1 Department of Pharmacology and Toxicology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205; and 2 Department of Pathology, University of Texas Health Science Center, Houston, Texas 77030
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ABSTRACT |
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The initiating step of neutrophil-induced
cytotoxicity in the liver is the recruitment of these phagocytes into
sinusoids. The aim of our study was to compare the efficacy of systemic
exposure with individual inflammatory mediators on neutrophil
activation and sequestration in the hepatic vasculature of C3Heb/FeJ
mice as assessed by flow cytometry and histochemistry, respectively. The CXC chemokine macrophage inflammatory protein-2 (MIP-2; 20 µg/kg)
induced a time-dependent upregulation of Mac-1 (318% at 4 h) and
shedding of L-selectin (41% at 4 h). MIP-2 treatment caused a
temporary increase of sinusoidal neutrophil accumulation at 0.5 h
[97 ± 6 polymorphonuclear leukocytes (PMN)/50 high-power fields
(HPF)], which declined to baseline (8 ± 2) at 4 h. The CXC
chemokine KC was largely ineffective in activating neutrophils or
recruiting them into the liver. Cytokines (tumor necrosis factor- and interleukin-1
) and cobra venom factor substantially increased Mac-1 expression and L-selectin shedding on neutrophils and caused stable sinusoidal neutrophil accumulation (170-220 PMN/50 HPF). Only cytokines induced venular neutrophil margination. Thus CXC chemokines in circulation are less effective than cytokines or complement in activation of neutrophils and their recruitment into the
hepatic vasculature in vivo.
complement activation; cytokines; adhesion molecules; L-selectin; Mac-1; CD11b/CD18
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INTRODUCTION |
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POLYMORPHONUCLEAR
LEUKOCYTES (neutrophils) contribute to liver injury in a number
of disease states including hepatic ischemia-reperfusion (21, 23), endotoxemia (24), sepsis
(36), alcoholic hepatitis (1), remote organ
injury (17), hemorrhagic shock (41), and
certain drug-induced liver injuries (7). The basic
mechanism of a neutrophil-mediated pathophysiology consists of three
steps: accumulation of activated neutrophils in the hepatic
vasculature, transmigration, and adherence-dependent cytotoxicity
against target cells (26). Prerequisite for toxicity is
the accumulation of these phagocytes in sinusoids and in postsinusoidal
or portal venules (4, 43). Previous investigations
demonstrated that systemic exposure to inflammatory mediators such as
complement factors (20, 44), tumor necrosis factor-
(TNF-
) (10, 14), interleukin-1 (10),
platelet-activating factor (3), or leukotriene C4 (3) can cause neutrophil sequestration in
the hepatic vasculature.
In recent years, CXC chemokines, which are potent chemotactic mediators for neutrophils, have been described (33). Antibodies against members of the CXC chemokine family were shown (6, 18, 32) to be protective against hepatic ischemia-reperfusion injury. Corresponding to the reduced injury, fewer neutrophils were found in the postischemic liver. Furthermore, substantial neutrophil accumulation in the liver was observed (40) in transgenic mice with a systemic overproduction of the CXC chemokine interleukin-8 (IL-8). In addition, transfection of the rat cytokine-induced neutrophil chemoattractant-1 (CINC-1) gene into hepatocytes resulted in hepatic neutrophil sequestration (34). These results suggested that CXC chemokines can be responsible for neutrophil recruitment into the liver (6, 18, 32, 34, 40). However, in a complex pathophysiology, neutrophils are exposed to local CXC chemokine gradients in the tissue and, at the same time, are also systemically exposed to a number of potent inflammatory mediators including CXC chemokines. Thus to determine which mediator(s) is potentially responsible for hepatic neutrophil recruitment, it is necessary to evaluate the systemic effects of chemokines and other inflammatory mediators separately from local tissue gradients.
