Endogenous ligands from damaged cells, so-called damage-associated molecular pattern molecules, can activate innate immunity via TLR4 signaling. Hepatic warm ischemia and reperfusion (I/R) injury and inflammation is largely TLR4 dependent. We produced TLR4 chimeric mice to assess whether the TLR4-dependent injury required TLR4 expression on liver parenchymal or nonparenchymal cells. Chimeric mice were produced by adoptive transfer of donor bone marrow cells into irradiated recipient animals using reciprocal combinations of TLR4 wild-type (WT; C3H/HeOuj) and TLR4 mutant (C3H/HeJ) mouse bone marrow. Wild-type chimeric mice bearing TLR4 mutant hemopoietic cells and TLR4 mutant mice transplanted with their own bone marrow-derived cells were protected from hepatic I/R and exhibited decreased JNK and NF-κB activation compared with WT chimeric mice transplanted with their own bone marrow. In contrast, TLR4 mutant mice transplanted with TLR4 WT bone marrow were not protected from liver I/R and demonstrated pronounced increases in JNK and NF-κB activation when compared with autochthonous transplanted mutant mice. In addition, depletion of phagocytes taking up gadolinium chloride failed to provide any additional protection to TLR4 mutant mice, but substantially reduced damage in WT mice after hepatic I/R. Together, these results demonstrate that TLR4 engagement on actively phagocytic nonparenchymal cells such as Kupffer cells is required for warm I/R-induced injury and inflammation in the liver.

SummaryNitric oxide (NO) is a short-lived biologic mediator that is shown to be induced in various cell types and to cause many metabolic changes in target cells. Inhibition of tumor cell growth and antimicrobial activity has been attributed to the stimulation of the inducible type of the NO synthase (NOS). However, there is limited evidence for the existence of such inducible NOS in a human cell type. We show here the induction of NO biosynthesis in freshly isolated human hepatocytes (HC) after stimulation with interleukin 1, tumor necrosis factor (TNF), IFN-% and endotoxin. Increased levels of nitrite (NO2-) and nitrate (N03-) in culture supernatants were associated with NADPH-dependent NOS activity in the cell lysates. The production of NO2-and N03-was inhibited by N6-monomethyl I.-arginine and was associated with an increase in cyclic guanylate monophosphate release. The data presented here provide evidence for the existence of typical inducible NO biosynthesis in a human cell type.A variety of cell types have been shown to produce nitric oxide (NO) from t-arginine by either a constitutive and/or an inducible enzyme (1). Recent reports provide strong evidence that vasorelaxation, induced through the constitutive and inducible NO pathway, might play an important role in the regulation of vascular tone in humans (2, 3). Little evidence for the existence of inducible NO enzyme activity in specific human cell types has been documented, despite its clear potential role in the elimination of tumor cells (4) and intra-and extracellular pathogens (recently reviewed in reference 5), as well as in the induction of sustained hypotension (6), as shown in animal models.We have shown that rat hepatocytes (HC) cocultured with Kupffer ceils and stimulated with LPS can produce large amounts of nitrite (NO2-) and nitrate (N03-), the stable end products of NO (7). Furthermore, we have shown that HC alone are capable of biosynthesis of NO when exposed to various immunostimuli (8-10). The expression of the inducible nitric oxide synthase (NOS) alters various HC functions in vitro. These include a substantial decrease in total protein synthesis (7, 11), the inhibition of mitochondrial aconitase (12), and the stimulation of cyclic guanylate monophosphate (cGMP) synthesis and release (13). In vivo studies suggest that hepatic NO can protect the liver from damage in sepsis (14). In this communication, we report that nitrogen oxides are also produced in large amounts by human HC in a reproducible manner, demonstrating that a human call type can, indeed, express an inducible NOS. Materials and MethodsCulture Medium. HC cultures were performed in Williams medium E (Gibco Laboratories, Grand Island, NY) supplemented with 10 .6 M insulin, 15 mM Hepes, t-glutamine, penicillin, streptomycin, and 10% dialyzed calf serum. Additional culture reagents included t-arginine hydrochloride (Gibco Laboratories) and N ~-monomethyl-r-arginine acetate (NMA), prepared by a modification of the method previously described (15).Hepatocyte Isolation. In acc...

