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Am J Physiol Gastrointest Liver Physiol 293: G663-G672, 2007. First published July 26, 2007; doi:10.1152/ajpgi.00070.2007
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LIVER AND BILIARY TRACT

Hepatic leukocyte recruitment in response to time-limited expression of TNF-{alpha} and IL-1beta

Amanda L. Patrick,1 Jacob Rullo,1 Suzanne Beaudin,2 Patricia Liaw,2 and Alison E. Fox-Robichaud1

1Department of Medicine, McMaster University, and 2The Henderson Research Center, Hamilton, Ontario, Canada

Submitted 8 February 2007 ; accepted in final form 18 July 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS AND MATERIALS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The development of chronic liver diseases is mediated by sustained hepatic inflammation. Our objective was to characterize the molecular mechanisms responsible for the hepatic inflammatory response to time-limited TNF-{alpha} and IL-1beta expression. C57Bl/6 mice were injected with 2 x 107 plaque forming units intraperitoneally of an adenoviral vector containing TNF-{alpha} or IL-1beta (AdTNF-{alpha} or AdIL-1beta). A nonreplicating adenoviral vector served as control. Four days later, under ketamine and xylazine anesthesia, the liver microvasculature was examined by intravital microscopy. In the postsinusoidal venules, leukocyte rolling increased significantly in response to both AdTNF-{alpha} and AdIL-1beta, compared with controls. This response was significantly reduced following injection of an anti-{alpha}4-integrin monoclonal antibody (MAb). Postsinusoidal rolling was further reduced to baseline following injection of an anti-P-selectin or anti-L-selectin MAb. Sinusoidal adhesion was greater in mice treated with AdIL-1beta than with AdTNF-{alpha}. Blocking {alpha}4-integrin, P-selectin, or L-selectin had no significant effect on sinusoidal or postsinusoidal adhesion. In separate experiments, we administered AdTNF-{alpha} or AdIL-1beta to mice deficient in ICAM-1. In ICAM-1–/– mice, postsinusoidal leukocyte rolling significantly increased following expression of IL-1beta but not TNF-{alpha}. AdIL-1beta- but not AdTNF-{alpha}-mediated sinusoidal adhesion was ICAM-1 dependent. AdTNF-{alpha}-induced sinusoidal adhesion was significantly reduced following 4 days of anti-MIP-2 MAb and anti-KC MAb. Prolonged expression of the cytokines TNF-{alpha} and IL-1beta increases hepatic leukocyte-endothelial cell interactions. Interestingly, the mechanisms through which these cytokines bring about adhesion within the sinusoids differ; AdIL-1beta sinusoidal adhesion uses an ICAM-1-dependent mechanism whereas AdTNF-{alpha}-mediated adhesion is ICAM-1 independent but CXC chemokine dependent.

chemokine; hepatic sinusoid; intravital microscopy; adhesion molecules

ENDOTHELIUM-DIRECTED INFLAMMATORY cell recruitment to an extravascular site of infection or injury is a four-stage paradigm involving tethering, rolling, and firm adhesion followed by emigration through cell junctions into the surrounding tissue. Three adhesion molecule families mediate neutrophil-endothelial cell interactions: the selectins, integrins, and members of the immunoglobulin superfamily (18, 28). The mechanisms for leukocyte recruitment have been well described for several vascular beds, including the cremaster muscle, the mesentery, and the dermis of many species. During the initial step of leukocyte recruitment, contact of circulating neutrophils with the endothelium at a site of injury is mediated by the selectins: leukocyte (L), platelet (P), or endothelial (E) selectin. L-selectin is expressed on circulating cells and released upon activation (20). E-selectin is expressed on endothelial cells (31). P-selectin is stored in Weibel-Palade bodies of endothelial cells and platelets, is expressed upon activation, and is the principal selectin mediating in many models of acute inflammation (7). Selectin expression is stimulated by proinflammatory mediators, including bacterial lipopolysaccharide, thrombin, tumor necrosis factor-{alpha} (TNF-{alpha}), and interleukin-1beta (IL-1beta) (9, 22, 49). Selectin-mediated rolling temporarily connects the leukocyte to the endothelial cells; therefore, for arrest and firm adhesion of neutrophils, integrins are upregulated either by a selectin-neutrophil encounter or by presentation of activating molecules (8). However, these principles of leukocyte recruitment may not apply to such organs as the lung (43) or liver (5, 15, 52).