Most compounds that recruit neutrophils into the liver are able to upregulate Mac-1 (CD11b/CD18) on neutrophils (19). However, whether CXC chemokines are able to activate neutrophils, as indicated by the increased expression of Mac-1, is still unclear. Previous in vitro experiments showed that human IL-8 (8), rat CINC-1 (48), and mouse KC (2) can increase Mac-1 expression on neutrophils. However, anti-CINC-1 antibodies did not affect endotoxin-induced upregulation of Mac-1 in vivo (48). This could mean that a temporary activation was missed or that CXC chemokines are not relevant for neutrophil activation in the presence of other, more potent inflammatory mediators. To clarify these issues, we compared the effects of individual cytokines, CXC chemokines, and complement on the systemic activation of neutrophils in vivo by flow cytometry and their effect on hepatic neutrophil sequestration in sinusoids and postsinusoidal venules by histochemistry. Our data show that CXC chemokines alone were only weak activators of neutrophils and therefore were only moderately effective in inducing neutrophil accumulation in the hepatic vasculature.
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MATERIALS AND METHODS |
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Animals.
Male C3Heb/FeJ (20-25 g body wt) were obtained from Jackson
Laboratories (Bar Harbor, ME). The animals had free access to food
(certified rodent diet no. 5002C, PMI Feeds, Richmond, IN) and water.
The experimental protocols followed the criteria of the University of
Arkansas for Medical Sciences and the Guide for the Care and Use
of Laboratory Animals [DHHS Publication No. (NIH) 85-23, Revised
1985, Office of Science and Health Reports, DRR/NIH, Bethesda, MD
20892]. Animals were treated with 8 or 20 µg/kg murine recombinant
macrophage inflammatory protein-2 (MIP-2), KC, TNF-, IL-1
(all
from Endogen, Woburn, MA), or cobra venom factor (CVF; 120 µg/kg;
Sigma, St. Louis, MO). Groups of animals were killed at various time
points after injection of an inflammatory mediator (0.5, 2, and 4 h). Blood was collected from the right ventricle into a heparinized
syringe. In some experiments, blood was obtained and allowed to clot.
Serum levels of MIP-2 and KC were measured with the respective ELISA
kits (R & D Systems, Minneapolis, MN). Sections of the liver were
frozen in liquid nitrogen or fixed in phosphate-buffered formalin for
histological analysis.
Histology. Formalin-fixed portions of the liver were paraffin embedded, and 5-µm sections were cut. Neutrophils were stained using the AS-D chloroacetate esterase technique as described in detail previously (22). Neutrophils were identified by positive staining and morphology and were counted in 50 high-power fields (×400) using a Nikon Labophot microscope. Only neutrophils present within sinusoids or extravasated into the tissue were counted; the number of neutrophils marginated within large vessels, e.g., hepatic venules, were evaluated separately (counted in 10 vessels of equal diameter). Cell damage was evaluated in parallel sections stained with hematoxylin and eosin. The percentage of necrosis was estimated by evaluating the number of microscopic fields with necrosis compared with the entire histological section. The pathologist (A. Farhood) performing the histological evaluation was blinded as to the treatment of animals.
Flow cytometric analysis of neutrophils. Peripheral blood neutrophils were incubated with FITC-conjugated RB6-8C5 (anti-Gr-1) and phycoerythrin (PE)-conjugated M1/70 (anti-CD11b) or PE-conjugated Mel-14 (anti-L-selectin) antibodies (Pharmingen, San Diego, CA) and stained using a whole blood lysis kit (Coulter Immunology, Hialeah, FL) as previously described in detail (9, 29). Two-color analysis of antibody binding to cells was analyzed by flow cytometry using a FACScan flow cytometer (Becton Dickinson, San Diego, CA). Peripheral blood neutrophils were gated by the forward and light angle scatter and Gr-1 FITC fluorescence. Nonspecific fluorescence was determined on cells incubated with isotype- and fluorochrome-matched control antibodies.
Nuclear extracts and electrophoretic mobility shift assay.
