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Published online before print December 12, 2003
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Department of Surgery, Malmö University Hospital, Lund University, Sweden
1Correspondence: Department of Surgery, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden. E-mail: henrikthorlacius{at}hotmail.com
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: adhesion • endotoxin • intravital microscopy • sepsis • transmigration
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Activation and tissue navigation of leukocytes are coordinated by secreted chemokines [9 10 11 ]. The chemokine family includes small peptides that are known to be key regulators of leukocyte activation and tissue accumulation [9 10 11 ]. The members of this family are subdivided into two main groups (CC and CXC) based on structural properties. In the mouse, the CXC chemokine family includes macrophage inflammatory protein-2 (MIP-2) [12 ] and cytokine-induced neutrophil chemoattractant (KC) [9 13 ]. MIP-2 and KC are known to be murine homologues of human growth-related oncogene chemokines [11 12 ]. The CXC chemokines are considered to attract predominately neutrophils [9 10 11 ] and have been implicated as important mediators of several severe conditions, such as endotoxemia-induced lung injury [14 ], glomerulonephritis [15 ], bacterial meningitis [16 ], and hepatic ischemia-reperfusion [17 ]. Although numerous studies have demonstrated clear-cut inductions of CXC chemokines in livers of endotoxemic animals [18 19 ], the literature on the functional role of CXC chemokines in endotoxin-provoked leukocyte recruitment and liver injury is limited and contradictory. For example, one study showed that an antibody directed against the rat CXC chemokine cytokine-induced neutrophil chemoattractant reduced hepatic accumulation of neutrophils in endotoxemic rats [18 ]. In contrast, a recent study reported that immunoneutralization of MIP-2 or KC was ineffective against endotoxin-induced recruitment of neutrophils in the liver [20 ]. Moreover, it was recently demonstrated that systemic injection of CXC chemokines is a weak inducer of neutrophil sequestration in liver sinusoids and, in fact, lacks the capacity to induce margination of neutrophils in postsinusoidal venules [21 ]. Thus, a role of CXC chemokines in endotoxin-induced leukocyte recruitment and tissue injury in the liver remains to be documented. One reason for the controversy discussed above may be related to the fact that those studies were limited to morphological assessment of leukocyte recruitment. In this context, it is important to note that intravital microscopy is an important tool to define the role of specific molecules in leukocyte-endothelium interactions, enabling direct observation and quantification of the multiple steps of the extravasation process of leukocytes, including leukocyte rolling and adhesion.
Based on the above considerations, we wanted to define the role of CXC chemokines in leukocyte extravasation, microvascular perfusion, hepatocellular injury, and apoptosis in endotoxemic mice. For this purpose, we used intravital fluorescence microscopy of the liver microcirculation in combination with molecular and morphological techniques.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experimental protocol
To delineate the role of MIP-2 and KC in endotoxin-induced leukocyte recruitment and liver injury, monoclonal antibodies (mAb) directed against MIP-2 (40 µg per mouse, clone 40605.111, rat IgG, R & D Systems Europe, Abingdon, Oxon, UK) and/or KC (40 µg per mouse, clone 48415.111, rat IgG, R & D Systems Europe) and a control antibody (R3-34, rat IgG, PharMingen, San Diego, CA) were given i.v. immediately before endotoxin challenge. In separate experiments, these doses of anti-MIP-2 and -KC antibodies blocked leukocyte recruitment induced by MIP-2 and KC, respectively, by more than 93% in the mouse cremaster muscle. Six hours before surgery and intravital observation, mice were pretreated i.p. with or without (negative-control animals) a combination of lipopolysaccharide (LPS; 10 µg/mouse; Sigma Chemical Co., St. Louis, MO) and D-galactosamine (Gal; 18 mg/mouse; Sigma Chemical Co.), dissolved in phosphate-buffered saline (PBS) to a total volume of 0.25 ml.
Surgical procedure
Five hours and 40 min after administration of test substances, animals were anesthetized, a transverse subcostal incision was performed, and the ligamentous attachments from the liver to the diaphragm and the abdominal wall were gently released. The animals were positioned on their left side, and the left liver lobe was carefully exteriorized onto an adjustable stage for analysis of hepatic microcirculation by use of intravital fluorescence microscopy. The liver surface was covered with a circular glass to avoid tissue drying and exposure to ambient oxygen. An equilibration period of 5 min was allowed before starting microscopical observation. Fifteen minutes later, after intravital observations, animals were killed by exsanguination, and blood was drawn from the heart for analysis of liver function tests, including alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using standard spectrophotometric procedures. Systemic leukocyte counts, including polymorphonuclear leukocytes (PMNL) and mononuclear leukocytes (MNL), were determined by use of a hematocytometer.
