Fig 1: Proteinase K-agarose digestion ablates HSP70 activity. (A) 200 μg rhHSP70 was incubated with digestion buffer (lane 1), Proteinase K-agarose beads (lane 2), or control agarose beads (lane 3) for 3 hr at 37°C, as described in Methods. Protein integrity and endotoxin content were determined by silver staining and the LAL assay, respectively. (B) 100 pg of ultrapure LPS, which is an approximation of the endotoxin content in 200 μg of rhHSP70 (see Results and Discussion), was incubated with Proteinase K-agarose beads or control agarose beads, under the conditions described in A. The ultrapure LPS treated with Proteinase K-agarose beads (PK) or control agarose beads (CTRL) were then used to stimulate primary human macrophages for 16 hr at 100 pg/mL LPS final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. (C) The HSP70 preparations in A were used to stimulate primary human macrophages for 4 hr at 10 μg/mL final concentration. Cell culture supernatants were collected and analyzed for TNF-α. Endotoxin levels shown at bottom are the calculated amounts in the macrophage cultures based on the LAL determinations in A. Untreated macrophage cultures produced ≤ 100 pg/mL of TNF-α under these conditions. Results shown are representative of two independent digestion and stimulation experiments. ***p < 0.0005.
Fig 2: The stimulatory effect of recombinant HSP70 on primary human macrophages is reduced by inhibition of TLR4, or by Polymyxin B treatment of the HSP70. (A) Primary human macrophages were left untreated (NT), or treated with 100 μg/mL of HTA125 or an isotype control antibody for 30 min. Macrophages treated with antibodies were then stimulated for 4 hr with rhHSP70 at a final concentration of 10 μg/mL. Cell culture supernatants were collected and analyzed for TNF-α. **p < 0.005. (B) 5 μg rhHSP70 was pretreated with buffer alone or 10 μg Polymyxin B (at 100 μg/mL) for 30 min on ice, and then used to stimulate primary human macrophages for 4 hr at 10 μg/mL rhHSP70 final concentration in each culture. Cell culture supernatants were collected and analyzed for TNF-α. *p < 0.05, ***p < 0.0005.
Fig 3: Upon Hsp70 engagement, Tlr4 initiates a pro-inflammatory response via Myd88- and Trif-dependent signaling cascades. A) Expression analysis of Tlr4 signaling cascade genes in Hsp70 (5 μg/ml) and PBS-treated glial cells demonstrated increased expression of Myd88 in wild type astrocytes (**P < 0.01, n = 6; WTa and WTm referred to WT astrocytes and WT microglia; Tlr4KOa and Tlr4KOm referred to Tlr4KO astrocytes and Tlr4KO microglia). B) A comparative analysis of Tlr4, Myd88, Trif, Ripk1 gene expression in wild type and Tlr4KO glial cells suggests the preferential activation of Myd88 signaling cascade. C) The amount of Tnf in culture media from wild type, Tlr4KO, Myd88KO and TrifKO glial cells treated with Hsp70 and PBS (control) (**P < 0.01, n = 6). D) Two main downstream branches of Tlr4 signaling. Hsp70, heat shock protein 70; PBS, phosphate buffering saline; WT, wild type.
Fig 4: Extracellular Hsp70 is accumulated in the vitreous humor of animals suffering from retinal ischemia: A) Level of Hsp70 in vitreous humor of sham-operated and ischemic eyes 6 and 24 hours after reperfusion. B) Representative western blot. h, hours; Hsp70, heat shock protein 70; IR, ischemic reperfusion.
Fig 5: PMB in Hsp70-treated astrocytes behaves as PKC inhibitor rather than endotoxin inhibitor. A) Pretreatment of astrocytes only with the highest PMB concentration (10 μg/ml) caused significant reductions in the pro-inflammatory activity induced by the recombinant Hsp70, while the pro-inflammatory response of LPS-treated astrocytes was significantly reduced by the addition of 1 and 10 μg/ml of PMB. Primary astrocytes were treated with Hsp70 (5 μg/ml) containing 1.5 pg/ml of endotoxins or LPS (2.5 ng/ml) in the presence or absence of PMB (**P < 0.01, n = 6). Differential expression of cytokines and chemokines was assessed in primary astrocytes after 24 hours by using quantitative RT-PCR. B) The amounts of Tnf in culture media from astrocytes treated with Hsp70 (5 μg/ml) or LPS (2.5 ng/ml) in the presence or absence of PMB were measured by using ELISA (**P < 0.01, *P < 0.05, n = 6). C) The level of Tnf was significantly reduced in media from astrocyte cultures treated with Hsp70 in the presence or absence of PKC inhibitor (Gö6976, 10 nM and 100 nM). Culture media was collected 24 hours after treatment, and then the Tnf level was detected using the ELISA kit (**P < 0.01, n = 6). Hsp70, heat shock protein 70; LPS, lipopolysaccharides PKC, protein kinase C; PMB, polymyxin B.
Supplier Page from Enzo Life Sciences, Inc. for HSP70 (low endotoxin) (human), (recombinant)