Human mast cells produce and release the cytotoxic lymphocyte associated protease granzyme B upon activation.

Mast cells are widely distributed throughout the body and express effector functions in allergic reactions, inflammatory diseases, and host defense. Activation of mast cells results in exocytosis of preformed chemical mediators and leads to novel synthesis and secretion of lipid mediators and cytokines. Here, we show that human mast cells also express and release the cytotoxic lymphocyte-associated protease, granzyme B. Granzyme B was active and localized in cytoplasmic granules, morphologically resembling those present in cytotoxic lymphocytes. Expression and release of granzyme B by mast cell-lines HMC-1 and LAD 2 and by cord blood- and mature skin-derived human mast cells depended on the mode of activation of these cells. In mast cell lines and cord blood-derived mast cells, granzyme B expression was mainly induced by non-physiological stimuli (A23187/PMA, Compound 48/80) and substance P. In contrast, mature skin-derived mast cells only produced granzyme B upon IgE-dependent stimulation. We conclude that granzyme B is expressed and released by human mast cells upon physiologic stimulation. This suggests a role for granzyme B as a novel mediator in mast cell biology

Proteasome Regulates the Delivery of LDL Receptor-related Protein into the Degradation Pathway

The low-density lipoprotein receptor (LDLR)-related protein (LRP) is a multiligand endocytic receptor that has broad cellular and physiological functions. Previous studies have shown that both tyrosine-based and di-leucine motifs within the LRP cytoplasmic tail are responsible for mediating its rapid endocytosis. Little is known, however, about the mechanism by which LRP is targeted for degradation. By examining both endogenous full-length and a minireceptor form of LRP, we found that proteasomal inhibitors, MG132 and lactacystin, prolong the cellular half-life of LRP. The presence of proteasomal inhibitors also significantly increased the level of LRP at the cell surface, suggesting that the delivery of LRP to the degradation pathway was blocked at a compartment from which recycling of the receptor to the cell surface still occurred. Immunoelectron microscopy analyses demonstrated a proteasomal inhibitor-dependent reduction in LRP minireceptor within both limiting membrane and internal vesicles of the multivesicular bodies, which are compartments that lead to receptor degradation. In contrast to the growth hormone receptor, we found that the initial endocytosis of LRP minireceptor does not require a functional ubiquitin–proteasome system. Finally, using truncated cytoplasmic mutants of LRP minireceptors, we found that a region of 19 amino acids within the LRP tail is required for proteasomal regulation. Taken together our results provide strong evidence that the cellular turnover of a cargo receptor, i.e., LRP, is regulated by the proteasomal system, suggesting a broader function of the proteasome in regulating the trafficking of receptors into the degradation pathway