Heat Shock Protein 27 and 60 Directly Activate Human Microglia

Purpose : We previously reported the first convincing evidence demonstrating that glaucoma has an autoimmune component caused by commensal bacteria-primed CD4+ T cells that entered the retina and cross-reacted with heat shock protein (HSP)-expressing neurons via a mechanism of molecular mimicry. Our subsequent studies implicate that microglial activation is a cause of the immune responses and retinal degeneration in glaucoma. Since the limited availability of primary human microglia, immortalized human microglia clone 3 cell line (HMC3) is useful for the examination of the microglia behavior under pathological conditions. To test if HSP27 and HSP60 could induce activation of microglia, we examined the response of cytokines expression and morphological changes of HMC3. Other known inflammatory stimulators were used as the positive control. Methods : HMC3 cell line (ATCC) were cultured in EMEM medium and challenged with 10ug/ml HSP27, 10ug/ml HSP60, 200ng/ml LPS or 100ng/ml LPS with or without 5mM ATP for additional 30 minutes. Cells received medium alone were used as controls. After 24 hours, RNAs of HMC3 cells were collected by ZYMO Research Quick-RNA Microprep Kit, and RNA reverse transcript was carried out by PrimeScript™ RT Master Mix. Sybr green RT-PCR mixtures containing different primers and cDNA samples were subjected to PCR using EP realplex real-time PCR system. Relative fold changes of mRNA transcripts were presented and compared with the control group. Moreover, low density HMC3 cell cultures were set up, and images of cell morphology were captured 24 hours after LPS, HSP27 or HSP60 treatment, and the morphology changes were quantified. Results : Our data showed that while LPS with or without ATP induced increased expression of pro-inflammatory cytokines such as IFNγ in HMC3, HSP27 and HSP60 could also activate HMC3 to express higher level of IFNγ and TNFα. Quantification of cell morphology showed shortened dendritic processes and enlarged round cell body size in LPS, HSP27 and HSP60 stimulated groups compared to vehicle controls (P<0.05). Conclusions : The present study revealed that HMC3 cells reacted similarly as primary microglia to the known inflammation stimulators. HSP27 and HSP60 could directly induce pro-inflammatory responses of HMC3 cells, supporting a notion that HSPs may induce microglial activation as an early cause of glaucoma-associated immune responses.