissue Site-Specific Requirements of Mast Cells and Complement Components C3/C5a Receptor in IgG Immune Complex-Induced Injury of Skin and Lung J. Immunol., July 15, 2001; 167(2): 1022 - 1027. [Abstract] [Full Text] [PDF]
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 R. L. Warner, L. Beltran, E. M. Younkin, C. S. Lewis, S. J. Weiss, J. Varani, and K. J. Johnson Role of Stromelysin 1 and Gelatinase B in Experimental Acute Lung Injury Am. J. Respir. Cell Mol. Biol., May 1, 2001; 24(5): 537 - 544. [Abstract] [Full Text]
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N. Chouchakova, J. Skokowa, U. Baumann, T. Tschernig, K. M. H. Philippens, B. Nieswandt, R. E. Schmidt, and J. E. Gessner Fc{{gamma}}RIII-Mediated Production of TNF-{{alpha}} Induces Immune Complex Alveolitis Independently of CXC Chemokine Generation J. Immunol., April 15, 2001; 166(8): 5193 - 5200. [Abstract] [Full Text] [PDF]
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R.-F. Guo, A. B. Lentsch, R. L. Warner, M. Huber-Lang, J. V. Sarma, T. Hlaing, M. M. Shi, N. W. Lukacs, and P. A. Ward Regulatory Effects of Eotaxin on Acute Lung Inflammatory Injury J. Immunol., April 15, 2001; 166(8): 5208 - 5218. [Abstract] [Full Text] [PDF]
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 S. Ikeda, H. Saito, K. Fukatsu, T. Inoue, I. Han, S. Furukawa, T. Matsuda, and A. Hidemura Dietary Restriction Impairs Neutrophil Exudation by Reducing CD11b/CD18 Expression and Chemokine Production Arch Surg, March 1, 2001; 136(3): 297 - 304. [Abstract] [Full Text] [PDF]
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S. Nataf, S. L. Carroll, R. A. Wetsel, A. J. Szalai, and S. R. Barnum Attenuation of Experimental Autoimmune Demyelination in Complement-Deficient Mice J. Immunol., November 15, 2000; 165(10): 5867 - 5873. [Abstract] [Full Text] [PDF]
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 A. Alonso, Y. Bayon, M. Renedo, and M. S. Crespo Stimulation of Fc{gamma}R receptors induces monocyte chemoattractant protein-1 in the human monocytic cell line THP-1 by a mechanism involving I{kappa}B-{alpha} degradation and formation of p50/p65 NF-{kappa}B/Rel complexes Int. Immunol., April 1, 2000; 12(4): 547 - 554. [Abstract] [Full Text] [PDF]
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M. S. Mulligan, A. B. Lentsch, M. Huber-Lang, R.-F. Guo, V. Sarma, C. D. Wright, T. R. Ulich, and P. A. Ward Anti-Inflammatory Effects of Mutant Forms of Secretory Leukocyte Protease Inhibitor Am. J. Pathol., March 1, 2000; 156(3): 1033 - 1039. [Abstract] [Full Text] [PDF]
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N. Davoust, S. Nataf, R. Reiman, M. V. Holers, I. L. Campbell, and S. R. Barnum Central Nervous System-Targeted Expression of the Complement Inhibitor sCrry Prevents Experimental Allergic Encephalomyelitis J. Immunol., December 15, 1999; 163(12): 6551 - 6556. [Abstract] [Full Text] [PDF]
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