Interleukin-1α is the major alarmin of lung epithelial cells released during photodynamic therapy to induce inflammatory mediators in fibroblasts

Abstract

Background: Photodynamic therapy (PDT) causes tissue damage that initiates a local inflammatory response. Post-PDT reactions are considered to assist in mobilising the immune system thereby affecting tumour recurrence. The initiating process of the PDT-dependent tissue reaction remains to be determined.

Methods: Primary cultures of human lung cells were established. The photoreaction mediated by pyropheophorbide-a, at specific subcellular sites and levels resulting in the release of alarmins by epithelial cells (Eps), was defined by immunoblot analyses and expression profiling. The activity of Ep-derived factors to stimulate expression of proinflammatory mediators, including IL-6, and to enhance neutrophil binding by fibroblasts (Fbs) was determined by functional bioassays.

Results: Epithelial cells release IL-1β as the primary Fb-stimulatory activity under basal conditions. Intracellular IL-1α, externalised following photoreaction, accounts for most of the PDT-mediated Fb activation. Expression of IL-1 is subject to increase or loss during oncogenic transformation resulting in altered alarmin functions mobilisable by PDT. Photoreaction by a cell surface-bound photosensitiser (PS) is 10-fold more effective than PSs localised to mitochondria or lysosomes. High-dose intracellular, but not cell surface, photoreaction inactivates IL-1 and reduces Fb stimulation.

Conclusion: These in vitro data suggest that the subcellular site and intensity of photoreaction influence the magnitude of the stromal cell response to the local damage and, in part, support the relationship of PDT dose and level of post-PDT inflammatory response observed in vivo.

Figure 1

Fibroblasts as target for irritants and alarmins. (A) Monolayers of primary cultures of N-Fb, passage 4, were treated for 15 min with the irritants or cytokines listed at the bottom. The changes in the phosphorylation of the signalling proteins indicated on the right were determined by western blotting. (B, C) N-Fb were treated for 24 h as indicated and then were exposed for 15 min to CSFE-stained neutrophils. Adherent neutrophils were imaged by CSFE-fluorescence (B, bottom panel, and C) and the change in the expression of ICAM-1 as well as the neutrophil-associated haptoglobin determined by western blotting (B, upper panels). (D) separate cultures of N-Fb were treated with the cytokines indicated at the bottom and analysed for the 15-min response to activate NFkB and p38 and for the 24-h response to induce ICAM-1 and binding of CSFE-stained neutrophils. (E) Baseline and IL-1β-inducible IL-6 production by early (P3–10) and late (P>10) passage culture of N-Fb and T-Fb were quantified by bioassay and Luminex immunobead-binding assays. Box plots compile data from 8 to 24 independent culture sets.

Figure 2

Effect of PDT on lung Eps. (A–E) Replicate N-Ep cultures, passage 2, in 6-well culture dishes were incubated for 30 min with the indicated concentrations of HPPH followed by 24 h incubation in HPPH-free medium. Mitochondrially localised HPPH was determined by fluorescent microcopy (A, × 100 magnification) and quantified (C, open circles). The cultures were exposed to 665 nm light. Cells from one culture set were immediately extracted and analysed for the indicated proteins by western blotting (B). The second set of cultures was incubated for an additional 24 h. The relative level of viable cells was determined (C, closed circle). Equivalent aliquots of culture supernatant and lysate of adherent cell material were analysed by western blotting for the indicated proteins (D). Additional aliquots of conditioned media were 10-fold diluted and used to treat N-Fb in 48-well plate for 24 h (E, F). The treatment-dependent change in the cellular expression of ICAM-1 was determined by western blotting (E), binding of neutrophils (F, open squares, LUx10⁷) and release of IL-6 activity determined on H-35 cell assay (F, closed squares). The same diluted EC-conditioned media were used to treat primary pulmonary macrophages for 24 h for determining the effect on IL-6 production (G). (H, I) Primary cultures of BEC (n=12), N-Ep (n=12), T-Ep (n=12) (H) and established Ep lines (I) were analysed for basal line and PDT-induced (50 nℳ HPPH or 100 nℳ HPPH-Gal) release of FSA during the 24-h post-PDT culture period. Box plots represent compilation of data derived from separate cell preparations representing passages 2 to 7 (H) and repeat (n=3–12) measurements on individual cultures of established lines for up to passage 90 (I).

