SHOCK, Vol. 19, No. 4, pp. 394–396, 2003

Letters to the Editor
involved in the direct adhesion of neutrophils to hepatocytes(7). The latter mechanism may be more relevant when theendothelial cell barrier is damaged, e.g., during hepatic ischemia-reperfusion injury (8).
In a recent issue of SHOCK, Sakamoto and coworkers (9)confirmed these previous in vivo findings. Using a controlledin vitro flow system, the authors studied the interactions of
TNF-stimulated cultured sinusoidal endothelial cells with activated neutrophils obtained from a peritonitis experiment. The
principal findings were that endotoxin stimulated Kupffer cells
to generate TNF-␣. This cytokine was then responsible forICAM-1 upregulation on the endothelial cells. Although
ICAM-1 expression supported increased neutrophil adhesion
and transmigration, all changes were less than 100% of baseline values, which is small compared with in vivo changes of>3000% during endotoxemia (3, 4). One reason for the limited
effects may be, in part, that ICAM-1 is constitutively expressedon sinusoidal endothelial cells. This low-level ICAM-1 expression may be sufficient to support adhesion and transmigrationof activated, i.e., ␤2 integrin-expressing, neutrophils. This
interpretation is supported by the findings that the antiICAM-1 antibody reduced neutrophil adhesion and transmigration below baseline levels. Another issue is that isolated
neutrophils had been subjected to chemotactic stimulation,adhesion, and transmigration in vivo several hours before theactual in vitro experiment. This may have affected neutrophil
behavior under these conditions. Furthermore, it is surprisingthat neutrophils transmigrate at all in these in vitro experiments
without the presence of a chemotactic gradient. These data arein contrast to in vivo findings where transmigration does notoccur without a signal from the extravascular space (5, 6).
The most controversial data are the increased adhesion ofneutrophils with increased ICAM-1 expression on sinusoidal
endothelial cells and the reversal of these effects by antibodies
against TNF or ICAM-1. Sakamoto et al. (9) concluded fromthese findings that “ICAM-1 may play a major role in thehepatic infiltration of neutrophils during endotoxic liver
injury.” I respectfully disagree with this conclusion. The data
only suggest that under the current experimental conditions invitro, ICAM-1 supports the adhesion of approximately
50%–60% of neutrophils. It remains unclear how the other
40%–50% of neutrophils adhere to these endothelial cells.
Furthermore, extrapolations from such in vitro experiments tothe in vivo situation are extremely difficult. The liver has two
principal sites of neutrophil accumulation. Neutrophils canadhere in sinusoids and venules (portal or postsinusoidal).
There is clear in vivo evidence that ICAM-1 supports adhesionin postsinusoidal venules (10, 11). However, during endotoxemia, neutrophils appear to actually transmigrate from sinusoids and not from postsinusoidal venules (5). On the otherhand, there is no evidence that neutrophils accumulate in sinusoids in an ICAM-1-dependent manner (3, 8, 10, 12). Furthermore, during systemic complement activation, neutrophils

The citation and abstract of the article discussed in the lettersbelow are as follows:
Involvement of Kupffer Cells in the Interaction Between
Neutrophils and Sinusoidal Endothelial Cells in Rats. Shock
18(2):152–157, 2002
During endotoxic liver injury, large numbers of neutrophils
infiltrate the liver and serum levels of tumor necrosis factor-␣
(TNF-␣) become elevated. The object of this study was toassess the roles of TNF-␣ secreted by Kupffer cells in theinteraction between neutrophils and sinusoidal endothelial cells(SECs). Rat neutrophils were perfused onto SECs that werestimulated with either TNF-␣ or supernatant from lipopolysaccharide (LPS)-stimulated Kupffer cells using an in vitro flowsystem. Numbers of adhered or migrated neutrophils werecounted, and the effect of an antibody against intercellularadhesion molecule-1 (ICAM-1) was studied. Compared withcontrols (200 ± 21 cells/mm2), neutrophil adhesion to SECs
was significantly increased by both TNF-␣ (342 ± 26 cells/mm2; P < 0.05) and LPS-stimulated Kupffer cell supernatant(331 ± 29 cells/mm2; P < 0.05). Anti-ICAM-1 significantly
inhibited neutrophil adhesion (139 ± 10 cells/mm2; P < 0.05).
LPS-stimulated Kupffer cells secreted TNF-␣ in an LPS dosedependent manner, and they significantly enhanced ICAM-1
expression on SECs (P < 0.05 vs. control). In addition, dexamethasone suppressed TNF-␣ production by LPS-stimulated
Kupffer cells and decreased ICAM-1 expression and neutrophil
adhesion on SECs. These findings suggest that Kupffer cells
are involved in neutrophil adhesion and migration in hepatic
sinusoids via TNF-␣ production and induction of ICAM-1
expression on SECs during liver injury associated with endotoxemia.
To the Editor: Polymorphonuclear leukocytes (neutrophils) areinvolved in causing acute liver cell damage induced by a variety of pathophysiological conditions, including endotoxinmediated liver injury (1). A low dose of endotoxin triggers
formation of TNF-␣, which is responsible for hepatic neutrophil accumulation (2, 3). TNF-␣ induces the transcriptional
activation of ICAM-1 expression on sinusoidal endothelial
cells and other liver cell types within 4 h (3, 4). However,
without further signals, the neutrophils will remain in the sinusoids and will not cause any injury (5). If galactosamine iscoadministered with endotoxin, TNF-␣ initiates selective
parenchymal cell apoptosis, which is responsible for neutrophil
transmigration and the severe aggravation of the injury byneutrophils (6). Blocking the function of ICAM-1 with amonoclonal antibody prevents neutrophil transmigration andeliminates the neutrophil-mediated injury phase (3). Thus,
when the sinusoidal endothelial cell layer is intact, the mainfunction of ICAM-1 in the pathophysiology is to facilitate
neutrophil transmigration. On the other hand, ICAM-1 is also394

