A century ago, when the digestive properties of pancreatic secretions became known, the morphological appearance of pancreatitis on autopsy sections suggested that the disease represents an autodigestive process (1
). This view has not changed, and according to our current understanding, the onset of the disease involves a premature and intrapancreatic activation of proteolytic enzymes (2
). Digestive proteases are, however, synthesized and secreted as inactive precursor zymogens, and the pancreatic acinar cell possesses a whole variety of protective mechanisms that either prevent premature zymogen activation or inhibit protease activity (41
). The question, therefore, remains as to which molecular factors either trigger the initial protease activation or permit the cellular defenses to be overcome. One of the hypotheses that have attempted to address this issue predicts that CTSB, a lysosomal cysteine proteinase, is critically involved in the initial trypsinogen activation (8
). This cathepsin B hypothesis is based on the observations that CTSB can activate trypsinogen in vitro (9
), that CTSB is redistributed to a zymogen-granule–enriched subcellular compartment (10
), and that lysosomal enzymes colocalize with digestive zymogens during the early course of experimental pancreatitis (11
). Although this cathepsin-hypothesis appears attractive from a cell biologic point of view, a number of experimental observations appear to be incompatible with its assumptions: (a) a colocalization of cathepsins with digestive zymogens has not only been observed in the initial phase of acute pancreatitis, but also under physiological control conditions and in secretory vesicles that are destined for regulated secretion from healthy pancreatic acinar cells (42
); (b) a redistribution of CTSB into a zymogen-enriched subcellular compartment can be induced in vivo by experimental conditions that interfere with lysosomal sorting and are neither associated with nor followed by the development of acute pancreatitis (43
); (c) the administration of potent lysosomal enzyme inhibitors in vivo does not prevent the onset of acute experimental pancreatitis (14
); (d) in experiments that used lysosomal enzyme inhibitors in vitro, an increase in the rate of intracellular trypsinogen activation, as well as a decrease in the rate of intracellular trypsinogen activation, has been reported (12
), and even a protective role against premature zymogen activation has been considered for CTSB (44
). In view of the limited specificity and bioavailability of the existing inhibitors for lysosomal hydrolases, the only remaining option to address the cathepsin-hypothesis conclusively was therefore to use CTSB-deficient animals and to study them in an experimental model of acute pancreatitis.
We have chosen this approach and have used a strain of mice in which the ctsb
gene was deleted by targeted disruption (15
). In an extensive anatomical and functional characterization, we could show that the resulting CTSB–/–
animals are phenotypically indistinguishable from their wild-type controls, that they reproduce normally, and that they carry no abnormalities in either their organ development or immune system. We used these CTSB-deficient animals to induce an experimental variety of acute pancreatitis. This secretagogue-induced model of the disease appears ideally suited to address the cathepsin-hypothesis for a number of reasons: (a) it is noninvasive and can be induced in mice and other small rodents (46
); (b) it is associated with significant intrapancreatic trypsinogen activation that precedes acinar cell injury (6
); and (c) many of the local and systemic alterations known to be relevant for the human disease are found in this animal model including hyperamylasemia, significant pancreatic tissue necrosis, systemic inflammatory response, and lung injury (26
The targeted disruption of the ctsb gene and the complete absence of functional CTSB in the pancreas of these mice altered the course of acute pancreatitis in a number of ways. The most dramatic change in comparison to wild-type control animals, and also the most relevant in regard to the cathepsin-hypothesis of acute pancreatitis, was a significant reduction in the premature and intrapancreatic trypsinogen activation. In terms of substrate-defined trypsin activity, this reduction amounted to more than 90% over the course of 24 hours, when the greater pancreatic trypsinogen content of CTSB–/– animals is taken into account.
This prevention of premature trypsinogen activation was, however, incomplete. In terms of the presence of TAP in the pancreas, which indicates the total amount of trypsinogen that has been activated over time rather than the actual activity at a given point in time, the reduction of trypsinogen conversion that can be attributed to the absence of CTSB was approximately 50% and thus corresponded to the reduction of acinar cell necrosis. CTSB is therefore clearly not the only factor involved in premature intrapancreatic trypsinogen activation or cell injury and additional mechanisms such as trypsinogen autoactivation or a trypsinogen activation by other lysosomal cysteine proteinases, e.g., cathepsin L, H, and S, must be considered as potential alternatives.
Although the reduction of trypsinogen activation corresponded to the decrease in acinar cell injury, the amount of cells undergoing apoptosis and, more surprisingly, the systemic inflammatory response did not. Neither the degree of leukocyte infiltration in the pancreas or lungs nor the histological degree of lung injury during pancreatitis was affected by the absence of CTSB. This may indicate that the presence or absence of CTSB is irrelevant for the development of systemic and inflammatory complications associated with acute pancreatitis. Alternatively, this observation might indicate that as long as pancreatic damage above a certain threshold level has occurred, the systemic complications of the disease evolve in a manner that is completely independent of the initial degree of trypsinogen activation or the extent of acinar cell injury.
In conclusion, we used CTSB-deficient mice to study whether CTSB is critically involved in premature zymogen activation and pancreatic damage in an experimental model of pancreatitis. Our results show that the presence or absence of CTSB is, indeed, an important factor that determines the degree of premature trypsinogen activation and the extent of acinar cell necrosis, but not the systemic inflammatory response associated with acute pancreatitis. To our knowledge, these data provide the first direct and conclusive experimental evidence for a role of cathepsin B in the onset of acute pancreatitis.