Neutrophil lysosomal dysfunctions in mutant C57 Bl/6J mice: interstrain variations in content of lysosomal elastase, cathepsin G and their inhibitors

In this paper we report the serum antiprotease screening and the biochemical and functional characteristics of neutrophils in a variety of mouse strains with different susceptibilities for developing a protease-mediated injury. C57Bl/6J mice and their mutants tight-skin and pallid have a lower serum elastase inhibitory capacity (-30, -65 and -70% respectively) than other inbred strains (i.e. NMRI and Balb/c, which both have similar values). We demonstrate that these values are a consequence of a decreased concentration of the alpha 1-protease inhibitor for elastase [PI(E)], which is the major serum inhibitor of elastase and cathepsin G. In addition, neutrophil lysosomal dysfunctions characterized by abnormally high contents of elastase and cathepsin G, or defective lysosomal secretion are observed in tight-skin and pallid mice respectively. Another C57Bl/6J mutant with lysosomal abnormalities is the beige mouse. Negligible amounts of elastase and cathepsin G, as well as defective neutrophil degranulation, have been described previously in this strain. We found, however, discrete amounts of a latent form of neutrophil elastase that undergoes a spontaneous activation by a protease-dependent mechanism. We also report that neutrophil cathepsin G in this mouse is tightly bound to lysosomal membranes, but is released in near normal quantities during exocytosis. Cytosolic elastase and cathepsin G inhibitors, which were previously reported as being specific for the beige neutrophils, have also been detected in all the examined strains. Neutrophil functions, lysosomal enzyme content and serum antiprotease screening may represent key elements in the protease-antiprotease balance and may explain the different interstrain susceptibility to developing lesions in which an elastolytic activity has been implicated.

Alpha 2-macroglobulin restricts plasminogen activation to the surface of RC2A leukemia cells.

Human RC2A myelomonocytic leukemia cells are able to activate the prourokinase (pro-u-PA) they secrete so that active u-PA is present both in serum-free conditioned medium from these cells, as well as on the cell surface. When the cells are grown in serum-containing medium, no u-PA activity can be found in the medium but active u-PA is found bound to the cell surface where it can generate bound plasmin. This distribution of u-PA activity was shown to be, first, the net result of slow inactivation of free active u-PA by serum inhibitor(s) and simultaneous rapid uptake of u-PA onto the cell surface. Binding to cells was at least six times faster than inactivation by 10% serum. The principal serum inhibitor of u-PA was identified as alpha 2-macroglobulin (alpha 2M), and prior inactivation of u-PA by purified human alpha 2M was also shown to prevent uptake of u-PA activity onto cells. Second, although endogenous u-PA could form covalent complexes with purified alpha 2M in the culture medium of RC2A cells, covalent alpha 2M complexes were not formed by u-PA on the cell surface; the u-PA taken up in this compartment was protected against alpha 2M inhibition. u-PA anchored to plastic surfaces via monoclonal antibodies to the amino-terminal region of u-PA was also protected against alpha 2M, suggesting that the protection of cell surface u-PA results from a steric effect. These results provide evidence as to how the active u-PA produced by leukemia cells can contribute to proteolytic activity on their cell surface in the presence of serum inhibitors.

Effect of serum from patients with essential hypertension on sodium transport in normal leucocytes

1. This study has confirmed that serum from patients with essential hypertension inhibits sodium transport and elevates intracellular sodium in normal human leucocytes in vitro when compared with that of well matched normotensive controls. 2. The magnitude of this effect was positively correlated with the diastolic blood pressure of the hypertensive patient. 3. The degree of sodium transport inhibition conferred by the hypertensive's serum was correlated with the abnormal sodium transport in the hypertensive's own leucocytes. 4. These results confirm the presence of a serum inhibitor of sodium transport in essential hypertension. The relationship between the inhibitory effect and severity of hypertension argues that it may be of mechanistic importance.