HSP70/J-protein chaperone machines: expression analysis in granulocytes

Background

Polymorphonuclear leucocytes (PMNLs) represent the first line of defence in innate immunity. PMNLs are the predominant cells accumulating in the synovial fluid of rheumatoid arthritis (RA) patients and play a crucial role in the development of tissue damage. Unstimulated PMNLs are postmitotic terminally differentiated cells whose default setting is death by apoptosis. Pathogens or inflammation induce a profound transcriptional and translational activity. Aberrant expression of members of the major chaperone family HSP70 occurs in synovial tissue of RA [1, 2] and in PMNLs subjected to stress in the form of exercise [3] or polytrauma [4]. Activity and substrate specificity of HSP70s is regulated by the family of J-proteins. HSP70s and J-proteins together generate important chaperone machines, which are not only involved in classical chaperone tasks like proper protein folding but also in fine tuning of molecules involved in signal transduction and apoptotic pathways. Hardly any data exist on J-protein expression in PMNLs.

Objectives

Establishment of the promyeloid precursor cell line HL60 as a model for studies on the expression of several members of the HSP70/J-protein families on the level of mRNA and protein. Investigation of PMNLs from healthy donors (HD) and RA derived from peripheral blood (PB) and synovial fluid regarding the presence of members of the HSP70/J-protein chaperone machines.

Methods

HL60 cells were cultivated in FCS-supplemented RPMI 1640 medium. PMNLs were separated by density gradient centrifugation followed by hypotonic lysis of erythrocytes. Total RNA was isolated and contaminating DNA was eliminated by Dnase digestion when necessary. Gene-specific primers complementary for various genes of HSP70s and J-proteins were designed and applied in RT-PCR. The presence and amount of several HSP70s and J-proteins were analysed by western blots, applying equal amounts of protein per lane.

Results

In unstimulated HL60 cells, transcripts of the constitutive member of the HSP70 family hspa8 (Hsc70) and several inducible HSP70s (hspa1b, hspa5, hspa6) could be verified. No hspa2 transcript was detected. The same expression pattern of these five genes was observed in PB-PMNLs from HD. RT-PCR revealed a clear signal for all J-proteins under analysis (Hdj2, HTid, Hsj1, Mdg1, Tpr2 and Sec63) in HL60. In contrast, we observed a complete absence of these J-protein transcripts in PB-PMNLs from HD. Interestingly, in PMNLs from RA some J-protein transcripts were expressed in PB-PMNLs or synovial fluid PMNLs. On the protein level, Hsp70, Hsc70 and BiP were detected in HL60, as well as Hdj2 and HTid. In contrast, Hsc70 and Hdj2 showed a reduced expression, and moreover BiP and HTid were under the level of detection in HD PB-PMNLs.

Conclusions

We observed a distinct expression of several members of the HSP70/J-protein chaperone machines in PMNLs from HD or RA, and HL60. We assume that the observed reduction in protein expression of the HSP70/J-protein chaperone machines and the complete missing of J-protein transcripts contributes to the pro-apoptotic state of unstimulated PMNLs in the periphery. We suggest that the reappearance of several J-protein transcripts may be involved in the altered phenotype and prolonged survival of PMNLs in RA.