HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/7/1230 00-0554fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SINDRAM, D. Articles by CLAVIEN, P.-A. Search for Related Content PubMed PubMed Citation Articles by SINDRAM, D. Articles by CLAVIEN, P.-A.

Synergism between platelets and leukocytes in inducing endothelial cell apoptosis in the cold ischemic rat liver: a Kupffer cell-mediated injury ^{1 ,2}

DAVID SINDRAM^*, ROBERT J. PORTE^*, MAUREANE R. HOFFMAN, REX C. BENTLEY and PIERRE-ALAIN CLAVIEN^*,3

Hepatobiliary and Liver Transplant Laboratory, Departments of
^* Surgery and
Pathology, Duke University Medical Center, Durham North Carolina, USA; and
Department of Visceral and Transplantation Surgery, University of Zurich, Switzerland

³Correspondence: Department of Visceral and Transplantation Surgery, Zurich University Hospital, 8091 Zurich, Switzerland. E-mail: clavien{at}chir.unizh.ch

SPECIFIC AIMS

The first aim of this study was to investigate the effects of platelets and leukocytes on sinusoidal endothelial cell apoptosis in the cold ischemic liver. Second, we studied the role of Kupffer cells (the resident macrophages in the liver) in this type of injury.

PRINCIPAL FINDINGS

1. Leukocytes induce sinusoidal endothelial cell (SEC) apoptosis
The effect of leukocytes on SEC apoptosis was assessed in the isolated perfused rat liver (IPRL) model using livers preserved for the shortest period of time in this model (30 min preservation) and a preservation time corresponding to the limit of graft survival after arterialized orthotopic liver transplantation (24 h). Leukocyte counts in the perfusate decreased by 22 ± 4% after 1 h of reperfusion in livers preserved for less than 30 min. These livers exhibit no detectable injury on H & E-stained biopsies, and aspartate amino transferase levels in the perfusate remained undetectable. Using the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay, we found no evidence of apoptosis in any of these control livers.

A significant increase in the loss of circulating leukocytes was observed in the perfusate when using livers preserved for 24 h. A sharp decrease of circulating leukocytes was seen within 10 min after reperfusion, after which a gradual decrease continued. SEC apoptosis was evaluated in these livers in the presence and absence of leukocytes in the perfusate. Sinusoidal lining cell apoptosis was detected in each experiment. The number of apoptotic cells was sixfold higher in livers reperfused with leukocytes when compared with those reperfused in absence of leukocytes (Fig. 1 ).

View larger version (15K): [in this window] [in a new window]   Figure 1. Number of apoptotic sinusoidal endothelial ells (SEC) in livers preserved for 24 h in cold UW (University of Wisconsin) solution and reperfused for 1 h in the IPRL. Reperfusion of grafts with leukocytes or platelets alone resulted in a significantly higher number of TUNEL positive SEC per high-power field (HPF; 400x) compared with controls (*P<0.01, ANOVA and Student’s t test). A threefold increase in the number of TUNEL-positive cells was noted in the presence of both platelets and leukocytes in the perfusate compared with perfusions with either leukocytes or platelets alone (**P<0.01, ANOVA and Student’s t test). This effect was abrogated by both methods of Kupffer cell inhibition, despite the presence of both platelets and leukocytes in the perfusate (P<0.01, ANOVA and Student’s t test) (L+P: leukocytes plus platelets).

2. Platelets induce SEC apoptosis
Platelet loss was minimal in livers preserved for less than 30 min. Upon reperfusion of 24 h preserved livers, platelets sequestered rapidly into the sinusoids; 29±4% of the platelets was lost from the circulation after 60 min of reperfusion. A sixfold increase in the number of apoptotic SEC was noted in the presence of platelets compared with the experiment without platelets. This figure was comparable to the experiments using only leukocytes in the perfusate (Fig. 1) .

3. Platelets and leukocytes synergistically exacerbate SEC apoptosis
In this set of experiments, livers were reperfused with both leukocytes and platelets. Although no injury was detected in 30 min preserved grafts, livers preserved for 24 h demonstrated increased SEC apoptosis. Compared with experiments using leukocytes or platelets alone, the combination of platelets and leukocytes was associated with a threefold increase in SEC apoptosis in TUNEL stained biopsies (Fig. 1) .

Next, we evaluated the effect of one population of blood elements on the adhesion of the other. The amount of leukocyte adhesion in 24 h preserved livers was comparable in livers perfused with leukocytes alone or with a perfusate containing leukocytes and platelets. Nor was the percentage of platelets lost from the circulation into livers perfused with platelets alone different from experiments using perfusate containing platelets and leukocytes. Leukocytes, but not platelets, contain a nucleus. Therefore, they could undergo apoptosis and contribute to the number of apoptotic cells detected with the TUNEL assay. However, double-labeling experiments excluded apoptosis of leukocyte.