The liver is a unique organ in that it receives not only a blood supply from hepatic arteries but also venous blood returning from the gut via portal veins, which drain into the sinusoids before terminating in the central (postsinusoidal) venules. Surrounding the sinusoids are endothelial cells that form a fenestrated sheath above the space of Disse, facilitating the exchange of metabolites and nutrients between the lumen and the liver parenchyma. Infectious agents can also be transported from the gut via portal blood to the liver, necessitating specialized mechanisms for immediate immune responses such as resident lymphocyte and macrophage populations (Kupffer cells) within the lumen of the sinusoids (6). Although there have been several studies investigating the molecular mechanisms in complex inflammatory models such as ischemia-reperfusion (14, 53), bile duct ligation (19), or endotoxin plus galactosamine-induced fulminate hepatic failure (10–12), our understanding of molecular mechanisms used by individual proinflammatory mediators is incomplete. In a previous study Fox-Robichaud and Kubes (15), using a single injection of TNF-{alpha}, demonstrated that both venular beds as well as the sinusoids support leukocyte endothelial interactions. Using mice deficient in P-selectin or both P- and E-selectin, they demonstrated that leukocytes use {alpha}4-integrin independent of selectins within the venules. However, they were unable to determine the mechanisms for leukocyte recruitment within the sinusoids. Less is known about the molecular mechanisms responsible for IL-1beta-mediated inflammation, particularly within the liver microcirculation.

Although TNF-{alpha} and IL-1beta are biochemically unique proteins, these monocyte-macrophage-derived cytokines elicit similar hormonelike activities on target cells that trigger proinflammatory events vital to host defense (29, 42). These cytokines act in conjunction with each other to produce clinical signs of a systemic inflammatory response, such as seen in severe sepsis, and may contribute to the organ dysfunction in early severe sepsis (4). Although the eventual proinflammatory reaction may be similar for these cytokines, it is likely that the exact molecular mechanism by which these molecules recruit leukocytes is different. In contrast to previous work (15), we compared the effects of sustained expression of TNF-{alpha} or IL-1beta on the liver of mice using adenovirus-mediated gene transfer. Adenoviral vectors produce a limited expression of the gene of interest over a period of days and thus this model is more clinically relevant to the study of the sustained inflammation such as in sepsis or chronic liver disease. We used intravital microscopy to visualize hepatic microcirculation using both specific monoclonal antibodies and adhesion molecule-deficient mice, to determine the molecular mechanisms mediating this leukocyte recruitment.


   METHODS AND MATERIALS
TOP
 ABSTRACT
 METHODS AND MATERIALS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Male C57Bl/6 mice (20–30 g body wt), Helicobacter hepaticus free, were obtained from Taconic (Germantown, NY). Male mice deficient in P-selectin were originally generated as described in Ref. 38 (Jackson Laboratories, Bar Harbor, ME). ICAM-1-deficient mice were originally generated as described in Ref. 48 (Jackson Laboratories). Both P-selectin- and ICAM-1-deficient animals have a C57BL/6 background; therefore male C57BL/6 mice (Taconic) served as controls. The animals had free access to certified irradiated food (Harlan Teklab 7904) and water and were housed in specific pathogen-free facilities. Animal protocols were approved by the University of McMaster Animal Care Committee and met the Canadian Guidelines for Animal Research.

Adenovirus. Replication-deficient type 5, E1-deleted, and E3-deficient adenoviral gene delivery vectors were used to deliver the cytokines to the peritoneum. Human TNF-{alpha} and IL-1beta genes were inserted into the E1 region of the viral constructs (AdTNF-{alpha} and AdIL-1beta, respectively). Human TNF-{alpha} is known to bind to the murine TNF receptor 1 and is capable of inducing hepatotoxicity in high doses (30). Similarly, human IL-1 binds to the murine IL-1 receptors (41) and produces expected hepatocellular responses (26). A blank E1 region vector (AdDL70) was used as control. The construction of the adenoviral vectors (AdTNF-{alpha}, AdIL-1beta, and AdDL70) has been previously described (2, 27, 36). They were a kind gift from Dr. Jack Gauldie (Centre for Gene Therapeutics, McMaster University).

Intravital microscopy. Mice were anesthetized with a mixture of ketamine (200 mg/kg ip) and xylazine (10 mg/kg ip). The right jugular vein was cannulated to maintain anesthesia and for injection of monoclonal antibodies. The abdomen was shaved and opened by a midline incision extending close to the costal margin. Mice were then placed on a Plexiglas microscope stage in a left lateral position and the left lobe of the liver was gently expressed onto the stage. To prevent dehydration, the liver was covered with plastic wrap (Saran Wrap). Body temperatures were maintained by use of an infrared heat lamp and a heated water-circulating jacket built into the Plexiglas stage and monitored via an intrarectal temperature probe (Traceable).