SHOCK APRIL 2003

LETTERS TO THE EDITOR

accumulate in sinusoids but not in postsinusoidal venules in theabsence of increased ICAM-1 expression (11). It was repeatedly argued that neutrophil accumulation in sinusoids is not
mediated by adhesion molecules, but is caused by passive trapping due to a combination of endothelial cell swelling, active
vasoconstriction, and enhanced stiffness of the neutrophil cellmembrane. Although the trapping hypothesis is difficult toprove, unless novel adhesive interactions are identified, it
currently represents the most likely explanation for the accumulation of neutrophils in sinusoids. Once neutrophils are
stopped in sinusoids, a chemotactic event will trigger ␤2 integrin-ICAM-1-dependent transmigration and adhesion to hepatocytes. Because of the dependence on ICAM-1 expression,transmigration will not occur until ICAM-1 is upregulated, inspite of the fact that neutrophils may be present in sinusoids
several hours earlier. This mechanism may actually be thereason why neutrophil cytotoxicity in the liver does not occur
until 6 h after endotoxin administration (1, 5).
In summary, extensive in vivo data clearly demonstrated thatneutrophil adhesion in postsinusoidal venules requires ICAM-1expression. In contrast, neutrophil accumulation in sinusoids isindependent of ICAM-1. On the other hand, transmigration andadhesion to hepatocytes are ICAM-1-dependent events. The invitro data presented by Sakamoto et al. (9) mimic more neutrophil adhesion mechanisms in postsinusoidal venules and do notreflect conditions in sinusoids, the most relevant location forneutrophil cytotoxicity during endotoxemia.
REFERENCES
1. Jaeschke H, Farhood A, Smith CW: Neutrophil-induced liver cell injury inendotoxin shock is a CD11b/CD18-dependent mechanism. Am J Physiol
261:G1051–G1056, 1991.
2. Schlayer HJ, Laaff H, Peters T, Woort-Menker M, Estler HC, Karck U,Schaefer HE, Decker K: Involvement of tumor necrosis factor in endotoxintriggered neutrophil adherence to sinusoidal endothelial cells of mouse liver andits modulation in acute phase. J Hepatol 7:239–249, 1988.
3. Essani NA, Fisher MA, Farhood A, Manning AM, Smith CW, Jaeschke H:
Cytokine-induced hepatic intercellular adhesion molecule-1 (ICAM-1) mRNA
expression and its role in the pathophysiology of murine endotoxin shock andacute liver failure. Hepatology 21:1632–1639, 1995.
4. Jaeschke H, Essani NA, Fisher MA, Vonderfecht S, Farhood A, Smith CW:
Release of soluble intercellular adhesion molecule-1 (sICAM-1) into bile andblood in murine endotoxin shock. Hepatology 23:530–536, 1996.
5. Chosay JG, Essani NA, Dunn CJ, Jaeschke H: Neutrophil margination andextravasation in sinusoids and venules of the liver during endotoxin-inducedinjury. Am J Physiol 272:G1195–G1200, 1997.
6. Jaeschke H, Fisher MA, Lawson JA, Simmons CA, Farhood A, Jones DA:
Activation of caspase-3 (CPP32)-like proteases is essential for TNF-␣-induced
hepatic parenchymal cell apoptosis and neutrophil-mediated necrosis in amurine endotoxin shock model. J Immunol 160:3480–3486, 1998.
7. Nagendra AR, Mickelson JK, Smith CW: CD18 integrin and CD54-dependentneutrophil adhesion to cytokine-stimulated human hepatocytes. Am J Physiol
272:G408–G416, 1997.
8. Farhood A, McGuire GM, Manning AM, Miyasaka M, Smith CW, Jaeschke H:
Intercellular adhesion molecule-1 (ICAM-1) gene expression and its role inneutrophil-induced ischemia-reperfusion injury in the liver. J Leukocyte Biol
57:368–374, 1995.
9. Sakamoto S, Okanoue T, Itoh Y, Nakagawa Y, Nakamura H, Morita A, Daimon
Y, Sakamoto K, Yoshida N, Yoshikawa T, Kashima K. Involvement of Kupffer
cells in the interaction between neutrophils and sinusoidal endothelial cells inrats. Shock 18:152–157, 2002.
10. Vollmar B, Senkel A, Menger MD: In vivo evidence that intercellular adhesion
molecule-1 does not mediate endotoxin-induced hepatic leukocyte-endothelial
cell interaction. J Hepatol 23:613–616, 1995.
11. Bajt, ML, Farhood A, Jaeschke H: Effects of CXC chemokines on neutrophil

