TLR2-induced calpain cleavage of epithelial junctional proteins facilitates leukocyte transmigration

Abstract

Recruitment of polymorphonuclear leukocytes (PMNs) into the lungs in response to inhaled pathogens is initiated by epithelial signaling, the activation of toll-like receptors (TLRs), and the production of the chemokine interleukin-8. To reach the site of infection, PMNs must be mobilized through epithelial junctions. Here, we demonstrate that Ca(2+) fluxes generated by TLR2 signals activate calpains, Ca(2+)-dependent cysteine proteases. These activated calpains cleave the transmembrane junctional proteins occludin and E-cadherin without breaching the integrity of the epithelial barrier. Calpain inhibitors decrease PMN transepithelial migration in response to TLR2 agonists both in vitro and in a mouse model of P. aeruginosa infection. Thus, TLR2 signaling in the airway not only induces chemokine expression to recruit PMNs, but also initiates cleavage of junctional proteins to accommodate transmigration of the recruited PMNs.

Figure 1

Epithelial cell junctions are affected by bacterial stimulation. Polarized 16HBE cells were incubated for 4 h with (A) media, (B) heat killed PAO1, (C) P3C or (D) thapsigargin. Cells were incubated with biotin detected with Alexa Fluor 555 conjugated streptavidin (red) and stained for ZO-1 (green). Apical to basolateral x-y scans are shown with corresponding z-sections below. Data is representative of at least three separate experiments.

Figure 2

Occludin and E-cadherin are targets for calpain cleavage. (A) Distribution of occludin (red) and E-cadherin (green) is altered following 6 h incubation with media, heat killed PAO1 or P3C. Cells were stained with DAPI (blue). Data is one representative of at least three separate experiments. (B) In the presence of 20 mM CaCl₂, 1 μg of exogenous human calpain 1 cleaved occludin and E-cadherin but not JAM-1 or claudin-1 immunoprecipitated from 1HAEo- cell lysates. The exogenous calpain 1 in these reactions is autolysed upon activation in the presence of Ca²⁺. * Shorter exposure time on the three right bands compared to the three left bands. Data is one representative of at least three separate experiments.

Figure 3

TLR2 mediated activation of calpain. (A)1HAEo- cells or (B) Human small airway epithelial cells in primary culture (SAEC) were loaded with a fluorogenic calpain substrate, t-BOC L-leucine L-methionine, and incubated with heat killed PAO1 or P3C in the presence of 20 μM calpeptin or DMSO vehicle control at the indicated times. (C) 1HAEo- cells expressing scrambled or TLR2 siRNA were stimulated with heat killed PAO1 at the indicated times. The fluorescence was quantified with ex 360 nm, em 465 nm and baseline fluorescence was subtracted. Data represents the mean ± s.d. of quadruplicate samples and is one representative of three independent experiments. (* P<0.05 compared with media alone controls; Student’s t-test).

Figure 4

Occludin is a calpain substrate. (A) Co-localization of calpain (green) and occludin (red) in polarized 16HBE cells following 1 h stimulation with heat killed PAO1 or P3C is shown in xy and xz sections. The adjacent panel shows an enlargement of white boxed area of the xy image. (B) Immunoprecipitation of occludin from 1HAEo- cell following stimulation with heat killed PAO1 or P3C and detection of occludin, pan-calpain, calpain 1 and calpain 2 by immunoblot. Data are representative of at least three separate experiments. (C) Immunoprecipitation of occludin from 1HAEo- cells following heat killed PAO1 stimulation and detection of the 80 kD hyperphosphorylated form of occludin, 60 kD full length form of occludin and 45kD cleavage fragment of occludin. (D) Detection of occludin cleavage in 1HAEo- cells overexpressing TLR2 WT or TLR2 Y616A/Y761A (TLR2YY). (E, F) Detection of occludin cleavage in cells treated with 6 μM BAPTA/AM, 20 μM ALLN, 20 μM GM6001, or 25 μM Z-DEVD-FMK. (G) Detection of occludin cleavage in P3C stimulated scrambled control or calpain 1 and 2 siRNA (CAPN 1 & 2) expressing cells. Silencing of calpain 1 and 2 expressions in scrambled control and siRNA expressing cells is shown in the adjacent panel. (H) Neutravidin immunoprecipitation (IP) from biotinylated 1HAEo- cells that were transfected (Txf) with C-terminal myc6 tagged occludin (Occ myc6) or N-terminal RFP tagged occludin (RFP Occ) and incubated with media alone (M) or heat killed PAO1 (PAO1) were immunoblotted (WB) with anti-myc or anti-RFP antibodies. Cartoon illustrates the forms of occludin that correspond with the bands on the immunoblot. (Data are representative of at least three separate experiments).

Figure 5

E-cadherin is a calpain substrate. (A) Co-localization of calpain (red) and E-cadherin (green) in polarized 16HBE cells following 1 h stimulation with heat killed PAO1, P3C amd Thaps is shown in xy and xz sections. The adjacent panel shows an enlarged version of the xy merged image. (B) Immunoprecipitation (IP) of E-cadherin from 1HAEo-cells incubated with media alone (M) thapsigargin (Thaps) or heat killed PAO1 and detection of E-cadherin and calpain by immunoblot. (C) Identification of the E-cadherin cleavage product in 1HAEo- cells following 4 h stimulation with thapsigargin as compared with media (M) control. (D) Detection of E-cadherin cleavage products in 1HAEo- cells following 4 h stimulation with heat killed PAO1 or P3C in the presence or absence of 20 μM calpeptin. Data is representative of at least three separate experiments.

Figure 6

PMN transmigration across airway epithelial monolayer is facilitated by calpain. (A) The number of PMNs that have migrated into the apical compartment of 16HBE monolayers stimulated with heat killed PAO1 or P3C in the presence or absence of calpeptin. (B) The migration of 10⁶ calcein-AM labeled PMNs across a Transwell in response to 10⁸ CFU/ml live PAO1, 10⁸ CFU/ml HKPAO1 or 10 nM fMLP was not affected by the presence of 20 μM calpeptin. Data is presented as number of PMNs multiplied by 10⁴ and represents mean ± s.d. of sextuplicate samples of one representative from three independent experiments. (P< 0.05, P<0.001; Student’s t-test).

Figure 7

Calpain activity contributes to PMN recruitment in response to P. aeruginosa infection. (A) The % of PMNs in a single cell suspension of lung was quantified in wild type or tlr2^−/− pups treated with i.p. calpeptin or vehicle (Un) and intranasally inoculated with 10⁸ CFU PAO1 or PBS. (B) Lung suspensions from representative mice in (A) were immunoblotted for occludin and E-cadherin cleavage products. (C) Adult wild type mice treated with i.p calpeptin or vehicle (Un) were intranasally inoculated with 10⁹ CFU PAO1 or PBS for 2 h. BAL and lung cell suspensions were obtained and absolute numbers of PMNs enumerated by flow cytometry. (D) Bacterial counts in CFU/ml were determined from whole lung suspension. (E) KC mRNA expression was quantified from lung suspensions and KC protein levels determined from BAL. (A,C) Individual mouse values are shown, and the short horizontal lines indicate the median values of each group. (*P <0.05, ** P <0.01 compared with wild type PAO1 infected mice; non-parametric Mann-Whitney test).