Neutrophil elastase converts human immature dendritic cells into transforming growth factor-beta1-secreting cells and reduces allostimulatory ability

Abstract

During microbial infection, neutrophils (polymorphonuclear leukocytes; PMNs) activate dendritic cells (DCs). However, early reports illustrated that neutrophil-derived mediators may suppress responses to mitogens. In the present study, we investigated the mechanism used by PMNs to modulate the immunostimulatory ability of DCs. Autologous syngeneic PMNs decreased T-cell proliferation induced by allogeneic DCs. Culture supernatant (CS) derived from PMNs also decreased allostimulation ability of immature DCs and increased the expression of transforming growth factor (TGF)-beta1 on DCs. A TGF-beta1 monoclonal antibody, a CD40 monoclonal antibody, or a serine protease inhibitor reversed the effect of PMN CS on DC allostimulatory ability. Furthermore, elastase reproduced the inhibitory effect of PMN CS on DC allostimulatory ability and the TGF-beta1 production. The role of elastase was confirmed by examining PMN CS from two patients with cyclic neutropenia, a disease due to mutations in the neutrophil elastase gene. These PMN CS samples had reduced elastase activity and were unable to increase DC TGF-beta1 production. Moreover, elastase and PMN CS induced IkappaBalpha degradation in DCs. We conclude that PMNs decrease DC allostimulatory ability via production of elastase leading to a switch of immature DCs into TGF-beta1-secreting cells.

Figure 1

Effect of PMNs on DC’s immunostimulatory ability. A: MLR induced by graded doses of viable allogeneic DCs in the presence of different concentrations of autologous (syngeneic) PMNs. Stimulator allogeneic DCs (1 to 8 × 10⁴) were incubated with 10⁵ responder PBMCs and 1.25 to 10 × 10⁴ syngeneic PMNs. Thus, responder cells (PBMCs) and PMNs were related to the donor and unrelated to DCs. B: MLR induced by viable allogeneic DCs (2 × 10⁴) in the presence of different concentrations of unprimed or LPS autologous (syngeneic) PMN-primed at two different time points. C: Allogeneic DCs (2 × 10⁴) were incubated with 10⁵ PBMCs, and 10⁵ syngeneic PMNs were added above or below a Transwell (TW) bare 0.4-μm polycarbonate filter. After 4 days, cells were pulsed with 1 μCi of [³H]thymidine per well for the last 16 hours of culture, harvested, and counted in a beta counter. Proliferation is depicted relative to the proliferation of MLR cultures in the absence of PMNs (100% = 30,739 ± 664 cpm). Relative proliferation is depicted as average means of triplicates ± SEM from a series of four experiments for A and three experiments for B and C. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with control group, using Dunnett’s multiple comparisons test.

Figure 2

Effect of PMN CS on DC’s immunostimulatory ability and surface molecule expression. A: MLR induced by allogeneic immature (iDCs) or previously LPS-treated DCs for 3 hours with unprimed PMN (uPMN) CS or pPMN CS. Priming was performed with IL-8, formyl-Met-Leu-Phe, or LPS followed by thorough washing. CS samples were harvested after 3 hours of culture of the PMNs. Next, CS samples were incubated with untreated or LPS-treated DCs for another 3 hours, and then the DCs were washed and cultured with 10⁵ PBMCs. After 4 days, cells were pulsed with 1 μCi of [³H]thymidine per well for the last 16 hours of culture, harvested, and counted in a beta counter. Proliferation is depicted relative to the proliferation of MLR cultures in the absence of PMNs (see Materials and Methods). Relative proliferation is depicted as average means of quadruplicates ± SEM from a series of five experiments *P < 0.05, **P < 0.01 compared with control group, using Dunnett’s multiple comparisons test. B: HLA class II, CD83, CD86, CD18, and CD40 expression on immature DCs untreated or treated with IL-8 pPMN CS. Immature DCs treated with IL-8 pPMN CS were stained with primary mAb and analyzed by flow cytometry as described in Materials and Methods. Shaded histograms are untreated DCs, which overlapped nearly completely the histogram of pPMN-CS-treated DCs. Data are representative of three independent experiments.