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activation and sequestration in the hepatic vasculature. Am J Physiol
281:G1188–G1195, 2001.
12. Jaeschke H, Farhood A, Fisher MA, Smith CW: Sequestration of neutrophils in
the hepatic vasculature during endotoxemia is independent of ␤2 integrins andintercellular adhesion molecule-1. Shock 6:351–356, 1996.

Dr. Hartmut Jaeschke
University of ArizonaCollege of Medicine
AHSC 24136
1501 N. Campbell Ave.
Tucson, AZ 85724

Reply: The contribution of ICAM-1 to neutrophil-SEC interaction is under controversy. In a recent issue, we reported that
ICAM-1 plays a crucial role in neutrophil adhesion to andmigration through the SEC monolayer in vitro (1). Jaeschke etal. (2, 3) previously reported that anti-ICAM-1 had no significant effect on hepatic accumulation of neutrophils in endotoxic
liver injury, and speculated that neutrophil accumulation iscaused by SEC and Kupffer cell swelling and the stiffness ofneutrophil cell membrane when exposed to endotoxin. In thisletter, they stated that the inhibition of ICAM-1 had only alimited effect in our in vitro study, and that the data obtained
under our experimental conditions could not be applied to thebehavior of neutrophils in hepatic sinusoid in vivo. However, incontrast to their observation, a few experiments in vivo showedthat ICAM-1 was involved in liver injury during endotoxemia(4, 5), which is consistent with our findings.
We can propose two reasons why their data in vivo differsfrom ours in vitro. First, they investigated the role of ICAM-1
in hepatic infiltration of neutrophils only 90 min after LPS
exposure in mice. As we stated in the issue, this time frame
would be too early for them to estimate the effect of antiICAM-1 antibody on neutrophil-SEC interaction because overexpression of ICAM-1 on SEC was not observed until 6 to 8 hafter LPS exposure in vivo (6, 7). In fact, Xu et al. (4) reported
that hepatic infiltration of neutrophils was significantly inhibited 24 h after LPS injection in ICAM-1-deficient mice, but
was not inhibited 2 h afterward. This in vivo data clearly
showed that ICAM-1 was involved in neutrophil accumulation
in ICAM-1-overexressing liver. We can agree with the data ofJaeschke that hepatic neutrophil accumulation was not mediated by ICAM-1 90 min after LPS exposure when ICAM-1 was
not enough upregulated on SEC. However, our experimentssuggest that ICAM-1 plays an important role in hepatic infiltration in a later stage, when the expression of ICAM-1 wasenhanced on SEC. The next reason is that they estimatedneutrophil accumulation by histochemical analysis. To investigate neutrophil-SEC interaction, we used an in vitro flow
system (IVFS) (1, 8), which allows of the dynamic analysis ofcell-to-cell interaction (9). IVFS enables us to investigate theeach step of infiltration and to distinguish adhered cells fromfloating or rolling cells on SEC monolayers.
In other words, we can count the actually adhered or
migrated cells by using our device, but by means of theirmethods, it is impossible to distinguish strictly neutrophil396

