Hyperthermia promotes and prevents respiratory epithelial apoptosis through distinct mechanisms

Abstract

Hyperthermia has been shown to confer cytoprotection and to augment apoptosis in different experimental models. We analyzed the mechanisms of both effects in the same mouse lung epithelial (MLE) cell line (MLE15). Exposing MLE15 cells to heat shock (HS; 42°C, 2 h) or febrile-range hyperthermia (39.5°C) concurrent with activation of the death receptors, TNF receptor 1 or Fas, greatly accelerated apoptosis, which was detectable within 30 minutes and was associated with accelerated activation of caspase-2, -8, and -10, and the proapoptotic protein, Bcl2-interacting domain (Bid). Caspase-3 activation and cell death were partially blocked by inhibitors targeting all three initiator caspases. Cells expressing the IκB superrepessor were more susceptible than wild-type cells to TNF-α-induced apoptosis at 37°C, but HS and febrile-range hyperthermia still increased apoptosis in these cells. Delaying HS for 3 hours after TNF-α treatment abrogated its proapoptotic effect in wild-type cells, but not in IκB superrepressor-expression cells, suggesting that TNF-α stimulates delayed resistance to the proapoptotic effects of HS through an NF-κB-dependent mechanism. Pre-exposure to 2-hour HS beginning 6 to16 hours before TNF-α treatment or Fas activation reduced apoptosis in MLE15 cells. The antiapoptotic effects of HS pretreatment were reduced in TNF-α-treated embryonic fibroblasts from heat shock factor-1 (HSF1)-deficient mice, but the proapoptotic effects of concurrent HS were preserved. Thus, depending on the temperature and timing relative to death receptor activation, hyperthermia can exert pro- and antiapoptotic effects through distinct mechanisms.

Figures

Figure 1.

Heat shock (HS) augments extrinsic apoptosis. (A) Mouse lung epithelial (MLE) cell line 15 (MLE15) cells were incubated with indicated concentration of recombinant mouse TNF-α for 24 hours at 37°C with or without a concurrent 2-hour 42°C HS, and survival was assessed by crystal violet staining residual adherent cells and measuring absorbance at 570 nm. Cell death was different between HS and no HS cells (P < 0.05 by MANOVA). (B) MLE15 cells were incubated with or without 0.3 ng/ml TNF-α for 24 hours at 37°C with 2-hour HS beginning at the indicated time after TNF-α; 37°C indicates no HS. (C) MLE15 cells were incubated for the indicated time at 37°C or 42°C with or without 2 ng/ml TNF-α, lysed, and immunoblotted for active caspase-3 (C3), poly ADP-ribose polymerase (PARP), and β-tubulin. PARP-CL, cleaved PARP; PARP-FL, full-length PARP. (D) MLE15 cells were treated as in (B), but with 2.5 μg/ml Jo2 anti-Fas antibody. (E and F) MLE15 cells were preconditioned with 2-hour HS beginning at the indicated time before induction of apoptosis with 0.3 ng/ml TNF-α (E) or 2.5 μg/ml Jo2 antibody (F), with or without concurrent 2-hour HS, and 24-hour survival assessed by crystal violet staining. (G) MLE15 cells without (left panel) or with 2-hour HS preconditioning beginning 16 hours before a 2-hour incubation at 37°C or 42°C, with or without 2 ng/ml TNF-α, were lysed and immunoblotted for active caspase-3 and PARP. All graphs show means (±SE) of six experiments. (B and D) *P < 0.05 versus TNF-α or Jo2 without HS. (E and F) †P < 0.05 and ¶P < 0.05 versus similarly preconditioned and treated cells without concurrent HS and cells that were sham (37°C) preconditioned. A570, absorbance at 570 nm.

Figure 2.

The effects of HS on TNF-α–induced initiator caspase and Bcl2-interacting domain (Bid) activation. (A–G) MLE15 cells were incubated with 2 ng/ml TNF-α at 37°C or 42°C and sequentially lysed for fluorimetric (A, C, and E) and immunoblot (B, D, and F) analysis of caspase-8 (A and B), caspase-10 (C and D), and caspase-2 (E and F), and for immunoblot analysis of Bid (G). Caspase and Bid cleavage products are indicated by arrows (B, D, F, and G). Full-length Bid (Bid-FL) and caspases are indicated by large arrowhead. All graphs show means (±SE) of four experiments; *P < 0.05 versus 37°C. (B, D, F, and G) Each panel is representative of four similar blots. (H) MLE15 cells were pretreated with inhibitors of caspase-2, -8, and -10 (50 nM each) alone or in combination for 40 minutes at 37°C, then were incubated at 42°C with or without 2 ng/ml TNF-α and immunoblotted for activated caspase-3. A representative of three similar blots is shown. tBid, truncated Bcl2-interacting domain.

Figure 3.

