Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway

Abstract

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.

Keywords: CD40; Per a 10; allergen; dendritic cell; proteolytic activity.

Fig 1

Effect of Per a 10 protease activity on CD40 expression by dendritic cells (DCs). (a) The histograms represent the CD40 expression by monocyte-derived DCs that were unstimulated (medium only), stimulated with proteolytically active or heat-inactivated Per a 10 and analysed by flow cytometry. (b) Mean fluorescence intensity (MFI) values of CD40 expression in unstimulated or stimulated DCs. The MFI value of samples is corrected with MFI of the isotype control. Data are representative of seven experiments. (c) Soluble CD40 level was estimated in culture supernatant of DCs stimulated with active or heat-inactivated Per a 10. Data (n = 7) are presented as mean ± standard deviation. *P < 0·05; ^†P < 0·05 in comparison to medium only.

Fig 2

Interleukin (IL)-12 secretion in the culture-supernatant of dendritic cells (DCs). (a) DCs (n = 7) were stimulated with proteolytically active or heat-inactivated Per a 10 for 48 h and evaluated for IL-12 secretion by enzyme-linked immunosorbent assay (ELISA). (b) DCs (n = 3) were pretreated with CD40 blocking antibody followed by stimulation with proteolytically active or heat-inactivated Per a 10 and tested for IL-12 secretion. Lipopolysaccharide (LPS) was used as a positive control. Data are presented as mean ± standard deviation (s.d.). *P < 0·05; ^†P < 0·05 in comparison to medium only.

Fig 3

Activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-kappa B (NF-κB) in Per a 10-stimulated dendritic cells (DCs). The DCs pretreated or not with inhibitor for (a) p38 MAPK or (b) NF-κB pathways and stimulated with active or heat-inactivated Per a 10 were analysed by Western blot. Well 1, immature DCs; 2, inhibitor alone; 3, inhibitor plus active Per a 10; 4, active Per a 10 alone; 5, inhibitor plus heat-inactivated Per a 10; and 6, heat-inactivated Per a 10 alone. (c) Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control and (d) expression of tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) in Well 1, immature DCs; 2, heat-inactivated Per a 10; and 3, inhibitor plus heat-inactivated Per a 10.

Fig 4

CD40 expression by dendritic cells (DCs) pretreated with (a) p38 mitogen-activated protein kinase (MAPK) inhibitor-SB203580 or (b) nuclear factor-kappa B (NF-κB) inhibitor Bay 11–7085 followed by stimulation with proteolytically active or heat-inactivated Per a 10. (c) Mean fluorescence intensity (MFI) values of CD40 expression of DCs pretreated with p38 MAPK or NF-κB inhibitor. Data are representative of 11 experiments performed. (d) Soluble CD40 levels in culture supernatant of DCs. ‘aPer a 10’ represents active Per a 10 and ‘hPer a 10’ represents heat-inactivated Per a 10. Data are representative of three experiments performed. *P < 0·05; ^†P < 0·05 in comparison to medium only.

Fig 5

Cytokine levels (a) interleukin (IL)-12, (b) interferon (IFN)-γ, (c) IL-6 and (d) tumour necrosis factor (TNF)-α in culture supernatant of mature dendritic cells (DCs) pulsed with proteolytically active or heat-inactivated Per a 10. DCs were pretreated with p38 mitogen-activated protein kinase (MAPK) inhibitor-SB203580 or nuclear factor-kappa B (NF-κB) inhibitor Bay 11–7085 and then stimulated with proteolytically active or heat-inactivated Per a 10. DCs treated with inhibitors alone were taken as internal control. Data (n = 11) are presented as mean ± standard deviation (s.d.). *P < 0·05; ^†P < 0·05 in comparison to medium only and heat-inactivated Per a 10 group.

Fig 6

Cytokine levels (a) interleukin (IL)-4, (b) IL-5, (c) IL-6, (d) IL-12p70, (e) IL-13, (f) interferon (IFN)-γ and (g) tumour necrosis factor (TNF)-α in culture supernatants of dendritic cell (DC)–T cell co-cultures. Autologous CD4⁺ T cells were co-cultured with proteolytically active or heat-inactivated Per a 10 [pretreated or not with signalling inhibitor (p38 MAPK inhibitor-SB203580 or nuclear factor-kappa B (NF-κB) inhibitor Bay 11–7085]-stimulated DCs. Data (n = 11) are presented as mean ± standard deviation (s.d.). *P < 0·05.

Fig 7

Diagrammatic representation of CD40-mediated signalling in human dendritic cells (DCs) in response to Per a 10 stimulation.