Hepatic vascular beds, specifically the sinusoidal and postsinusoidal venules, were visualized by microscope (Zeiss Inverted Axiovert S100) under a x40 objective lens. Images of the hepatic microcirculation were captured with an attached camera (Newvicon, DAGE-MTI) and projected onto a monitor (Panasonic CT-2086YD). The images were recorded on a videocassette recorder (Panasonic PV-VS4820-K) for offline analysis. Within each field of view, ~6–10 sinusoids were observed or one postsinusoidal venule. The number of rolling and adherent leukocytes was quantified during video playback analysis. Leukocytes considered rolling were defined as those tethering to a given venule with a torsional motion. Adherent leukocytes were defined as those that adhered to the venular or sinusoidal endothelium and remained stationary for a minimum of 30 s. Following isolation of postsinusoidal or sinusoidal vessels, images were recorded at 0 and 10 min for a duration of 2 min at each time point to obtain an average of cells per minute per field of view.

Following completion of intravital microscopy, blood was collected from the right ventricle via cardiac puncture into a heparinized syringe. Blood obtained was allowed to clot at room temperature. Remaining whole blood was centrifuged at 13,000 rpm for 10 min and the serum was removed and stored at –70°C for cytokine analysis. Total leukocyte count was determined with a sample of whole blood mixed with 3% acetic acid and 1% crystal violet (5:44:1) in a hemocytometer. Tissue samples of liver were frozen in liquid nitrogen or fixed in 10% phosphate-buffered formalin.

Serum chemistry. To assess for potential hepatocellular injury or dysfunction blood was collected into neonatal collection tubes to determine serum alanine aminotransferase (ALT) and bilirubin levels. Serum chemistry was performed at the Hamilton Regional Laboratory Medicine Program in the department of Laboratory Medicine at the McMaster Medical Centre with standard automated equipment.

Experimental plan. In the first set of experiments, the first group (n = 6) of C57BL/6 mice received an intraperitoneal injection of AdTNF-{alpha}. The second group (n = 6) received AdIL-1beta and the third group (n = 6) received null control virus (AdDL70) for comparison. All were administered adenovirus at a dose of 2 x 107 plaque-forming units (pfu) in 100 µl sterile saline on day 0. Animals were studied at day 4 postinjection. In a subsequent experiment, following AdTNF-{alpha} or AdIL-1beta injection, at 4 days mice (n = 3) received 50 µg iv monoclonal antibody (MAb) anti-{alpha}4-integrin (R1-2, BD Biosciences, Mississauga, ON, Canada), following baseline assessment. A 20-min response time was given before vessels were recorded. The experiment was repeated (n = 4) using a different anti-{alpha}4-integrin clone (PS/2, 50 µg iv, Southern Biotech, Birmingham, AL) to test the validity of R1-2 in response to AdTNF-{alpha} at 4 days. Following recordings of the response to anti-{alpha}4-integrin clone (PS/2), anti-L-selectin MAb (MEL-14, 3 mg/kg iv, BD Biosciences) was introduced and the microcirculation was recorded 20 min later. For comparison, in a separate experiment (n = 3), 4 days following intraperitoneal AdTNF-{alpha} or AdIL-1beta injection, 20 µg of P-selectin MAb (CD62P, clone RB40.34, BD Biosciences) was injected immediately following initial baseline recording. The response to the P-selectin antibody was recorded 5 min later. To confirm the potential importance of P-selectin, we also examined the response to our adenoviral vectors in P-selectin-deficient mice as above.

ICAM-1-deficient mice. Viral response in ICAM-1-deficient mice (n = 4 AdTNF-{alpha},n = 4 AdIL-1beta, n = 3 AdDL70) was observed at 4 days postinjection. Following baseline assessment, these mice received 50 µg iv MAb {alpha}4-integrin (R1-2). A 20-min response time was given before vessels were recorded. Subsequently, anti-P-selectin MAb was injected (RB40.34, 20 µg iv) immediately following {alpha}4-integrin response recordings. Similarly, E-/P-/L-selectin-deficient mice (n = 4 AdTNF-{alpha},n = 4 AdIL-1beta, n = 4 AdDL70) and their controls (n = 3 AdTNF-{alpha},n = 4 AdIL-1beta, n = 5 AdDL70) received 50 µg iv anti-{alpha}4-integrin MAb following baseline assessment at 4 days.

In a subsequent experiment, ICAM-1-deficient animals (n = 3) initially received a dose of AdTNF-{alpha} (2 x 107 pfu) and were subsequently injected with an anti-MIP-2 (macrophage inflammatory protein-2, murine CXCL2) MAb (40 µg ip, R&D Systems, Hornby, ON, Canada) with anti-KC (keratinocyte chemokine, murine CXCL1) MAb (40 µg ip, R&D Systems) for 4 days and were studied on day 4.