Nuclear extracts were prepared from frozen liver sections as described
in detail previously (11). Gel shift reagents were used
according to the manufacturer's suggested protocol (Promega, Madison,
WI). Double-stranded nuclear factor-B (NF-
B) consensus oligonucleotide probe (5'-AGTTGAGGGGACTTTCCCAGGC-3') was
end-labeled with [
-32P]ATP (10 µCi at 222 TBq/mmol;
Amersham, Arlington Heights, IL). Binding reactions, containing 35 fmol
(~1 × 105 dpm) of oligonucleotide and 5 µg of
nuclear protein were conducted at room temperature for 20 min in a
total volume of 10 µl in binding buffer [10 mM Tris · HCl,
pH 7.5, 50 mM NaCl, 1 mM MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol, 4% glycerol (vol/vol), and 0.5 µg poly(dI-dC)]. For
competition reactions, unlabeled oligonucleotide was added 5 min before
addition of radiolabeled probe. After the binding reactions, 1 µl of
10× gel loading buffer was added, and the reaction was subjected to
nondenaturing 4% PAGE in low-ionic-strength buffer (45 mM Tris-borate
and 1 mM EDTA) at 100 V/20 mA for ~2 h (13).
Gels were vacuum dried and exposed to X-ray film (Hyperfilm MP;
Amersham) at
70°C.
Northern blot analysis.
Total cellular RNA was isolated from liver tissue as previously
described in detail (9). RNA was quantified
spectrophotometrically, and equal amounts of RNA samples were
electrophoresed on denaturing agarose-formaldehyde gels and transferred
to Gene Screen Plus hybridization membranes (NEN Research Products,
Boston, MA) (10, 11). RNA was cross-linked by
baking the membranes at 80°C for 2 h under vacuum. Mouse
intercellular adhesion molecule-1 (ICAM-1) hybridization probe was
prepared by PCR using a mouse ICAM cDNA (16) and the
following oligonucleotides: 5'-TGGAACTGCACGTGCTGTAT-3' and
5'-ACCATTCTGTTCAAAAGCAG-3' encompassing nucleotides 500-900. The
probe for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was prepared with a PCR-Amplimer kit (Clontech, Palo Alto, CA). Purified fragments were radiolabeled with
[-32P]dCTP using a random hexanucleotide primer kit
(Stratagene, La Jolla, CA). Transfered membranes were prehybridized
with Rapid-Hyb buffer (Amersham) at 65°C for 2 h and then
hybridized with labeled probes overnight at 65°C. Membranes were
washed in 1× SSC (1× SSC is 0.15 M sodium chloride and 0.015 M sodium
citrate, pH 7.0) containing 0.1% SDS for 15 min at room temperature.
Membranes were washed twice at 55°C in 0.2× SSC containing 0.1% SDS
for 30 min. The washed blots were exposed to Hyperfilm MP X-ray film (Amersham) at
80°C.
RNase protection assay.
All protocols followed the instructions of the RiboQuant multiprobe
RNase protection assay system (PharMingen, San Diego, CA). With the use
of an in vitro transcription kit and a customized template set
(containing mouse MIP-2, mouse KC, and L32), a radiolabeled probe set
was synthesized using [-32P]UTP
(30). These probes were hybridized with total RNA
isolated from liver tissue for 16 h. After digestion of
nonhybridized RNA with RNase, the protected probes were separated on a
denaturing acrylamide gel. The gel was dried and then exposed to X-ray
film (Kodak X-OMat, Fisher Scientific, Pittsburgh, PA) for 12 h at
80°C. The developed X-ray films were scanned using a calibrated imaging densitometer (GS-710, Bio-Rad Laboratories, Hercules, CA) and
Quantity-One software (Bio-Rad).