Figure 3

Kinetics of FSA and DAMP release by PDT-treated T-Ep. Adenocarcinoma-derived T-Ep, passage 4, in a 6-well plate was treated with the indicated concentrations of HPPH for 30 min followed by a 24-h culture period. After light treatment, the cultures were incubated for additional 24 h with periodic collection of CM and replacement with fresh media. Aliquots of the culture media were three-fold diluted, used to determine FSA by treating N-Fb for 24 h and determining the IL-6-dependent induction of GFP in H-35 cells (A). Equal aliquots of Ep-conditioned media were analysed for the indicated proteins by western blotting (B).

Figure 4

Size separation of proteins in CM of PDT-treated T-Ep. Duplicate cultures of T-Ep, passage 3, in a 10-cm-diameter culture dish were treated either as light control or with 25 nℳ HPPH-PDT. Conditioned medium 2 and 24 h post PDT were concentrated and subjected to chromatography on a G-150 Sephadex column. Proteins in each fraction were analysed for the indicated proteins (A, upper panel), FSA in the coupled Fb-H-35 cell assay (B) and for induced neutrophil binding to the treated Fb (C). Detection of the FSA activity by chromatographed COS-1 cell-derived IL-1α is included in (B and C). The centrifuged homogenate of untreated T-Ep from one 10-cm dish was similarly size fractionated and the entire fractions were analysed for IL-1α protein by western blotting (A, lower panel). The elution positions of the molecular size markers (in kDa) are indicated at the top of the column elution profile.

Figure 5

Identification of IL-1α and IL-1β as Ep-derived FSA and alarmin. Conditioned medium of control Ep cultures (Ep-PDT) and PDT-treaded Ep cultures (Ep+PDT), whole-cell homogenate of control Ep and Sephadex G-150-purified 17 kD FSA were reacted with antibodies against IL-1α or IL-1β as indicated and then tested for the ability to activate signalling (A), to enhance IL-6 that induces GFP expression in H-35 cells (B) and to increase neutrophil binding (C).

Figure 6

Enhanced cell damage and release of FSA by cell surface-restricted photoreaction. A, 100 nℳ HPPH-Gal was bound to the cell surface of T-Ep, passage 3, during 30-min incubation at 4° C followed by internalisation and lysosomal deposition during a 24-h incubation at 37° C. Fluorescent micrographs of the cultures at × 100 magnification are shown. (B, C) Replicate sets T-Ep cultures in 6-well dishes were reacted with the indicated concentrations of HPPH-Gal restricted to cell surface or to be internalised. The cell-associated fluorescence was quantified (B, open circles) and the cells in all sets were treated on ice with 665 nm light. One set of cultures was immediately lysed and analysed by western blotting to the extent of STAT3 crosslinking and loss of EGFR (C). The other set was cultured for an additional 24 h to determine survival rate (B, closed circle). (D, E) Duplicate set of T-Ep, passage 5, were subjected to HPPH-Gal binding to cell surface or lysosomal accumulation at the indicated HPPH-Gal dose. The photoreaction was carried out at 0° C and the cultures were incubated for an additional 24 h. Conditioned media collected after 2 h and 24 h were analysed for FSA on N-Fb cultures (D). Equivalent aliquots of cell lysates and conditioned media were analysed by western blotting for the indicated DAMPs (E). (F, G) Replicate cultures of T-Ep in 6-well plates were subjected to high-dose mitochondrial HPPH-PDT (F) or cell-surface HPPH-Gal PDT (G) with light treatment carried out on ice. After 4-h post-PDT incubation, the adherent cells, combined with cellular debris recovered by centrifugation of CM, were homogenised. Aliquots of the cell homogenates and cell-free-CM were analysed for the amount of IL-1 activity per culture.