Cytometric bead array. All protocols followed the instructions of the BD Cytometric Bead Array (CBA) (BD Biosciences), to generate a cytokine profile for serum and liver cell samples. All livers collected were stored at –70°C before cytokine analysis. A specific amount of lysis buffer (49 parts BD Insect Cell Lysis Buffer to 1 part Protease Inhibitor Cocktail, BD Biosciences) was added to each sample based on tissue weight (mg). The liver sample was homogenized for 1 min and spun in a microcentrifuge at 13,000 rpm for 15 min, the supernatants were collected, and the remaining pellets were discarded. Liver homogenates were filtered through a 0.2-nm filter (Pall). The CBA was used to capture and quantify an array of soluble analytes by using a fluorescence-based flow cytometry (Becton Dickenson FACSCalibur) and a dual laser (488-nm and 635-nm excitation wavelength). We used a Mouse Inflammation Kit, which is able to simultaneously measure IL-6, IL-10, MCP-1, IFN-{gamma}, TNF-{alpha}, and IL-12p70. Data was analyzed by BD Biosciences Immunocytometry Systems Analysis Software.

ELISA. For quantitative determinations of TNF-{alpha} and Il-1beta concentrations in blood serum and liver cells, serum and liver homogenates were analyzed by a Human TNF-{alpha}/TNFSF1A Quantikine HS ELISA (3rd Generation, catalog number HSTA00C) and a Human IL-1beta/IL-1F2 Quantikine HS ELISA Kit, 2nd Generation, respectively (R&D Systems).

Statistical analysis. Data is expressed below as means ± SD, and n is the number of animals per experimental group. Statistical differences between the experimental groups were calculated by using computer software packages (Excel and KaleidaGraph 3.6) with Student's t-test (assuming equal variances) and ANOVA with Bonferroni's correction for multiple comparisons. Statistical significance was set at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Adenoviral dose and inflammatory response. Our initial challenge was to find the correct adenoviral dose of AdTNF-{alpha} that stimulated an immune response within the hepatic microvasculature without causing hepatic ischemia. A dose-response curve was generated to determine the optimal dose. Viral dose treatments ranged from 5 x 106 to 5 x 108 pfu ip. At 5 x 106 and 1 x 107 pfu the hepatic leukocyte-endothelial cell interactions were not above baseline (i.e., naive) in all the vascular beds. Doses above 5 x 107 pfu all caused hepatic ischemia with increasing areas of pale necrosis on gross examination and absence of microcirculatory flow when examined by intravital microscopy (data not shown). An AdTNF-{alpha} dose of 2 x 107 pfu proved optimal at 4 days. Although the adenoviral vector expressing IL-1beta did not cause ischemia up to 5 x 108 pfu, we chose to use equivalent pfu for control vector and both cytokines.

Human TNF-{alpha} and IL-1beta were present in the liver and serum at 4 days. Wild-type mice showed higher levels of expression of TNF-{alpha} than ICAM-1-deficient mice (Table 1). Liver expression of both cytokines was also greater than serum levels in wild-type and knockout mice. The mouse inflammation panel CBA was used to screen for secondary cytokine and chemokine production. Levels of the CC chemokine MCP-1 were increased in the liver of adenoviral-treated mice (AdDL70: 54.2 ± 5.5 pg/ml, AdTNF-{alpha}: 125.5 ± 70.5 pg/ml, AdIL-1beta: 23.7 ± 30.0 pg/ml). Although the amount of MCP-1 produced in the liver was significantly greater in response to AdTNF-{alpha} treatment compared with AdIL-1beta, neither was significantly different from the AdDL70-treated mice. There was no IL-6, IL-10, IFN-{gamma}, or IL-12p70 produced in the liver at 4 days in all groups (data not shown).


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  		Table 1. Expression of human TNF-{alpha} and human IL-1beta in the serum and liver

 
White blood cell counts were significantly higher in AdIL-1beta- and AdTNF-{alpha}-stimulated C57BL/6 and ICAM-1-deficient mice compared with their respective controls. Interestingly, all mice treated with AdIL-1beta showed higher leukocyte counts than AdTNF-{alpha}-stimulated mice (Table 2). This data suggests that both cytokine-containing adenoviral vectors caused a small but significant increase in systemic leukocyte counts.


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  		Table 2. Leukocyte counts and serum biochemistry

 
To assess for evidence for hepatocellular injury and hepatocellular dysfunction, we measured serum ALT and bilirubin levels, respectively. As shown in Table 2, all groups of mice were within the normal distribution of ALT levels, suggesting no hepatocellular injury with our dose of adenoviral vectors. Serum bilirubin levels were elevated outside the normal range in the AdDL70- and AdIL-1beta- but not AdTNF-{alpha}-treated mice, suggesting some element of hepatocellular dysfunction with the adenoviral treatment.

