Nuclear factor-kappaB regulates inflammatory cell apoptosis and phagocytosis in rat carrageenin-sponge implant model

Abstract

In the present study we investigated whether apoptosis and phagocytosis are regulated by nuclear factor (NF)-kappaB in a model of chronic inflammation. The subcutaneous implant of lambda-carrageenin-soaked sponges elicited an inflammatory response, characterized by a time-related increase of leukocyte infiltration into the sponge and tissue formation, which was inhibited by simultaneous injection of wild-type oligodeoxynucleotide decoy to NF-kappaB. Molecular and morphological analysis performed on infiltrated cells demonstrated: 1) an inhibition of NF-kappaB/DNA binding activity; 2) an increase of polymorphonuclear leukocyte apoptosis correlated either to an increase of p53 or Bax and decrease of Bcl-2 protein expression; and 3) an increase of phagocytosis of apoptotic polymorphonuclear leukocytes by macrophages associated with an increase of transforming growth factor-beta1 and decrease of tumor necrosis factor-alpha as well as nitrite/nitrate production. Our results, showing that blockade of NF-kappaB by oligodeoxynucleotide decoy increases inflammatory cell apoptosis and phagocytosis, may contribute to lead to new insights into the mechanisms governing the inflammatory process.

Figure 1

Leukocyte infiltration (A) and granulomatous tissue formation (B). Data are expressed as mean ± SEM of 15 to 20 sponges from 9 to 10 rats. °°°, P < 0.0001 versus saline; ***, P < 0.0001 versus λ-carrageenin alone.

Figure 2

Morphological summary depicting cell migration, tridimensional organization, and activation status in the sponge after treatments. A: Light microscopy: relationship between spaces invaded by cells (outlined areas) and exudate as well as debris. B: Scanning electron microscopy: presence of cells organized even in clusters with debris (black asterisk) and exudate (white asterisk) areas. C: Transmission electron microscopy: ultrastructural features of cell populations with cytosol suggestive of phagocytosis as well as autophagy (open arrows) and nuclei involution (filled arrows) Fields are representative of three experiments. Original magnifications: ×100 (A); ×2400 (B); ×6400 to 8900 (C).

Figure 3

Leukocyte population infiltrated into the sponge. Data are expressed as mean ± SEM of three sponges from three rats. Cell counting was performed as reported in Materials and Methods.

Figure 4

A: Representative electrophoretic mobility shift assay showing NF-κB/DNA binding activity in nuclear extract from inflammatory cells. Data are from a single experiment and are representative of six separate experiments. B: Characterization of NF-κB/DNA complex was performed on λ-carrageenin-treated cells harvested on 1 day after implant. In competition reaction nuclear extracts were incubated with radiolabeled W.T. ODN probe in the absence or presence of identical but unlabeled oligonucleotide (W.T., 50×) or mutated nonfunctional ODN probe (Mut, 50×). In addition, nuclear extracts were incubated with radiolabeled NF-κB probe in absence or presence of: identical but unlabeled oligonucleotide (W.T., 50×), mutated nonfunctional NF-κB probe (Mut, 50×) or unlabeled oligonucleotide containing the consensus sequence for Sp-1 (Sp-1, 50×). In supershift experiments nuclear extracts were incubated with antibodies against p52, c-Rel, RelB, p50, and p65 15 minutes before incubation with radiolabeled NF-κB probe. Data are from a single experiment and are representative of six separate experiments. C: Representative Western blot of p50 and p65 nuclear level from cells collected on 1 day after implant. Histone 1 expression is shown as control. Data are from a single experiment and are representative of three separate experiments. D: Immunohistochemical analysis of p65 protein expression in cells from saline (a), λ-carrageenin (b), W.T. ODN (c), and Mut ODN (d) decoy-treated sponges harvested on 1 day after implant. Fields are representative of three experiments. Original magnifications, ×320 (D).

Figure 5

A: Morphological features of apoptosis (arrows) in PMNs from λ-carrageenin (a–c) and W.T. ODN decoy-treated sponges (d–f). Fields are representative of three experiments. B: PMN apoptosis was evaluated by FACS using annexin V staining. Data are expressed as mean ± SEM of three to five sponges from three to five rats. **, P < 0.001; ***, P < 0.0001 versus λ-carrageenin alone. Original magnifications, ×320 (A).

Figure 6

Representative Western blot of p53 (A), Bax, and Bcl-2 (B) protein expression in nuclear and cytosolic extracts from inflammatory cells. Histone 1 and β-actin expression are shown as control, respectively. Data are from a single experiment and are representative of three to four separate experiments.

Figure 7

Immunohistochemical summary of p53 (A), Bax (B), and Bcl-2 (C) protein expression in inflammatory cells after treatments. Fields are representative of three experiments. Original magnifications, ×320.

Figure 8

A: Morphological appearance of MΦ phagocytosis (arrows) from λ-carrageenin (a–c) and W.T. ODN decoy-treated sponges (d–f). Fields are representative of three experiments. B: Phagocytosis was evaluated as phagocytic percent. Data are expressed as percentage of mean ± SEM of MΦ containing at least one ingested apoptotic PMN (n = 3 sponges from three rats). *, P < 0.05; ***, P < 0.0001 versus λ-carrageenin alone. Original magnifications, ×320 (A).

Figure 9

Effect of W.T. ODN decoy on TGF-β1 (A), TNF-α (B), and NOx (C) production by inflammatory exudate. Data are expressed as mean ± SEM of three experiments in triplicate. °, P < 0.05; °°, P < 0.001; °°°, P < 0.0001 versus saline; *, P < 0.05; **, P < 0.001; ***P < 0.0001 versus λ-carrageenin alone.