Caspase-1 Regulates Escherichia coli Sepsis and Splenic B Cell Apoptosis Independently of Interleukin-1[beta] and Interleukin-18

More than 500,000 people develop sepsis annually and 175,000 of them die in the United States alone (1). Septic shock activates numerous proinflammatory mediators, which can result in multiple organ injury (2, 3). In addition, executioner cysteineaspartate proteases (caspases) play a key role in the disassembly of cells during septic shock via various proapoptotic stimuli. Pharmacologic blockade of caspase activation improves organ function and survival in animal models of sepsis and ischemia reperfusion injury (4). Interleukin 1ß (IL-1ß) is one of the major proinflammatory cytokines known to be produced in sepsis (5-8). It is synthesized as an inactive 31-kD precursor that requires a unique cysteine protease, IL-1ß-converting enzyme (caspase-1), to generate biologically active 17-kD IL-1ß (9, 10).

Although caspase-1 plays no part in the spontaneous apoptosis of monocytes and macrophages (11), its activation via intracellular pathogens can induce macrophage apoptosis (12, 13) and its deletion has been linked to survival in animal models of endotoxin shock (14). This protective effect could logically be attributed to caspase-1's role in activating the precursor, proIL-1ß. Unexpectedly, however, active IL-1ß does not regulate survival from endotoxin shock, because IL-1ß knockout animals are not protected from endotoxin-induced death (15). This difference may hold an important key to understanding the role of caspase-1 in host responses. Importantly, prior caspase-1 knockout experiments have not analyzed the apoptotic role of caspase-1 in sepsis. Furthermore, it is important to expand the model to a live bacterial challenge because IL-1ß may be critical to coordinating the more complex host eradication of pathogens (16, 17).

The present study was designed to determine the mechanisms responsible for the caspase-1 knockout protection from the sepsis response to live intraperitoneal Escherichia coli injections. Mice were genetically deficient in caspase-1, IL-1ß, or IL-1ß and IL-18 or pharmacologically deficient in functional caspase-1. Our studies confirm for the first time that the reported differences in survival between caspase-1 and IL-1ß knockout animals are translatable to complex live-infection models of sepsis. We show that the protection is unique to caspase-1 and not IL-1ß or IL-18. Furthermore, we show that, although the IL-1ß/IL-18 double knockout mice are not protected from the E. coli challenge, these mice are protected by a synthetic caspase inhibitor. We also show that the caspase-1 knockout state or the use of a synthetic caspase inhibitor prevents splenic B cell apoptosis. Finally, we demonstrate that apoptotic splenic B lymphocytes induce macrophages to assume an inhibitory phenotype. These results are particularly relevant because they support the hypothesis that inhibition of apoptosis can promote sepsis survival (18) and that caspase-1 may be a critical determinant of the apoptosis response in sepsis. Some of the results of these studies have been previously reported in the form of an abstract (19).

METHODS

Mice

All animal experiments performed were done according to animal protocols approved by the Animal Care Use Committee of the Ohio State University College of Medicine. Caspase-1 knockout (caspase-1^sup -/-^) and IL-1ß knockout (IL-1ß^sup -/-^) mice of B10.RIII background were generated by Merck Research Laboratory (Rahway, NJ) (20). IL-1ß/ IL-18 double knockout mice of C57B16 background were obtained from Dr. A. Zychlinsky, Max Planck Institute, Berlin (authorized by Dr. S. Akira, Japan). Age-matched control mice were purchased from Jackson Laboratory (Bar Harbor, ME).

Genotyping of Mice

Based on the maps of the wild-type and caspase-1, IL-1ß, and IL-1ß/ IL-18 knockout mice, primers were designed for genotyping. Before each experiment, polymerase chain reaction of tail snip DNA was performed to identify the specific genotypes. Details of primer designs are provided in the online supplement.

Mouse Sepsis Survival