Leukocyte-endothelial cell interactions in response to AdTNF-{alpha} and AdIL-1beta in C57BL/6 mice. Leukocyte-endothelial cell interactions were increased in the hepatic microcirculation 4 days post-AdTNF-{alpha} and AdIL-1beta treatment. As shown in Fig. 1A, leukocyte rolling flux increased significantly in response to both TNF-{alpha} and IL-1beta compared with controls. In addition, the leukocyte rolling was greater in mice treated with vector expressing TNF-{alpha} than those injected with AdIL-1beta. There was, however, no significant difference in central venular adhesion between the mice given the control vector and the mice receiving either AdTNF-{alpha} or AdIL-1beta (Fig. 1B). Within the sinusoids adhesion significantly increased in response to both TNF-{alpha} and IL-1beta containing adenoviral vectors compared with controls at 4 days. Although expression of both cytokines increased adhesion, IL-1beta stimulation caused a significantly greater response (Fig. 1C).


Figure 1
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  		Fig. 1. Hepatic leukocyte endothelial cell interactions in response to AdTNF-{alpha} and AdIL-1beta in C57BL/6 mice at 4 days posttreatment. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; +P < 0.05 compared with AdTNF-{alpha}.

 
Overlapping roles of {alpha}4-integrin and P-selectin in AdTNF-{alpha}-treated C57BL/6 mice. The increase in leukocyte flux seen in the postsinusoidal vessels in response to AdTNF-{alpha} treatment was reduced by 60% following injection of an anti-{alpha}4-integrin MAb (R1-2) and completely eliminated by following injection of an anti-P-selectin Ab (Fig. 2A). Consistent with previous findings using a single TNF-{alpha} injection (15), AdTNF-{alpha}-induced sinusoidal adhesion was not reduced by anti-{alpha}4-integrin or anti-P-selectin treatment (Fig. 2C). The postsinusoidal venular leukocyte adhesion is shown for completeness (Fig. 2B).


Figure 2
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  		Fig. 2. Hepatic leukocyte endothelial cell interactions in AdTNF-{alpha}-treated C57BL/6 mice in response to anti-{alpha}4-integrin MAb (R1-2) and anti-P-selectin MAb (RB40.34). Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; +P < 0.05 compared with AdTNF-{alpha}.

 
Validity of anti-{alpha}4-integrin R1-2 clone and role of L-selectin in AdTNF-{alpha}-induced C57BL/6 mice. Given some concerns about the mechanisms by which the anti-{alpha}4-integrin clone R1-2 may alter the integrin (34), we repeated these experiments using the other available MAb (PS/2). The increase in leukocyte flux seen in the postsinusoidal vessels in response to AdTNF-{alpha} treatment was again significantly reduced following injection of the anti-{alpha}4-integrin MAb (PS/2) (Fig. 3A). The addition of a monoclonal antibody against L-selectin (MEL-14) following PS/2 injection was used to determine whether there were additional overlapping roles for the selectins and {alpha}4-integrin for leukocyte rolling as we saw with P-selectin. AdTNF-{alpha}-induced postsinusoidal rolling was further significantly reduced when the anti-L-selectin antibody was added to the {alpha}4-integrin MAb (Fig. 3A). Postsinusoidal adhesion is again shown for completeness (Fig. 3B). Similarly, AdTNF-{alpha}-induced sinusoidal adhesion was not reduced following injection of the second MAb against {alpha}4-integrin or with the addition of the L-selectin MAb (Fig. 3C). Finally, we confirmed the importance of P-selectin in the AdTNF-{alpha}-induced hepatic leukocyte rolling by repeating these experiments in the P-selectin-deficient mice (Fig. 4). There was no postsinusoidal venular rolling (Fig. 4A) and postsinusoidal adhesion was at baseline levels (Fig. 4B). Sinusoidal adhesion was not significantly different between AdTNF-{alpha}-treated P-selectin-deficient mice compared with the AdTNF-{alpha}-treated C57Bl/6 mice (Fig. 4C).


Figure 3
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  		Fig. 3. Hepatic leukocyte endothelial cell interactions in response to anti-{alpha}4-integrin (PS/2) and anti-L-selectin in AdTNF-{alpha}-treated C57Bl/6 mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; #P < 0.05 compared with AdTNF-{alpha}; +P < 0.05 compared with AdTNF-{alpha} + PS/2.

 

Figure 4
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  		Fig. 4. Hepatic leukocyte endothelial cell interactions in response to AdTNF-{alpha} in P-selectin-deficient mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls.

 
Overlapping roles of {alpha}4-integrin and P-selectin in AdIL-1beta-induced C57BL/6 mice. To determine whether the overlapping role for the ligands mediating leukocyte rolling was cytokine specific, we repeated these experiments using AdIl-1beta. As shown in Fig. 5A, leukocyte rolling in the postsinusoidal venules in response to AdIL-1beta was significantly reduced following intravenous injection of the {alpha}4-integrin MAb (R1-2) and eliminated following the addition of the anti-P-selectin MAb. Neither central venular adhesion (Fig. 5B) nor sinusoidal adhesion (Fig. 5C) was affected by the addition of the two antibodies.


