IHC of human skin fibroblasts (Left: control, Right: 24 hours after 7th passage of senescence), using Anti-Hsp60 (clone: LK1)
Evidence has accumulated to demonstrate that the cytosolic location of Hsp60 does not only result from mitochondrial export but also independently of such release 174. As demonstrated previously by Chandra and colleagues, apoptosis induction in LNCaP prostate cancer cells leads to cytosolic Hsp60 accumulation without any apparent mitochondrial release 174. Concomitantly, cytosolic residing Hsp60 which has not been imported into mitochondria before contains the mitochondrial localization signal (MLS) 174. From the cytosol, Hsp60 may reach various cellular compartments in malignant 3, 175 and nonmalignant cells 171, 172. Increased cell surface expression of Hsp60 serves a danger signal for the immune system culminating in the stimulation of dendritc cells (DCs) and, ultimately the induction of T cell-mediated anti-tumor immune responses 4, 5, 176, 177.
Apart from its intracellular and surface localization, Hsp60 has also been identified in the extracellular space and the circulation. The mechanisms underlying its export involve an active secretory process with the participation of exosomes and lipid rafts 3, 178, 179. Hsp60 can also be released via the classical ER/Golgi protein secretory pathway 74. This finding is supported by the fact that Hsp60 secretion is sensitive to Brefeldin A treatment, a classical inhibitor of ER/Golgi-dependent secretory pathways. Moreover, the Golgi transport inhibitor monensin exhibited a similar effect and induced the relocation of Hsp60 to the Golgi 74.
Extracellular Hsp60 is also able to enter the circulation and thus can be found in the plasma of healthy individuals 180,181,182,183. Mass spectrometric analysis of circulating Hsp60 revealed that the circulating chaperonin is predominantly the intact protein without MLS implying that this circulating protein originated from mitochondria 183. In some cases, serum Hsp60 has been reported at levels able to elicit biological activity in vitro 184,185,186. The ability of cells to release Hsp60 appears to depend on the cell type, as the level of extracellular Hsp60 generated by human neuroblastoma cells is ten times higher than that generated by keratinocytes 187. Studies by several groups exhibited a highly variable log normal distribution profile of circulating Hsp60 in healthy individuals. It is worth mentioning that individuals segregate into those with high plasma and those with low plasma levels of Hsp60 180, 188, 189. However, the molecular mechanisms affecting the profile of circulating Hsp60 levels have not been identified up to date, but variations in transcriptional activity do not appear to be involved 190.
Various factors such as stress have been identified to affect circulating levels of Hsp60. In this context, the Whitehall cohort studies (I and II) of British civil servants provided evidence of an association between high Hsp60 plasma levels and psychosocial measures including socioeconomic status, psychological distress, and social isolation 180. Age is a further crucial factor impacting peripheral Hsp60 levels, since Hsp60 levels have been found to progressively decline with increasing age 191, 192. In pathologic situations such as acute myocardial infarction (AMI), elevated circulating Hsp60 levels 189 might be attributed to necrosis of cardiomyocytes and concomitant endothelial dysfunction 193, 194. In this regard, the group of Georg Wick observed an AMI-mediated release of highly immunogenic and cross-reactive Hsp60 into the circulation in response to myocardial ischemia and myocardiocyte injury 195. Higher shear stress might also contribute to AMI-mediated Hsp60 release as demonstrated previously 196, 197. Although cellular necrosis inevitably results in the unspecific release of Hsp60, the biological consequences of Hsp60 alone and Hsp60 exported in the absence of concomitant necrosis might vary considerably. The finding of a necrosis-independent release of Hsp60 and its appearance in the peripheral circulation of healthy individuals reflects an essential function. Meanwhile, the role of circulating levels of Hsp60 in numerous disease states is well known and has been summarized by Frostegård and Pockley already in 2005 198.
Work from different laboratories has shown that in the periphery of normal individuals as well as in patients with autoimmune and cardiovascular diseases anti-Hsp60 antibodies are present. Unpublished observations by the group of A. Graham Pockley suggest that Hsp60 might also exist as soluble immune complexes.