Intravital microscopy
For observations of the liver microcirculation, we used a modified Olympus microscope (BX50WI, Olympus Optical Co. GmbH, Hamburg, Germany) equipped with different water-immersion lenses (x40 NA 0.75/x63 NA 0.9). The image was televised (Sony Trinitron) using a charge-coupled device video camera (FK 6990 Cohu, Pieper GmbH, Schwerte, Germany) and recorded on videotape (Panasonic SVT-S3000 S-VHS recorder) for subsequent off-line evaluation. Perfusion within individual microvessels was studied after contrast enhancement by fluorescein isothiocyanate-dextran (0.1 ml, 2 µmol/kg; Sigma Chemical Co.). In vivo labeling of leukocytes with rhodamine-6G (0.1 ml, 0.05 mg/ml; Sigma Chemical Co.) enabled quantitative analysis of leukocyte flow behavior in sinusoids and postsinusoidal venules. Quantification of microcirculatory parameters was performed off-line by frame-to-frame analysis of the videotaped images. Five postsinusoidal venules with connecting sinusoids were evaluated in each animal. Microcirculatory analysis included determination of the number of perfused sinusoids given as a percentage of the total number of sinusoids observed (i.e., sinusoidal perfusion). Within postsinusoidal venules, leukocyte rolling was measured by counting the number of cells rolling in the venule during 30 s and is expressed as cells/min. Leukocyte adhesion was measured by counting the number of cells that adhered along the venular endothelium and remained stationary during the observation period of 30 s and is expressed as cells/mm venule length. Blood-flow velocities were measured off-line by use of CapImage software (Zeintl, Heidelberg, Germany). The velocity was calculated as a mean value from five measurements per venule and is expressed as mm/s. Venular wall shear rate was determined based on the Newtonian definition: Wall shear rate = 8 [(red blood cell velocity)/venular diameter], as described previously [22
]. Wall shear stress was calculated according to the wall shear stress = wall shear rate x 0.025 (dyn/cm), assuming a blood viscosity of 0.025 poise. Hepatocyte apoptosis was measured in the same microscopic setup as above. For this purpose, the fluorochrome Hoechst 33342 (Hoechst, 0.02 ml, 0.2 µg/ml; Molecular Probes, Leiden, the Netherlands) was topically applied onto the liver surface for staining of hepatocyte DNA. Hoechst is a fluorescent dye that has been widely used for analysis of nuclear morphology, e.g., nuclear condensation and fragmentation in cultured hepatocytes and endothelial cells [23
]. After exsanguination and 5 min of incubation, six microscopical fields (using a x63 lens) were recorded for off-line quantification of hepatocyte nuclei showing signs of apoptosis (chromatin condensation and fragmentation). Hepatocyte apoptosis is given as the percentage of the number of hepatocyte nuclei showing apoptotic features from the total number of hepatocyte nuclei observed.
Reverse transcriptase-polymerase chain reaction (RT-PCR)
Three hours after challenge with PBS or LPS/Gal, total RNA was extracted from whole-liver tissue using an acid guanidinium-phenol-chloroform method (TRIzol reagent) and was treated with RNase-free DNase (DNase I), according to the manufacturer’s protocol, to remove potential contamination from genomic DNA. RNA concentrations were determined by measuring the absorbance spectrophotometrically at 260 nm. RT-PCR was performed with the SuperScript one-step RT-PCR system. Each reaction contained 500 ng total liver RNA as template and 0.2 µM each primer in a final volume of 50 µL. Mouse ß-actin served as an internal control gene. The RT-PCR profile was one cycle of DNA synthesis at 50°C for 30 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C, and extension at 72°C for 1 min, followed by one cycle of final extension at 72°C for 10 min. After RT-PCR, aliquots of the RT-PCR products were separated on 2% agarose gel containing ethidium bromide and were photographed. The primer sequences of KC, MIP-2, and ß-actin were as follows: KC (forward) 5'-GCC AAT GAG CTG CGC TGT CAA TGC-3', KC (reverse) 5'-CTT GGG GAC ACC TTT TAG CAT CTT-3'; MIP-2 (forward) 5'-GCT TCC TCG GGC ACT CCA GAC-3', MIP-2 (reverse) 5'-TTA GCC TTG CCT TTG TTC AGT AT-3'; ß-actin (forward) 5'-ATG TTT GAG ACC TTC AAC ACC-3', ß-actin (reverse) 5'-TCT CCA GGG AGG AAG AGG AT-3'.