Figure 5
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  		Fig. 5. Hepatic leukocyte endothelial cell interactions in response to anti-{alpha}4-integrin (R1-2) and anti-P-selectin (RB40.34) in AdIL-1beta-treated C57Bl/6 mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; +P < 0.05 compared with AdIL-1beta.

 
Role of ICAM-1 in AdTNF-{alpha}- and Ad IL-1beta-induced leukocyte-endothelial interactions. As shown above, in both the AdTNF-{alpha}- and AdIL-1beta-treated mice there was no evidence that sinusoidal adhesion was dependent on VCAM-1 (the principal ligand for {alpha}4-integrin). We therefore turned our attention to the role of ICAM-1. Similar to the response seen in the C57Bl/6 mice, leukocyte rolling in the ICAM-1-deficient mice increased in response to AdTNF-{alpha} (Fig. 6A). As before, this rolling was still dependent on both {alpha}4-integrin and P-selectin. There was no leukocyte adhesion in the postsinusoidal vessels of the ICAM-1-deficient mice (Fig. 6B). However, sinusoidal leukocyte adhesion in the ICAM-1-deficient mice was no different from the wild-type mice (Fig. 6C), suggesting that ICAM-1 was not responsible for the AdTNF-{alpha}-induced response in the sinusoids.


Figure 6
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  		Fig. 6. Hepatic leukocyte endothelial cell interactions in response to anti-{alpha}4-integrin (R1-2) and anti-P-selectin (RB40.34) in AdTNF-{alpha}-treated ICAM-1 deficient (ICAM-1–/–) mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls, +P < 0.05 compared with AdTNF-{alpha} in ICAM-1–/– mice; #P < 0.05 compared with AdTNF-{alpha} in ICAM-1–/– mice + R1-2.

 
In contrast, when AdIL-1beta was given to the ICAM-1-deficient mice the flux of rolling leukocytes in the postsinusoidal venules greatly increased compared with the wild-type mice given AdIL-1beta. As shown in Fig. 7A, the subsequent administration of the {alpha}4-integrin MAb reduced 85% of the rolling interactions and the addition of anti-P-selectin antibody completely eliminated them. Central venular adhesion was again absent in the ICAM-1-deficient mice (Fig. 7B). Finally, in contrast to the results with AdTNF-{alpha}, sinusoidal leukocyte adhesion in the ICAM-1-deficient mice given the vector containing IL-1beta was significantly decreased compared with wild-type controls (Fig. 7C). This reduction in sinusoidal leukocyte adhesion in the ICAM-1-deficient mice likely accounts for the subsequent marked increase in leukocyte rolling in the downstream central venules seen in Fig. 7A.


Figure 7
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  		Fig. 7. Hepatic leukocyte endothelial cell interactions in response to anti-{alpha}4-integrin (R1-2) and anti-P-selectin (RB40.34) in AdIL-1beta-treated ICAM-1–/– mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; &P < 0.05 compared with AdIL-1beta-treated C57BL/6 mice; +P < 0.05 compared with AdIL-1beta-treated ICAM-1–/– mice; #P < 0.05 compared with AdIL-1beta in ICAM-1–/– mice + R1-2.

 
Role of MIP-2 and KC in AdTNF-{alpha}-mediated sinusoidal adhesion. Because the AdTNF-{alpha}-mediated sinusoidal leukocyte was not dependent on either ICAM-1 or {alpha}4-integrin, we examined the possibility that the recruitment was independent of the traditional adhesion molecules. Recent work by Li and colleagues (32) and ourselves (H. Kumar Ondiveeran, H. Fong, and A. Fox-Robichaud, unpublished observations) has suggested that leukocyte recruitment within the sinusoids may occur directly through the chemokines. With that knowledge, we tested the hypothesis that the AdTNF-{alpha}-mediated sinusoidal adhesion was dependent on the CXC chemokines MIP-2 and KC. As shown in Fig. 8, ICAM-1-deficient mice were given daily injections of MAbs against MIP-2 and KC. Postsinusoidal venular leukocyte rolling was not reduced with the addition of the chemokine antibodies (Fig. 8A). As shown above, the ICAM-1-deficient mice have minimal venular adhesion (Fig. 8B). However, for the first time, sinusoidal adhesion in the AdTNF-{alpha}-treated mice was reduced to the same level as control vector (Fig. 8C). This suggests that the low-flow hepatic sinusoid chemokines, likely presented on the endothelial surface, are capable of causing leukocyte arrest without the need for the "traditional" adhesion molecules.