Figure 3

Expression of TGF-β1 on DCs treated with pPMN CS. A: Intracellular levels of TGF-β1 in untreated (dotted line histograms) and IL-8 pPMN-CS-treated (thick line histogram) DCs. Cells were treated as described for Figure 2. Afterward, DCs were incubated with brefeldin A (10 μg/ml, 3 hours, 37°C), fixed, and stained with anti-TGF-β1 R-phycoerythrin or control isotype Ab (filled histogram) and analyzed by flow cytometry. Similar results were obtained in three additional experiments. B: TGF-β1 concentration in DC-derived CS. After 3 hours of DC treatment with PMN CS, the cells were washed and incubated for another 24 hours in RPMI-HSA medium. Then the supernatants were recovered, diluted, and assessed for TGF-β1 by an ELISA. Data represent the mean ± SEM of five experiments. **P < 0.01 compared with untreated DCs using post hoc Student-Newman-Keuls analysis. C and D: Reverse transcription-polymerase chain reaction analysis for TGF-β1 and β-actin mRNA in untreated and IL-8 pPMN-CS-treated DCs. After 3 hours of treatment with pPMN CS, RNA was extracted from DCs. Data represent one representative experiment of three for C and the mean ± SEM for D. E: Effect of mAb to CD40 on TGF-β1 induced by DCs treated with IL-8 pPMN CS. After DCs were treated with IL-8 pPMN CS, the cells were incubated with CD40 mAb for another 3 hours. Then the supernatants were recovered, diluted, and assessed for TGF-β1 by ELISA. Data represent the mean ± SEM of four experiments. *P < 0.05; **P < 0.01 compared with untreated DC group using post hoc Student-Newman-Keuls analysis.

Figure 4

TGF-β1 activity. A: TGF-β1 bioactivity was assessed using the mink lung cell assay as described in Materials and Methods. Mink lung cells (20 × 10³ or 40 × 10³ cells/well) were incubated with DC-derived CS. After 22 hours, the incorporated [³H]thymidine (used as a marker of DNA synthesis) was measured. Data represent the mean ± SEM of five experiments; **P < 0.01 Student’s t-test. B: Effect of TGF-β1 and a mAb against TGF-β1 on DC-induced allogenic lymphoproliferation. DCs were untreated or treated for 3 hours with IL-8 pPMN CS. Afterward, the cells were washed and incubated with allogenic lymphocyte in the presence of 20 μg of TGF-β1 mAb, isotype control mAb, or 10 μg of TGF-β1. After 4 days, cells were pulsed with 1 μCi of [³H]thymidine per well for the last 16 hours of culture, harvested, and counted in a beta counter. Proliferation is depicted relative to the proliferation of MLR cultures induced by untreated DCs (100%). Relative proliferation is depicted as average means of quadruplicates ± SEM from a series of four experiments. ^#P < 0.05 compared with untreated DC group, and *P < 0.05 compared with isotype control mAb, using Dunnett’s multiple comparisons test.

Figure 5

Effect of elastase on DC lymphocyte allostimulatory ability. A: Elastase activity of PMN CS. PMNs from two healthy donors were primed or not primed with IL-8 for 5 minutes. Afterward, the cells were thoroughly washed and cultured in RPMI-HSA medium for another 3 hours. Then the supernatants were recovered, diluted, and assessed for elastase activity. B: TGF-β production and MLR induced by allogeneic immature DCs previously treated with elastase. Different concentration of elastase was incubated with DCs for 3 hours and then thoroughly washed and cultured in RPMI-HSA medium for 48 hours for assay of TGF-β or incubated with 10⁵ PBMCs for the MLR. Proliferation is depicted relative to the proliferation of MLR cultures induced by untreated DCs (100%). Relative proliferation is depicted as average means of quadruplicates ± SEM from a series of four experiments. *P < 0.05; **P < 0.01 compared with control group, using Dunnett’s multiple comparisons (cpm and TGF-β1 from untreated DCs was 30,739 ± 664 cpm and 3 ± 0.2 ng/ml, respectively). C: To evaluate the effect of SLPI on PMN-CS-treated DC lymphocyte allostimulatory ability and TGF-β1 production, DCs (3 × 10⁴) were incubated for 3 hours with PMN CS and different concentrations of SLPI. Then, cells were thoroughly washed and incubated with 10⁵ PBMCs for the MLR (dotted line) or cultured in RPMI-HSA medium for 48 hours for assay of TGF-β1 production (solid line). Proliferation is depicted relative to the proliferation of MLR cultures induced by untreated DCs (100%). Relative proliferation is depicted as average means of quadruplicates ± SEM from a series of four experiments *P < 0.05 and **P < 0.01 compared with control group, using Dunnett’s multiple comparisons test.

Figure 6

Effect of PMN CS from cyclic neutropenic patients on elastase activity and TGF-β1 production. Elastase activity of uPMN CS and TGF-β production by uPMN CS were measured as described for Figure 5, A and C. In A, the data represent the CS from two cyclic neutropenic patients. In B, the TGF-β1 data represent the mean ± SD of one representative experiment of three. *P < 0.05 and **P < 0.01 compared with control group, using Dunnett’s multiple comparisons test. C: To examine the expression of IκBα by Western blot, DCs were untreated (column 1) or treated with LPS (columns 2 and 7), elastase (columns 3 and 8), uPMN CS (columns 4 and 9), SLPI (column 5), or uPMN CS + SLPI (column 6). Cells were treated for 10 and 20 minutes and processed as described in Materials and Methods. Data are representative of three independent experiments.