Peritonitis experiments To test the chemotactic effects of CXC chemokines, KC or MIP-2 (8 µg/kg) was injected intraperitoneally (5). Controls received 10 ml/kg PBS. After 2 or 4 h, the animals were killed and the peritoneal cavities lavaged twice with 2 ml of PBS. The lavage fluids were centrifuged (1,000 g) for 10 min to sediment the neutrophils. The pellets were resuspended in Tris-buffered 0.75% NH4Cl for 10 min to lyse erythrocytes. After centrifugation, the pellets were resuspended in detergent buffer (50 mM phosphate buffer containing 0.5% cetyltrimethylammonium bromide), briefly sonicated, and freeze-thawed twice. Myeloperoxidase (MPO) activity as an index for neutrophil accumulation was determined spectrophotometrically in 50 mM phosphate buffer (pH 6.0) containing 0.165 mg/ml o-dianisidine hydrochloride and 0.15 mM hydrogen peroxide. The change in absorbance was determined at 460 nm.
Statistics. All data are given as means ± SE. Comparisons between multiple groups were performed with one-way ANOVA followed by a Bonferroni t-test. P < 0.05 was considered significant.
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RESULTS |
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CXC chemokines and neutrophil activation.
Flow cytometric analysis of peripheral blood neutrophils indicated that
intravenous administration of a high dose of MIP-2 caused a progressive
increase in Mac-1 expression on circulating neutrophils. Expression of
Mac-1 reached values of 318% of baseline levels at 4 h (Fig.
1A). At the same time,
L-selectin was shed, resulting in a 41% loss of the adhesion molecule
at 4 h (Fig. 1B). MIP-2 administration induced a
transient accumulation of neutrophils in sinusoids with a maximum at
0.5 h (Fig. 2). All neutrophils were
located within sinusoids; no extravasation was detected. In contrast to
MIP-2, KC caused only a very moderate and transient increase in Mac-1
expression and L-selectin shedding at 0.5 h after administration
of the CXC chemokine (Fig. 1). Similarly, only a very minor increase in
the number of sinusoidal neutrophils was observed at 0.5 h (Fig.
2). No margination of neutrophils was found in postsinusoidal venules
after KC or MIP-2 injection during the 4-h time course of the
experiment (data not shown).
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Chemotaxis of CXC chemokines in vivo.
Because of the substantial difference between KC and MIP-2 in
activating neutrophils and causing hepatic neutrophil sequestration, we
evaluated the chemotactic potential of both chemokines in vivo. Intraperitoneal injection of 8 µg/kg of KC or MIP-2 resulted in peritoneal neutrophil accumulation as indicated by the increased MPO
activity of lavaged cells (Fig. 3).
However, there was no significant difference in neutrophil recruitment
between KC and MIP-2 (Fig. 3).
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Neutrophil activation by cytokines and complement.
To evaluate the capacity of other proinflammatory mediators to activate
neutrophils and cause hepatic neutrophil recruitment, murine TNF- or
IL-1
(20 µg/kg) was injected intravenously or complement was
maximally activated in vivo by injecting 120 µg/kg CVF. All three
mediators increased Mac-1 expression on circulating neutrophils by
500-700% above baseline values (Fig.
4A). However, there was a
substantial difference in the time course. Animals treated with CVF or
TNF-
showed a rapid onset of the response and a consistent increase
over the entire observation period. In contrast, IL-1
injection
resulted in a delayed increase at 2 h and a reduction of the
activated Mac-1 levels at 4 h (Fig. 4A). Although
L-selectin shedding after treatment with these mediators supported the
concept that circulating neutrophils are activated, there are
differences from the response of Mac-1 expression (Fig. 4B).
TNF-
and IL-1
were equally potent in inducing L-selectin shedding. Only a minor delay was observed with IL-
. However, activation of complement was significantly less potent in inducing L-selectin shedding despite the fact that it was the most potent activator of Mac-1 expression (Fig. 4B). All three mediators
induced sinusoidal neutrophil sequestration at 2 and 4 h but had
no effect at 0.5 h (Fig.