Figure 8
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  		Fig. 8. Hepatic leukocyte endothelial cell interactions in response to a 4-day treatment of anti-MIP-2 and anti-KC MAbs in AdTNF-{alpha}-treated ICAM-1–/– mice. Shown are leukocyte rolling (A) and adhesion (B) in the hepatic postsinusoidal venules and leukocyte adhesion in sinusoids (C). *P < 0.05 compared with AdDL70 controls; #P < 0.05 compared with AdTNF-{alpha}-treated ICAM-1–/– mice.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS AND MATERIALS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We have used time-limited expression of two common proinflammatory cytokines, TNF-{alpha} and IL-1beta, to examine their effects on the hepatic microcirculation. When examining mechanisms of leukocyte recruitment into vascular beds, most studies with a particular cytokine usually employ a single injection whereas clinically cytokine expression occurs over a period of hours to days. An alternative method to investigate cytokine expression is the use of adenoviral vectors for viral-mediated gene transfer therapy. However, it has been established that the effectiveness may be limited by host immune defenses, demonstrated by rapid neutrophil-endothelial interactions induced by the adenoviral vectors (33). In our study, we chose a concentration of vector that is two- to fourfold lower than is usually reported. The result was that at the end of our 4 days of treatment we found no rise in systemic leukocyte counts and no evidence of hepatocellular injury with the adenoviral vector. Having demonstrated the presence of the human cytokine gene product in both the circulation and the liver, we proceeded to investigate the molecular mechanisms of the cytokine-mediated hepatic inflammatory response.

The supposition has been that TNF-{alpha} and IL-1beta produce an equivalent immune response using the same molecular mechanisms. Evidence suggests that the cytokines TNF-{alpha} and IL-1beta induce chemotactic activity and emigration of neutrophils and act synergistically to produce clinical signs of sepsis despite binding to different cellular receptors (50). Subsequently, TNF-{alpha} and IL-1beta also activate leukocytes to produce a cascade of anti-inflammatory cytokines, such as IL-13, IL-10, and TGF-beta (40, 46). In this study we describe leukocyte-endothelial cell interactions primarily within the sinusoids and to a lesser extent within postsinusoidal venules of the hepatic microvasculature. We have shown that both AdTNF-{alpha} and AdIL-1beta elicit an immune response as shown by the increased leukocyte rolling in the postsinusoidal venules and firm adhesion within the sinusoids. This observation of leukocyte adhesion primarily within the hepatic sinusoids may be a limitation of our low doses of vector or a result of the intraperitoneal (rather than intravenous; Ref. 33) injection. It may also be a reflection of the chronic expression of a single cytokine, in contrast to other models of hepatic injury in which multiple cytokines are activated. At our chosen dose of vector, AdTNF-{alpha}-induced rolling was higher in the postsinusoidal venules compared with the AdIL-1beta immune responses. We have also shown that sinusoidal adhesion was higher in response to AdIL-1beta at this low dose of vector. Interestingly, the molecular mechanisms through which these cytokines bring about the firm adhesion within the sinusoids differ.

Adhesion molecule ICAM-1 plays different roles in the hepatic microcirculation in response to the two cytokines. In other tissues, ICAM-1 is upregulated and expressed on the endothelium following stimulation by proinflammatory mediators TNF and IL-1beta (16). It is the recognition and subsequent ligation of the beta2-integrins to ICAM-1 that ceases rolling and initiates firm adhesion of leukocytes prior to transmigration (23). In models of hepatic injury such as fMLP (formyl-Met-Leu-Phe) (52), bile duct ligation (19), ischemia-reperfusion (14), and endotoxin plus galactosamine-induced fulminate hepatic failure (11), ICAM-1 is implicated in sinusoidal leukocyte adhesion. In those models, there is, however, stimulation of multiple cytokines. In our study, AdTNF-{alpha}-induced adhesion within the sinusoids used an ICAM-1-independent mechanism whereas AdIL-1beta-induced leukocyte adhesion was dependent on ICAM-1. This is in contrast to an in vitro study examining the role of TNF on isolated rat sinusoidal endothelial cells (39) but consistent with work by others that suggests that ICAM-1 induction in the liver may differ depending on the stimuli (13, 51). Moreover, there was no difference in leukocyte adhesion in TNF-{alpha}-stimulated ICAM-1-deficient mice and wild type (31).