4. Kupffer cells mediate the effects of platelets and leukocytes on SEC apoptosis
Kupffer cells were selectively deleted by intravenous injection of gadolinium chloride 24 h before explantation of the liver. Then the effect of Kupffer cell depletion was evaluated in livers reperfused with platelets and leukocytes. A dramatic reduction in the number of apoptotic SEC was noted in livers from animals treated with gadolinium chloride (Fig. 1) .

Kupffer cell blockade did not interfere with the magnitude of leukocyte adhesion but was associated with a significant decrease in platelet entrapment, reducing the platelet adhesion to levels comparable to nonischemic controls. Whereas the selective destruction of Kupffer cells, may also be associated with transient increased of cytokines and other mediators of inflammation. To further evaluate the Kupffer cells in our model, we pretreated animals with pentoxifylline, a methylxanthine phosphodiesterase inhibitor that effectively inhibits the release of cytokines such as tumor necrosis factor (TNF-), IL-6, and IL-1, and oxygen radicals by Kupffer cells. This drug has been used extensively as a selective Kupffer cell inhibitor. The effects of pentoxifylline on cell adhesion were consistent with the experiments using gadolinium chloride. SEC apoptosis was also significantly reduced in animals pretreated with pentoxifylline (Fig. 1) .

CONCLUSIONS

Three novel findings emerge from this study. First, we have shown that leukocytes, like platelets, induce SEC apoptosis after reperfusion of the cold preserved liver. Second, we found that leukocytes and platelets synergistically exacerbate SEC apoptosis. Finally, we found that Kupffer cells are involved in the mechanisms of injury mediated by leukocytes and platelets.

Our aim was to investigate the role of platelets and leukocytes in mediating reperfusion injury of the cold ischemic liver in a whole organ system and in the absence of coagulation or other plasma factors. Therefore, we used an IPRL model in which all glass components were coated with Teflon^® to prevent cell adhesion and activation by the system itself. To optimize graft oxygenation, a perfusate was chosen based on Krebs-Henseleit buffer and isolated red cells (9% hematocrit). Control experiments without liver in the circuit showed minimal adhesion of leukocytes or platelets and no evidence of platelet activation during 3 h of perfusion. Furthermore, the model was validated by the absence of detectable injury when livers were preserved for a short period (30 min).

In previous experiments we had shown that the adhesion of platelets into 24 h cold preserved liver leads to increased injury of the graft through the induction of SEC apoptosis. Here we studied the effect of leukocyte sequestration and the interaction between leukocytes and platelets on the induction of SEC apoptosis. The data demonstrated that leukocyte adhesion after reperfusion is associated with increased apoptosis of the SEC. Although the degree of apoptosis in the presence of leukocytes alone was similar to experiments using platelets alone, a threefold increase in SEC apoptosis was found when platelets and leukocytes were added to the perfusate simultaneously. These data suggest that platelets and leukocytes act synergistically in the induction of graft injury. We ruled out apoptosis of leukocytes as a source of increased TUNEL staining by performing double staining experiments that label leukocytes with a fluorescent dye and the TUNEL assay. However, the mechanisms by which leukocytes and platelets synergistically induce SEC apoptosis remain unclear. Since platelet and leukocyte adhesion were not increased in the presence of both cells, increased cell adhesion could be excluded as a single reason for this injury.

A close interaction between platelets and leukocytes has been described in inflammation and coagulation. In the present study, we analyzed the effects of leukocytes and platelets as well as their interactions in the ischemic liver. When only leukocytes were added to the perfusate, the degree of leukocyte sequestration into the liver was similar as in previous reports. The addition of platelets into the perfusate did not significantly change the degree of leukocyte adhesion. Similar findings were noted regarding the effects of leukocytes on platelet adhesion in this model. The absence of effects of one circulating element on the adhesion of the other suggests that different binding sites are involved in the adhesion of platelets and leukocytes.

In the current experiments, the deletion of Kupffer cells resulted in a marked reduction in the sequestration of platelets but not of leukocytes. A similar effect of Kupffer cell inhibition on platelet sequestration has been described by others in a model of lipopolysacharide-induced inflammatory injury of the liver. This finding further supports the hypothesis that platelets and leukocytes do not share the same final binding sites. Various adhesion molecules from the selectin and integrin family are involved in the rolling and early adhesion of leukocytes and platelets to the sinusoidal lumen. Studies evaluating the expression of these adhesion molecules may provide further insight into the mechanisms of interaction between platelets, leukocytes, and Kupffer cells.

Studies of inflammatory injury of the liver have shown that Kupffer cells, the resident macrophages of the liver, closely interact with circulating blood cells. Morphologic studies have suggested that Kupffer cells are rapidly activated after reperfusion of the ischemic liver. Of note, the deleterious effects of Kupffer cells have been documented only in models using leukocytes and platelets. In contrast, investigators using models with blood-free perfusate have failed to identify a contribution of Kupffer cells to reperfusion injury. Therefore, we speculated that Kupffer cells interact with platelets and leukocytes in mediating injury. More specifically, we postulated that such an interaction is active in causing SEC apoptosis upon reperfusion of cold preserved liver. We inhibited Kupffer cells using two different modalities: gadolinium chloride, which destroys Kupffer cells; and pentoxifylline, a phosphodiesterase inhibitor that prevents the release of proinflammatory molecules from the Kupffer cells. These two approaches minimize the possibility of effects unrelated to Kupffer cell inhibition.