Enzyme-linked immunosorbent assay (ELISA)
The right liver lobe was weighed and washed in PBS containing PEST (1% penicillin and streptomycin) and fungizone (100 U/ml) and was then kept cool in cold, serum-free Dulbecco’s modified Eagle’s medium (DMEM). To eluate KC and MIP-2 from the liver tissue, 50 mg of the lobe was carefully sectioned and incubated in 1 ml DMEM solution containing 10% fetal calf serum and PEST at 37°C in a 12-well plate for 24 h. The cultured medium was harvested and stored in –20°C until analysis of KC and MIP-2 by using double antibody Quantikine ELISA kits (R & D Systems Europe), using recombinant murine KC and MIP-2 as standards. The minimal detectable protein concentrations are less than 0.5 pg/ml.
Measurement of caspase-3 protease activity
Caspase-3 is an intracellular cysteine protease that becomes activated during the cascade of events associated with and required for the execution of apoptosis. Caspase-3 protease activity in the liver tissue was measured using a caspase-3 colorimetric assay kit (R & D Systems Europe) according to the manufacturer’s instructions. Briefly, after homogenization of whole-liver tissue in cell lysis buffer, homogenates were centrifuged for 1 min at 10,000 g, and the supernatant was incubated with DEVD-peptide nucleic acid (pNA) and reaction buffer for 1.5 h at 37°C. Levels of the chromophore pNA released by caspase-3 activity were quantified spectrophotometrically. The data are given as fold-increases in caspase-3 activity of test livers relative to PBS-treated control livers.
Histology
Samples were taken from the left lobe of liver and fixed in 4% formaldehyde phosphate buffer overnight. Dehydrated, paraffin-embedded, 6-µm sectionswere stained with hematoxylin and eosin and analyzed under light microscopy. The number of extravascular leukocytes was randomly quantified in five high-power fields (x400) and expressed as number of cells/mm2.
Immunohistochemistry
Liver samples were frozen (–70°) overnight in optimal cutting temperature compound (Tissue-Tek, Sakura Finetek Inc., Torrance, CA), sectioned (7 µm), and fixed in acetone. Staining of MIP-2 and KC was performed by the indirect immunoperoxidase method using a horseradish peroxidase–3'-diaminobenzidine tetrahydrochloride kit (CTS008, R & D Systems Europe) according to the manufacturer’s protocol. Tissues were incubated with 15 µg/ml primary antibody (rat anti-mouse MIP-2 and rat anti-mouse KC, R & D Systems Europe). Slides were counterstained with Mayer’s hematoxylin (Sigma Chemical Co.).
Statistical analyses
Data are presented as mean values ± SEM. Statistical evaluations were performed using Kruskal-Wallis one-way ANOVA on ranks followed by multiple comparisons versus control group (Dunn’s method). P < 0.05 was considered significant, and n represents the number of animals.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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It is widely held that infiltration and activation of leukocytes constitute a rate-limiting step in endotoxin-induced liver injury [1
2
3
]. We have recently shown that leukocyte recruitment is a multistep process, initiated by selectin-mediated rolling [3
], mainly P-selectin [4
], in postsinusoidal venules in response to endotoxemia. Moreover, it has been documented that CD18 mediates stationary adhesion of leukocytes to the endothelium in hepatic venules [3
]. In this context, it should be mentioned that some studies have suggested that also the associated sinusoidal sequestration of leukocytes may contribute to hepatocellular liver injury, although a definitive contribution of sinusoidal leukocytes to the tissue damage remains controversial [3
8
24
25
]. An accumulating body of evidence suggests that chemokines are the main group of molecules regulating tissue trafficking of leukocytes in several disease models [10
14
15
16
17
]. Considering that MIP-2 and KC are up-regulated in endotoxemia, as shown herein and previously [18
19
], it is imperative to learn more about the role of these CXC chemokines, not only for a deeper pathophysiologic understanding but also as such knowledge is critical in the development of novel, anti-inflammatory strategies directed against septic liver injury. However, the limited published literature on the role of MIP-2 and KC in endotoxin-induced leukocyte infiltration is complex and partly contradictory. For example, one group has observed that administration of an antibody directed against the CXC chemokine, cytokine-induced neutrophil chemoattractant reduces LPS-provoked neutrophil accumulation in the rat liver [18
], whereas others have reported that immunoneutralization of MIP-2 and KC has no inhibitory effect on LPS-induced recruitment of neutrophils in the liver [20
]. These discrepancies cannot be resolved at present but may be related to the use of different species or antibodies. In the present study, we found that passive immunization against MIP-2 and KC effectively reduced hepatic infiltration of leukocytes. Although pretreatment of mice with the anti-MIP-2 and anti-KC antibodies had no effect on endotoxin-induced leukocyte rolling and adhesion inside the liver microvessles, we found that they abolished (83% reduction) extravascular accumulation of leukocytes in the liver elicited by LPS. One feasible explanation is that the anti-CXC chemokine antibodies break the chemotactic gradient created by hepatocytes in response to endotoxin challenge, which in turn disturbs the extravasation process of leukocytes. Indeed, this notion is supported by our observation that CXC chemokines are mainly expressed in hepatocytes and not on the endothelium in endotoxemia. Thus, these findings suggest that intravascular leukocyte-endothelium interactions are not dependent on CXC chemokines but may be triggered by other leukocyte-activating substances, such as platelet-activating factor and products of the complement system [26
27
]. Alternatively, CXC chemokines may not be sufficiently potent to trigger leukocyte adhesion inside the liver microvasculature. In fact, the latter hypothesis is supported by a previous study reporting that tumor necrosis factor 
and interleukin-1 are markedly more potent inducers of venular leukocyte accumulation than CXC chemokines in the liver [21
].