We then searched for an alternative mechanism for the AdTNF-{alpha}-mediated sinusoidal adhesion. In vitro work had previously suggested that chemokines such as MCP-1 and IL-8, under low shear, are capable of firmly adhering leukocytes in the absence of "traditional" adhesion molecules (17), and it is now known that certain chemokines, such as IL-8, are likely presented on the endothelial surface, attached to proteoglycans such as heparan sulfate and syndecan (21, 45, 37). Recent work in our laboratory (H. Kumar Ondiveeran, H. Fong, and A. Fox-Robichaud, unpublished observations) had shown CXC chemokine-dependent adhesion in the sinusoids of septic mice or mice given a single injection of TNF-{alpha}. This direct adhesive effect of the CXC chemokines is supported by data from Li and colleagues using the endotoxin plus galactosamine model of liver failure (32) as well as in the lung (1) and kidney (44) injury models. In the present study we injected ICAM-1-deficient mice with MAbs against the murine CXC chemokines MIP-2 (CXCL2) and KC (CXCL1) over the 4 days of AdTNF-{alpha} expression. This resulted in a reduction in sinusoidal leukocyte adhesion to control levels, demonstrating that in the low shear of the hepatic sinusoids, chemokines can arrest leukocytes without the need for selectins and independent of integrins. This agrees with recent work by Bonder and colleagues (3) demonstrating that other leukocytes such as Th2 lymphocytes can use novel adhesion molecules, in that instance VAP-1, to adhere within the hepatic sinusoids.

This study also identifies the overlapping roles for selectins and {alpha}4-integrin in postsinusoidal rolling during prolonged cytokine expression. Upon activation, surface expression of the P-selectin protein on endothelial cells occurs within minutes, indicating that P-selectin is involved in early mediation of leukocyte recruitment. In previous work using a single injection of TNF-{alpha}, leukocyte rolling in the portal and central venules was independent of P-selectin but dependent on {alpha}4-integrin (15), a molecule expressed on the surface of stimulated neutrophils (24) that has been shown to support nonselectin leukocyte rolling. In our study, introduction of the anti-{alpha}4-integrin MAb did not completely abolish leukocyte rolling within the postsinusoidal venules in response to AdTNF-{alpha} or AdIL-1beta, suggesting partial dependence on {alpha}4-integrin. However, lack of P-selectin inhibited all rolling, indicating that the rolling was predominately mediated by P-selectin. Kerfoot and Kubes (25) showed a similar overlapping role for P-selectin and {alpha}4-integrin within the brain microcirculation. Following anti-{alpha}4-integrin Ab treatment, we found up to a 60% reduction of leukocyte rolling with cessation of all rolling following the addition of anti-P-selectin whereas anti-P-selectin could block all rolling in the absence of anti-{alpha}4-integrin. Our data with ICAM-1-deficient mice suggests that AdTNF-{alpha}-elicited leukocyte rolling along the venules is also dependent on L-selectin. Although blocking P-selectin, L-selectin, and {alpha}4-integrin reduced rolling, only the lack of ICAM-1 was able to reduce venular adhesion completely. In contrast, it has been reported that P-selectin-dependent rolling is a precondition for leukocyte adhesion in TNF-{alpha}-stimulated cremaster muscle microcirculation (35). Leukocyte adhesion in the postsinusoidal venules was at baseline levels prior to antibody introduction, which could account for not visualizing any effect of anti-P-selectin. Our data does suggest, however, that sinusoidal leukocyte adhesion in a TNF-{alpha}- or IL-1beta-elicited immune response is not dependent on P-selectin, L-selectin, or {alpha}4-integrin.

Our study focused on the molecular mechanisms in response to these cytokines in the hepatic microcirculation only. However, following cytokine stimulation the nonadherent leukocytes exiting the liver venules will be still activated as they migrate into the pulmonary microvasculature. In a murine model examining the microvessels of a revascularized lung allograft, Sikora and colleagues (47) have shown that receptors for L- and P-selectin (alone and in combination with E-selectin) play a predominant role in TNF-{alpha}-induced leukocyte rolling in the lung microcirculation whereas {alpha}4-integrin played only a small part. The downstream effects of leukocyte activation in our model needs to be examined further.

In conclusion, our model provides a method to study the effects of particular cytokines within the hepatic microcirculation. Although the study is limited to a single dose of vector, on the basis of our initial responses to AdTNF our study demonstrates that cytokines TNF-{alpha} and IL-1beta elicited an immune response as shown by the increased leukocyte rolling in the postsinusoidal venules and firm adhesion in the sinusoids. TNF-{alpha}- and IL-1beta-induced postsinusoidal leukocyte rolling is principally mediated by P-selectin; however, {alpha}4-integrin was shown to play a partial role. TNF-{alpha}-induced hepatic response within the sinusoids uses an ICAM-1-independent, CXC chemokine-dependent mechanism whereas IL-1beta-induced leukocyte adhesion is dependent on ICAM-1.


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This work was supported by a grant from the Canadian Institutes of Health Research. A. Fox-Robichaud is supported by a Career Research Award from Rx & D Health Research Foundation and the Canadian Institutes of Health Research.


   FOOTNOTES
 

Address for reprint requests and other correspondence: A. Fox-Robichaud, Dept. of Medicine, HSC 4N52, McMaster Univ., 1200 Main St. West, Hamilton, ON, Canada L8N 3Z5 (e-mail: afoxrob{at}mcmaster.ca)

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.


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