5A). TNF-
and IL-1
were
most potent with a 27- to 29-fold increase over baseline. In contrast,
complement activation induced only a 10-fold increase at 2 h. The
response after TNF-
injection was more stable than after IL-1
or
CVF (Fig. 5A). Neutrophil margination in postsinusoidal
venules was absent in controls and at 0.5 h after injection of
cytokines (Fig. 5B). However, at 2 and 4 h a
substantial number of neutrophils adhered in these vessels. With the
exception of a minor increase at 2 h, CVF did not cause venular
neutrophil margination.
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Administration of TNF- and IL-1
induced formation of MIP-2
and KC.
Serum levels of MIP-2 increased 2,700- and 6,500-fold 1 h after
TNF-
and IL-1
administration, respectively (Fig.
6). Compared with MIP-2, the absolute
increase of KC serum levels was much higher after cytokine injection.
However, due to the higher baseline, there was only a 236- and 525-fold
increase with TNF-
and IL-1
, respectively (Fig. 6). CVF did not
increase serum levels of KC or MIP-2 (data not shown). To verify that
at least part of the chemokines measured in plasma were generated in
the liver, mRNA levels of KC and MIP-2 were evaluated by RNase
protection assay in controls and 1 h after TNF-
injection (Fig.
7A). In controls, mRNA levels
of both CXC chemokines were almost undetectable. However, TNF-
induced a substantial increase in MIP-2 and in particular in KC mRNA.
Densitometric analysis indicated that the KC-to-L32 (housekeeping gene)
ratio increased by 280-fold and the MIP-to-L32 ratio increased by
16-fold (Fig. 7B). Similar results were obtained when the
CXC chemokine mRNA levels were compared with GAPDH (data not shown).
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TNF- but not complement activates NF-
B and ICAM-1 mRNA
formation.
Because CVF caused margination of neutrophils in the sinusoids but not
in venules, we tested whether CVF could activate the transcription
factor NF-
B and induce ICAM-1 mRNA formation. In livers of control
animals, only minor amounts of NF-
B are present in the nucleus and
only traces of ICAM-1 mRNA were detectable (Fig.
8). CVF induced neither the translocation
of NF-
B into the nucleus nor ICAM-1 transcription at 1 h. In
contrast, administration of TNF-
caused a dramatic activation of
NF-
B and induction of ICAM-1 mRNA formation (Fig. 8).
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DISCUSSION |
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The objective of this study was to evaluate the capacity of high systemic levels of CXC chemokines to activate neutrophils as indicated by Mac-1 upregulation and L-selectin shedding and to induce neutrophil sequestration in sinusoids and venules as assessed by histochemistry. Our results showed that a high dose of the CXC chemokine KC caused only a minor, temporary activation of circulating neutrophils in vivo. This temporary activation correlated with a minor, transient accumulation of neutrophils in sinusoids. On the other hand, the CXC chemokine MIP-2 induced a sustained activation of circulating neutrophils but also only a temporary accumulation of neutrophils in sinusoids. These data suggest that sinusoidal neutrophil sequestration requires some degree of neutrophil activation. However, systemic neutrophil activation does not necessarily lead to a sustained accumulation of these phagocytes in sinusoids. Moreover, the magnitude of neutrophil activation as determined by Mac-1 expression and L-selectin shedding does not correlate with sinusoidal neutrophil sequestration. This effect was not only observed with CXC chemokines but also with activated complement. CVF induced the largest increase in Mac-1 expression but triggered only a moderate response of neutrophil accumulation in sinusoids. Thus systemic neutrophil activation may trigger neutrophil accumulation in the liver. However, it is neither qualitatively nor quantitatively a reliable assessment of hepatic neutrophil accumulation. This finding is of particular importance for human pathophysiologies in which, in most cases, the activation status of circulating neutrophil can be evaluated at different time points but liver tissue is only rarely available.
