Response to the letter by Dr. Jaeschke (2004)

We appreciate the comments by Jaeschke and Bajt about our paper, showing a critical role of CXC chemokines in endotoxin-induced liver injury [1], and we are happy to reply to these comments as follows. To measure apoptosis, there are many different techniques, which will give different rates of cellular apoptosis in any given tissue depending in part on the fact that apoptosis is a dynamic biochemical and morphological process. Herein, we used a biochemical (caspase-3) and morphological (Hoechst 33324) approach to determine apoptosis. When it comes to the role of tumor necrosis factor α (TNF-α), we agree that numerous studies have documented a dominant role of TNF-α in endotoxin-induced liver injury. However, the important issue is that TNF-α is an upstream mediator regulating leukocyte recruitment into the liver; i.e. TNF-α contributes to the endotoxin-provoked liver injury by an indirect, leukocyte-dependent mechanism [2] and that inhibition of leukocyte recruitment by targeting specific adhesion molecules markedly protects against TNF-α- and endotoxin-induced hepatocellular damage and apoptosis [3, 4]. In this paper [1], we have gone a step further by demonstrating that CXC chemokines regulate the extravasation of leukocytes in endotoxin-induced liver injury and that inhibition of CXC chemokine function protects against the hepatic damage. Thus, it is important to note that CXC chemokine-induced leukocyte recruitment is a mechanism downstream of TNF-α function in endotoxin-induced liver injury. Regarding the specificity of the anti-macrophage-inflammatory protein-2 (MIP-2) and anti-keratinocyte (KC) antibodies [rat immunoglobulin G (IgG)] used herein, we have shown in separate experiments that these antibodies block the function of their specific target. It is important that we have also included a control rat IgG, which had no effect on endotoxin-induced leukocyte recruitment and liver injury. Thus, the argument made by Jaeschke and Bajt that the observed effects may be a result of unspecific effects of the anti-CXC chemokine antibodies on TNF-α formation, based on a study using 300 mg/kg rat IgG [5], a dose more than 150 times higher than that used in our paper may not be applicable here. In this context, it is also important to note that the anti-MIP-2 and anti-KC antibodies used herein had no effect on endotoxin-induced leukocyte rolling and adhesion, which further argues against any unspecific effects of these antibodies on TNF-α-dependent events. There has been some discussion in the literature about the relative importance of sinusoids and postsinusoidal venules in the recruitment process of leukocytes to the liver parenchyma. Jaeschke and Bajt forward the view, “As has been shown in this model, neutrophil transmigration occurs from sinusoids and not from venules”, based on a single reference [6]. However, the vast literature about this subject has demonstrated that inhibition of P-selectin, which is not expressed on sinusoidal but is expressed on postsinusoidal venular endothelial cells [7], significantly reduces leukocyte recruitment and hepatocyte damage in this model of endotoxin-induced liver injury [4, 8]. In fact, the list of models in which inhibition of P-selectin function protects against leukocyte recruitment and liver injury is long and includes ischemia/reperfusion injury [910111213], colitis-associated hepatitis [14], concanavalin A-induced liver injury [15], and liver damage induced by hemorrhagic shock [16]. Taken together, the overall literature suggests that P-selectin, which is expressed on endothelial cells in postsinusoidal venules but not in sinusoids, is a critical determinant of leukocyte recruitment and plays a central role in several models of liver injury, including endotoxin-induced liver injury. Jaeschke and Bajt find it “puzzling” that inhibition of MIP-2 or KC significantly and similarly decreases endotoxin-induced leukocyte extravasation. However, this is not so difficult to understand given that there is a synergistic and potentiating interaction between MIP-2 and KC on leukocyte recruitment [17], analogous to that of cytokines such as TNF-α and interleukin-1 so that inhibition of either one markedly decreases the leukocyte response. Their own results, mentioned by Jaeschke and Bajt in their letter, are difficult to comment on, as these have only been published as an abstract, but we are convinced that these data will contribute to the understanding of CXC chemokine endotoxin-induced liver injury once published in a full paper. In summary, in light of the reasoning above, we are confident that our data support the concept that CXC chemokines play a critical role in endotoxin-induced liver injury.