The effects of leukocytes and platelets on SEC apoptosis were almost completely abrogated after Kupffer cell depletion or inactivation. In addition, we found no detrimental effects of Kupffer cells when platelets or leukocytes were absent from the perfusate (control experiments). These observations possibly explain the existing controversy on the role of Kupffer cells in reperfusion injury of the cold preserved liver. These findings point to a formerly unrecognized triangular interaction between Kupffer cells, platelets, and leukocytes in the mechanisms of reperfusion injury. Platelets and leukocytes need functional Kupffer cells to mediate injury. Similarly, Kupffer cells are harmless in the absence of these cells.

Kupffer cells, for example, are known to release several factors (like oxygen radicals, IL-1, and TNF-) that, after an ischemic period, could further activate already sensitized SEC, leading to increased expression of adhesion molecule. Alternatively, Kupffer cell products like free radicals and TNF- have been shown to directly induce endothelial cell apoptosis. Studies of the processes underlying inflammation have shown that platelets and leukocytes cooperate in the production of various proinflammatory factors, such as platelet factor 4, platelet-activating factor, and cathepsin G. Release of these factors often requires direct cellular contact through selectin or integrin adhesion molecules. These factors can lead to further activation of inflammatory cells, including Kupffer cells, resulting in a self-amplifying loop and an enormous increase in the release of reactive oxygen intermediates and TNF-. The crucial role of cytokines released from Kupffer cells, with a key role for TNF-, should be investigated further as a potential mechanism of SEC apoptosis in the post-ischemic liver.

In summary, we demonstrate that leukocytes and platelets induce SEC apoptosis upon reperfusion of the cold ischemic liver. Platelets and leukocyte enhance this injury in a synergistic manner. The injury also depends on Kupffer cell activation, highlighting the need for active leukocytes, platelets, and Kupffer cells for full expression of the injury. These findings have important implications for the interpretation of previous results and open new avenues for future research. Intervention in the platelet–leukocyte-Kupffer cell interactions might provide new strategies in preventing injury in the ischemic liver.

View larger version (9K): [in this window] [in a new window]   Scheme 1. No caption available..

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0554fje ; to cite this article, use FASEB J. (March 12, 2001) 10.1096/fj.00-0554fje

² The work on leukocyte and platelet synergism was presented at the American Association for the Study of Liver Diseases (AASLD), Dallas, November 1999, and received a AASLD Research Award. The Kupffer cell data was presented during the Plenary session at the AASLD meeting, Dallas, October 2000.

This article has been cited by other articles:

				M. T. de Boer, M. C. Christensen, M. Asmussen, C. S. van der Hilst, H. G. D. Hendriks, M. J. H. Slooff, and R. J. Porte The Impact of Intraoperative Transfusion of Platelets and Red Blood Cells on Survival After Liver Transplantation Anesth. Analg., January 1, 2008; 106(1): 32 - 44. [Abstract] [Full Text] [PDF]

				M. Lesurtel, R. Graf, B. Aleil, D. J. Walther, Y. Tian, W. Jochum, C. Gachet, M. Bader, and P.-A. Clavien Platelet-derived serotonin mediates liver regeneration. Science, April 7, 2006; 312(5770): 104 - 107. [Abstract] [Full Text] [PDF]

				H Suetsugu, Y Iimuro, T Uehara, T Nishio, N Harada, M Yoshida, E Hatano, G Son, J Fujimoto, and Y Yamaoka Nuclear factor {kappa}B inactivation in the rat liver ameliorates short term total warm ischaemia/reperfusion injury Gut, June 1, 2005; 54(6): 835 - 842. [Abstract] [Full Text] [PDF]

				C. Eipel, R. Bordel, R. M. Nickels, M. D. Menger, and B. Vollmar Impact of leukocytes and platelets in mediating hepatocyte apoptosis in a rat model of systemic endotoxemia Am J Physiol Gastrointest Liver Physiol, May 1, 2004; 286(5): G769 - G776. [Abstract] [Full Text] [PDF]

				P. AUKRUST, T. WAeHRE, J. K. DAMAS, L. GULLESTAD, and N. O. SOLUM Inflammatory role of platelets in acute coronary syndromes Heart, December 1, 2001; 86(6): 605 - 606. [Full Text] [PDF]

This Article Full Text (PDF) All Versions of this Article: 15/7/1230 00-0554fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by SINDRAM, D. Articles by CLAVIEN, P.-A. Search for Related Content PubMed PubMed Citation Articles by SINDRAM, D. Articles by CLAVIEN, P.-A.