The comparison between the effects of CXC chemokines and other
mediators known to systemically activate neutrophils and cause accumulation in the liver suggests that TNF, IL-1, and activated complement factors are substantially more potent in causing Mac-1 upregulation and L-selectin shedding on circulating neutrophils. The
response to IL-1 was slightly delayed, which may explain why in
previous studies (10) IL-1 was thought to be unable to
increase Mac-1 expression. TNF, IL-1, and to some degree activated
complement induced a pronounced and a sustained accumulation of
neutrophils in sinusoids. Interestingly, the sequestration of
neutrophils in sinusoids after cytokine administration and complement
activation was delayed compared with the effect of CXC chemokines
despite the fact that all mediators rapidly upregulated Mac-1. In
general, it is assumed that neutrophil accumulation in sinusoids is
independent of adhesion molecules (19). There is no
rolling in sinusoids (43, 45), and consequently, P
(12, 45)-, L (29)- and E-selectin (14,
29) did not affect neutrophil accumulation in sinusoids.
Furthermore, antibodies against 2-integrins
(22), ICAM-1 (10, 22), or vascular cell
adhesion molecule-1 (9) had no effect on the initial
sinusoidal neutrophil sequestration induced by cytokines. After
injection or generation of these inflammatory mediators, neutrophils
may be trapped because of reduced deformability (46),
sinusoidal cell swelling (35), and active vasoconstriction (47) rather than the interaction of adhesion molecules.
The different behavior of neutrophils after CXC chemokine injection compared with cytokines and complement suggests potentially different mechanisms of accumulation. However, this mechanism requires further study to be understood.
In contrast to sinusoids, the adherence of neutrophils in
postsinusoidal venules requires adhesion molecules. Selectins are involved in rolling, and ICAM-1/integrin interactions mediate firm
adhesion (12, 14, 29, 39, 42) similar to the general mechanism of neutrophil adhesion in postcapillary venules
(15). Our results showed that CXC chemokines, in contrast
to TNF and IL-1, did not induce any margination of neutrophils in
postsinusoidal venules. P-selectin is not expressed in the hepatic
vasculature of control animals (12). Adhesion in these
vascular beds requires either recruitment of P-selectin from
Weibel-Palade bodies (39) or transcriptional upregulation
of P-selectin as an initiating step (12). The induction of
P-selectin in postsinusoidal venules is mediated by cytokines and
dependent on NF-B activation (12). Only TNF or IL-1 can
activate NF-
B and upregulate adhesion molecules and therefore only
cytokines induced venular adherence. Because neither CXC chemokines
(27, 37) nor activated complement (Fig. 8) can trigger
these effects, these mediators were unable to cause relevant neutrophil
adherence in postsinusoidal venules. CXC chemokine formation is
regulated by the transcription factor NF-
B (28, 38).
Thus TNF and IL-1 induced the synthesis of KC and MIP-2 in vivo (Fig.
6). At least part of the chemokines was generated in the liver as
indicated by the increase in mRNA levels (Fig. 7). However, these
chemokines had only a moderate, transient effect on neutrophil
accumulation in sinusoids and did not trigger venular adherence. These
data suggest that the effects of TNF and IL-1 on neutrophil activation
and their sequestration in different vascular beds of the liver were
not secondary to CXC chemokine formation.
An important issue regarding our study is the relevance of the effect of systemic administration of inflammatory mediators to their mechanism of action in vivo. In general, neutrophil accumulation in liver sinusoids requires either systemic activation by inflammatory mediators and/or a local gradient of a chemotactic factor. During endotoxemia, high systemic levels and local gradients of CXC chemokines are present at the same time. In addition, high levels of cytokines are generated, and depending on the dose of endotoxin, complement can be activated. To investigate the role of CXC chemokines in this pathophysiology, we address one of several aspects in this study: the relative importance of systemic levels of CXC chemokines compared with cytokines and complement in activating neutrophils and inducing their accumulation in sinusoids. Our results suggest that CXC chemokines are of limited relevance for neutrophil recruitment into the liver through this mechanism. This conclusion is also supported by preliminary data that show no effect of antibodies against CXC chemokines on hepatic neutrophil accumulation during endotoxemia (25). However, in the absence of elevated systemic cytokine levels and activated complement, local gradients of chemotactic factors such as CXC chemokines may become important for neutrophil recruitment into the liver. Alternatively, local gradients of CXC chemokines may be important for directing movement of already sequestered neutrophils in the liver. Because transmigration is necessary for neutrophil cytotoxicity in the liver (4), CXC chemokines may be more relevant for extravasation.