Tissue recruitment of leukocytes is a key component in immune surveillance and host-defense reactions [28 ]. However, under various circumstances, the activation and infiltration of leukocytes result in tissue damage in certain diseases, including ischemia-reperfusion injury, graft rejection, and endotoxemia [5 ]. In the present study, we found that immunoneutralization of MIP-2 and KC not only abrogated leukocyte extravasation but also decreased hepatocellular injury by 82–94% and restored sinusoidal perfusion, suggesting that CXC chemokine-mediated leukocyte recruitment plays a critical role in septic liver injury. Considering that MIP-2 and KC bind to the same receptor, CXC chemokine receptor 2, it was of interest to test combined inhibition of MIP-2 and KC in this model. We found that blocking both CXC chemokines was similarly effective against endotoxin-induced leukocyte recruitment as inhibition of MIP-2 or KC alone, suggesting that MIP-2 and KC are necessary for the liver phenotype in endotoxemia. A key role of leukocytes has been documented previously by demonstrating that neutrophil depletion markedly attenuates LPS-provoked liver injury [29 ]. A number of mechanisms of neutrophil-mediated tissue injury have been forwarded. For example, neutrophils are potent producers of reactive oxygen species (ROS), such as hydroxyl radicals and superoxide, which exert harmful effects on hepatocytes and endothelial cells in the liver [30 ]. Indeed, we have recently observed that inhibition of ROS production protects against LPS-induced liver injury [31 ]. It is interesting that we also found that ROS regulates CXC chemokine expression in septic liver injury [31 ], emphasizing the importance of oxidant stress in endotoxemia. In this context, it is noticeable that we observed that inhibition of CXC chemokines reduced leukocyte extravasation (83% reduction) without affecting intravascular accumulation of leukocytes. Thus, our data demonstrate that CXC generation induced by endotoxin challenge is mainly important for the transmigration of leukocytes in the liver. These findings are compatible with the concept that extravasation out of the vessels is necessary for leukocytes to cause injurious impact on the liver parenchyma [8 ].
Hepatocyte apoptosis is a prominent and important feature in septic liver injury. In this context, it is interesting to note that one recent investigation has shown that engagement of Fas receptors not only causes cell death but also stimulates local production of CXC chemokines in the liver [32 ]. Moreover, Jaeschke and co-workers [24 ] have forwarded the possibility that endotoxin challenge provokes apoptosis before the onset of leukocyte recruitment. It is interesting that we found that immunoneutralization of MIP-2 and KC significantly decreased endotoxin-induced apoptosis by more than 58% in the liver, indicating that CXC chemokine-mediated neutrophil infiltration contributes to apoptosis in septic liver injury. Considered together, it may be suggested that there is an escalating mechanism in the liver, involving chemokine-induced neutrophil recruitment, which causes apoptosis on one hand, and apoptosis-induced secretion of chemokines, which causes neutrophil infiltration on the other.
Taken together, these novel results demonstrate that CXC chemokines constitute an integral part of the pathophysiology in endotoxemic liver injury by triggering leukocyte extravasation. Inhibition of MIP-2 and KC function attenuated not only tissue recruitment of leukocytes but also preserved microvascular perfusion and reduced hepatocellular injury and apoptosis. Conversely, intravascular accumulation of leukocytes is independent of MIP-2 and KC function. In conclusion, our data indicate that CXC chemokines may be useful targets to protect against excessive inflammation in septic liver injury.
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ACKNOWLEDGEMENTS |
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Received June 27, 2003; revised October 8, 2003; accepted October 21, 2003.
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B-independent, caspase-3-dependent pathway J. Biol. Chem. 276,49077-49082Related Articles
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