Objective. To test the hypothesis that nitric oxide (NO) is involved in the pathophysiology of arthritis.Methods. Arthritis was induced in male Lewis rats by the injection of adjuvant into the base of the tail. The NO synthase (NOS) inhibitor, NG-monomethyl-Larginine (L-NMA), was administered daily by the oral route for 19 days. Paw swelling, plasma fibrinogen levels, and urinary NOJNO, levels were measured to assess the effect of L-NMA on the arthritic response and whole-body NO production, respectively. On day 20, the ankle joints were processed for histopathologic evaluation.Results. The onset of clinical symptoms was preceded by elevated biosynthesis of NO. In a dosedependent manner, L-NMA inhibited both NO biosynthesis and paw swelling; histopathologic changes in the ankle joints were also prevented. D-NMA had no effect on the development of arthritis, while L-arginine reversed the effects of L-NMA. Fibrinogen levels in rats with arthritis were unaffected by L-NMA.Conclusion. NO is critical to the development of both the inflammatory and erosive components of adjuvant arthritis in rats. There may be a future clinical role for suitable inhibitors of NO production or activity.Rheumatoid arthritis is a chronic, incurable disease characterized by joint swelling, synovial in- flammation, and cartilage destruction. Antagonism of the mediators which drive these pathophysiologic changes represents one approach to improving the treatment of this condition. Such an endeavor requires the prior identification of the appropriate mediators. In a recent review (I), it was suggested that nitric oxide (NO) should be investigated in this regard; the present paper reports the results of a study that was designed to do so.In vitro studies have demonstrated that both the resident mesenchymal cells of the joints and the leukocytes which enter joints during inflammation have the capacity to synthesize considerable amounts of NO. Thus, cultures of articular chondrocytes (2,3) and synoviocytes (4) produce high levels of NO following activation by interleukin-1, a cytokine present in rheumatoid synovial fluids. Macrophages (9, polymorphonuclear leukocytes (6), mast cells (7), and, possibly, lymphocytes (8) are additional potential sources of NO in inflamed joints. Analysis of synovial fluids (9,lO) from arthritic joints has confirmed the presence of elevated amounts of nitrate and nitrite, the stable end products to which NO spontaneously oxidizes in vivo. Previous animal studies have implicated NO in inflammation (1 I), immunity (12), and tissue destruction (13).NO is synthesized by the enzyme NO synthase (NOS), which exists as several isozymes (14), the main distinction being between constitutive (cNOS) and inducible (iNOS) forms. Cells that contain cNOS quickly and transiently produce small amounts of NO in response to agonists which raise intracellular Ca2' concentrations, while cells with iNOS produce large amounts of NO for a prolonged period following a lag of several hours during which the enzyme is induced.Chondrocytes (2),...

Successful engraftment of organ transplants has traditionally relied on preventing the activation of recipient (host) T cells. Once T-cell activation has occurred, however, stalling the rejection process becomes increasingly difficult, leading to graft failure. Here we demonstrate that graft-infiltrating, recipient (host) dendritic cells (DCs) play a key role in driving the rejection of transplanted organs by activated (effector) T cells. We show that donor DCs that accompany heart or kidney grafts are rapidly replaced by recipient DCs. The DCs originate from non-classical monocytes and form stable, cognate interactions with effector T cells in the graft. Eliminating recipient DCs reduces the proliferation and survival of graft-infiltrating T cells and abrogates ongoing rejection or rejection mediated by transferred effector T cells. Therefore, host DCs that infiltrate transplanted organs sustain the alloimmune response after T-cell activation has already occurred. Targeting these cells provides a means for preventing or treating rejection.

The migration of effector or memory T cells to the graft is a critical event in the rejection of transplanted organs. The prevailing view is that the key steps involved in T cell migration -integrin-mediated firm adhesion followed by transendothelial migration -are dependent on the activation of Gα i -coupled chemokine receptors on T cells. In contrast to this view, we demonstrated in vivo that cognate antigen was necessary for the firm adhesion and transendothelial migration of CD8 + effector T cells specific to graft antigens and that both steps occurred independent of Gα i signaling. Presentation of cognate antigen by either graft endothelial cells or bone marrow-derived APCs that extend into the capillary lumen was sufficient for T cell migration. The adhesion and transmigration of antigen-nonspecific (bystander) effector T cells, on the other hand, remained dependent on Gα i , but required the presence of antigen-specific effector T cells. These findings underscore the primary role of cognate antigen presented by either endothelial cells or bone marrow-derived APCs in the migration of T cells across endothelial barriers and have important implications for the prevention and treatment of graft rejection.