Cytotoxic effect of HS persists when NF-κB signaling is blocked. (A) MLE15 cells stably transfected with IκB superrepressor (IκBSR) expression plasmid (upper panel) or empty plasmid (lower panel) were treated with 0.05 to 2 ng/ml TNF-α at 37°C for 24 hours with or without a concurrent 2-hour 42°C HS and survival assessed by crystal violet staining. (B) IκBSR-expressing MLE15 cells were incubated with 2 ng/ml TNF-α at 37°C or 42°C, sequentially lysed, and immunoblotted for active caspase-3. (C) MLE15 cells stably transfected with IκBSR expression plasmid (upper panel) or empty plasmid (lower panel) were incubated at 37°C for 24 hours with or without 0.3 ng/ml TNF-α and with 2-hour 42°C HS initiated at the times indicated, and survival was assessed by crystal violet staining. Control cells received no HS. (D) Wild-type (WT) and IκBSR-expressing MLE15 cells incubated with or without 0.3 ng/ml TNF-α were exposed to 2-hour 42°C HS initiated at the times indicated. Cells were lysed at the end of HS and immunoblotted for active caspase-3. Control cells (WT-37°C and IκBSR-37°C) were incubated in parallel without HS. All graphs show means (±SE) of four experiments; (A) 42°C is significantly different from no HS control by MANOVA (P < 0.0001); (C) *P < 0.05, †P < 0.05, and §P < 0.05 versus no–TNF-α control, the same HS exposure without TNF-α, and TNF-α without HS, respectively.

Figure 4.

HSF1 is required for cytoprotective effect of HS pre-conditioning but not for the cytotoxic effect of concurrent HS and TNF-α treatment. (A) WT and HSF1-knockout (KO) mouse embryonic fibroblasts (MEFs) were preconditioned with 2-hour 42°C HS beginning at the indicated time, treated with 4 ng/ml TNF-α and concurrent 2-hour HS, incubated for an additional 2 hours at 37°C, and cell survival was assessed by crystal violet staining. Control-treated cells were incubated in parallel at 37°C without TNF-α or HS. (B) WT and HSF1-KO MEFs were incubated with or without 4 ng/ml TNF-α and with or without a concurrent 2-hour 42°C HS, incubated at 37°C for an additional 2 hours, and cell survival was assessed by crystal violet staining. Means (±SE) of four experiments are shown. (A) WT is significantly different from HSF1-KO (P = 0.04 by MANOVA). (B) *P < 0.05 versus all three other treatments.

Figure 5.

Concurrent exposure to febrile-range hyperthermia (FRH) (39.5°C) augments extrinsic apoptosis through NF-κB– and HSF1-independent pathways. (A) MLE15 cells were incubated for 24 hours at 37°C or 39.5°C with the indicated doses of TNF-α and cell survival was measured by crystal violet staining. (B) MLE15 cells were treated with 2 ng/ml TNF-α at 37°C or 39.5°C for the indicated time, and cells were lysed and immunoblotted for active caspase-3, PARP, and β-tubulin. (C) IκBSR-expressing MLE15 cells were treated as described in (A) and cell survival assessed by crystal violet staining. (D) WT and HSF1-KO MEFs were incubated for 4 hours at 37°C or 39.5°C with or without 4 ng/ml TNF-α and cell survival was assessed by crystal violet staining. (E–K) MLE15 cells were incubated with 2 ng/ml TNF-α at 37°C or 39.5°C and sequentially lysed for fluorimetric (E, G, and I) and immunoblot (F, H, and J) analysis of caspase-8 (E and F), caspase-10 (G and H), and caspase-2 (I and J), and for immunoblot analysis of Bid (K). (F, H, J, and K) Caspase and Bid cleavage products are indicated by arrows. Full-length Bid and caspases are indicated by large arrowhead. All graphs show means (±SE) of four experiments; *P < 0.05 versus 37°C. (A and C) There was a difference between 37°C and 39.5°C and between TNF-α–treated and untreated at 37°C (P < 0.0001 by MANOVA). (F, H, I, J, and K) Each panel is representative of four similar blots. (L) MLE15 cells were pretreated with inhibitors of caspase-2, -8, and -10 (50 nM) alone or in combination for 40 minutes at 37°C, then were incubated at 39.5° with or without 2 ng/ml TNF-α and immunoblotted for activated caspase-3. A representative of three similar blots is shown.

Figure 5.

Concurrent exposure to febrile-range hyperthermia (FRH) (39.5°C) augments extrinsic apoptosis through NF-κB– and HSF1-independent pathways. (A) MLE15 cells were incubated for 24 hours at 37°C or 39.5°C with the indicated doses of TNF-α and cell survival was measured by crystal violet staining. (B) MLE15 cells were treated with 2 ng/ml TNF-α at 37°C or 39.5°C for the indicated time, and cells were lysed and immunoblotted for active caspase-3, PARP, and β-tubulin. (C) IκBSR-expressing MLE15 cells were treated as described in (A) and cell survival assessed by crystal violet staining. (D) WT and HSF1-KO MEFs were incubated for 4 hours at 37°C or 39.5°C with or without 4 ng/ml TNF-α and cell survival was assessed by crystal violet staining. (E–K) MLE15 cells were incubated with 2 ng/ml TNF-α at 37°C or 39.5°C and sequentially lysed for fluorimetric (E, G, and I) and immunoblot (F, H, and J) analysis of caspase-8 (E and F), caspase-10 (G and H), and caspase-2 (I and J), and for immunoblot analysis of Bid (K). (F, H, J, and K) Caspase and Bid cleavage products are indicated by arrows. Full-length Bid and caspases are indicated by large arrowhead. All graphs show means (±SE) of four experiments; *P < 0.05 versus 37°C. (A and C) There was a difference between 37°C and 39.5°C and between TNF-α–treated and untreated at 37°C (P < 0.0001 by MANOVA). (F, H, I, J, and K) Each panel is representative of four similar blots. (L) MLE15 cells were pretreated with inhibitors of caspase-2, -8, and -10 (50 nM) alone or in combination for 40 minutes at 37°C, then were incubated at 39.5° with or without 2 ng/ml TNF-α and immunoblotted for activated caspase-3. A representative of three similar blots is shown.