Another important aspect of our study was the finding that there are substantial differences in the degree of neutrophil activation and hepatic neutrophil sequestration between individual CXC chemokines. MIP-2 and KC are murine CXC chemokines, which are related to the Gro family of human CXC chemokines (33). Both chemokines act on the only CXC chemokine receptor in mice, CXCR2 (31), and trigger neutrophil chemotaxis and intracellular Ca2+ fluxes (31). However, MIP-2 appears to be ~10-fold more potent than KC in triggering these effects in isolated murine neutrophils (31). However, the 10-fold higher generation of KC compared with MIP-2 in response to cytokines in vivo may compensate for the lower efficacy of KC at the receptor. Our in vivo data also indicate that MIP-2 was more potent than KC in causing systemic neutrophil activation, but we observed a similar chemotactic response in the peritonitis experiments. However, the difference in neutrophil chemotaxis between MIP-2 and KC was only observed at very low concentrations (0.5 nM) (31). The dose used in our in vivo experiment resulted in considerably higher concentrations, which may explain the similar response to both chemokines. On the other hand, our data on neutrophil activation suggest that even with these high doses of CXC chemokines, which should have resulted in initial serum levels of one to two orders of magnitude beyond peak levels after cytokine administration (Fig. 6), there was still a difference in the neutrophil response to KC and MIP-2. This suggests that for realistic in vivo levels of these chemokines, only MIP-2 may be of importance for systemic neutrophil activation and hepatic neutrophil recruitment. Furthermore, low concentrations of CXC chemokines effectively trigger a chemotactic response (31) but do not upregulate Mac-1. This indicates that CXC chemokines may be more important for directing neutrophil movement than for activation.
In summary, our investigation demonstrated that the CXC chemokine MIP-2 has the capacity to activate neutrophils at high concentrations in vivo and induce a transient neutrophil accumulation in sinusoids but not in venules. The CXC chemokine KC is much less effective in triggering these effects. In contrast, the cytokines TNF and IL-1 and activated complement factors are potent mediators for upregulation of Mac-1 and shedding of L-selectin. These mediators also induce a sustained accumulation of neutrophils in sinusoids. In addition, cytokines cause adherence in postsinusoidal venules due to their capacity to induce upregulation of adhesion molecules. Thus CXC chemokines are less effective than cytokines or complement in activation of neutrophils and their recruitment into the hepatic vasculature in vivo. This suggests that local gradients of CXC chemokines may direct neutrophil movement within the liver. Systemic CXC chemokines may only be relevant to hepatic neutrophil sequestration if more potent mediators are absent.
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ACKNOWLEDGEMENTS |
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We thank Dr. Naeem Essani (Pharmacia) for preparing the murine ICAM-1 hybridization probe and Robert Hopper for expert technical assistance.
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FOOTNOTES |
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This work was supported in part by National Institute of Environmental Health Sciences Grant ES-06091 and National Institute on Alcohol Abuse and Alcoholism Grant AA-12916.
Address for reprint requests and other correspondence: H. Jaeschke, Dept. of Pharmacology and Toxicology, Univ. of Arkansas for Medical Sciences, 4301 W. Markham St. (Mailslot 638), Little Rock, AR 72205-7199 (E-mail: JaeschkeHartmutW{at}uams.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 14 March 2001; accepted in final form 6 August 2001.
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