SHOCK VOL. 19, NO. 4

adhesion from leukostasis, which was secondarily induced bycirculatory disturbance. Vollmar et al. (10) reported that thenumber of nonperfused sinusoids correlated with the number ofneutrophils in sinusoids during endotoxemia. Indeed, our invitro data showed that once a neutrophil adhered to SEC, thenext adhesive reaction occurred around the adhered neutrophil
at first. This finding may reveal that the adhered cells change
the flow rate and make turbulent flow, which enhances additional adhesive reaction (preliminary data). Besides, neutrophil-SEC interaction mediated by ICAM-1 induces SEC injury
by activated neutrophils (8). We do not deny the possibility
that neutrophil accumulation is caused by passive trapping due
to SEC swelling or active vasoconstriction; however, our findings revealed that anti-ICAM-1, in part but at least significantly, inhibited an initial adhesive reaction that induces thesecondary leukostasis and SEC injury in hepatic sinusoids invivo. Considering these points, even if anti-ICAM-1 inhibits
the adhesion of only 50%–60% of neutrophils, we canconclude that ICAM-1 plays a major role in the hepatic infiltration of neutrophils during endotoxic liver injury.
REFERENCES
1. Sakamoto S, Okanoue T, Itoh Y, Nakagawa Y, Nakamura H, Morita A, Daimon
Y, Sakamoto K, Yoshida N, Yoshikawa T, Kashima K: Involvement of Kupffer
cells in the interaction between neutrophils and sinusoidal endothelial cells inrats. Shock 18:152–157, 2002.
2. Jaeschke H, Farhood A, Fisher MA, Smith CW: Sequestration of neutrophils in
the hepatic vasculature during endotoxemia is independent of ␤2 integrins andintercellular adhesion molecule-1. Shock 6:351–356, 1996.
3. Essani NA, Fisher MA, Farhood A, Manning AM, Smith CW, Jaeschke H:LETTERS TO THE EDITOR
Cytokine-induced upregulation of hepatic intercellular adhesion molecule-1
messenger RNA expression and its role in the pathophysiology of murine
endotoxin shock and acute liver failure. Hepatology 21:1632–1639, 1995.
4. Xu H, Gonzalo JA, St Pierre Y, Williams IR, Kupper TS, Cotran RS, SpringerTA, Gutierrez-Ramos JC: Leukocytosis and resistance to septic shock in intercellular adhesion molecule 1-deficient mice. J Exp Med 180:95–109, 1994.
5. Kamochi M, Kamochi F, Kim YB, Sawh S, Sanders JM, Sarembock I, GreenS, Young JS, Ley K, Fu SM, Rose CE Jr: P-selectin and ICAM-1 mediate
endotoxin-induced neutrophil recruitment and injury to the lung and liver. Am
J Physiol 277:L310–L319, 1999.
6. Ahmad N, Gardner CR, Yurkow EJ, Laskin DL: Inhibition of macrophages
with gadolinium chloride alters intercellular adhesion molecule-1 expression inthe liver during acute endotoxemia in rats. Hepatology 29:728–736, 1999.
7. Ohira H, Ueno T, Torimura T, Tanikawa K, Kasukawa R: Leukocyte adhesion
molecules in the liver and plasma cytokine levels in endotoxin-induced rat liver
injury. Scand J Gastroenterol 30:1027–1035, 1995.
8. Sakamoto S, Okanoue T, Itoh Y, Sakamoto K, Nishioji K, Nakagawa Y,Yoshida N, Yoshikawa T, Kashima K: Intercellular adhesion molecule-1 andCD18 are involved in neutrophil adhesion and its cytotoxicity to cultured sinusoidal endothelial cells in rats. Hepatology 26:658–663, 1997.
9. McIntire LV, Eskin SG: Mechanical and biochemical aspects of leukocyte
interactions with model vessel walls. Kroc Found Ser 16:209–219, 1984.
10. Vollmar B, Glasz J, Senkel A, Menger MD, Messmer K: Role of leukocytes in
the initial hepatic microvascular response to endotoxemia. Zentralbl Chir
118:691–696, 1993.

Dr. Takeshi Okanoue
Dr. Shinichi Sakamoto
Kyoto Prefectural University ofMedicine
The Third Department of Internal
Medicine
Kawaramachi-Hirokoji,Kamigyo-ku, Kyoto
602-8655, Japan