ADAM17 deficiency by mature neutrophils has differential effects on L-selectin shedding

Abstract

L-selectin directs neutrophils to sites of inflammation, and upon their activation, surface expression of the receptor is rapidly down-regulated by ectodomain shedding. Tumor necrosis factor-alpha-converting enzyme (TACE, or ADAM17) is a sheddase of L-selectin; however, Adam17 gene targeting (ADAM17(DeltaZn/DeltaZn)) in mice is perinatal lethal and its role in L-selectin shedding by mature neutrophils has not been determined. This was addressed here by using radiation-chimeric mice reconstituted with ADAM17(DeltaZn/DeltaZn) fetal liver cells. ADAM17-deficient neutrophils, monocytes, and lymphocytes failed to shed L-selectin in response to PMA, as did neutrophils infiltrating the inflamed peritoneum. In addition, the absence of functional ADAM17 resulted in significantly increased levels of L-selectin surface expression by peripheral-blood leukocytes, indicating the sheddase also plays a role in the constitutive cleavage of L-selectin. Interestingly, not all manners of L-selectin turnover required ADAM17. Plasma L-selectin levels were similar between ADAM17(DeltaZn/DeltaZn)-chimeric and control mice, as was the shedding of L-selectin by neutrophils undergoing spontaneous apoptosis. The latter process, however, was diminished by a metalloprotease inhibitor, indicating the role of a sheddase other than ADAM17. Together, our data reveal that L-selectin's surface density on neutrophils is regulated by ADAM17, but homeostatic L-selectin cleavage is not.

Figure 1.

Induced L-selectin shedding by neutrophils from ADAM17^ΔZn/ΔZn- and ADAM17^+/+-chimeric mice. (A) Peripheral-blood leukocytes from the chimeric mice were treated with or without PMA for 20 minutes, as indicated, and then double-stained for surface expression of L-selectin (left row) or Mac-1 (right row) and neutrophil marker Ly-6G. (B) Peripheral-blood (pb) and peritoneal-cavity (pc) neutrophils from the chimeric mice following 2 hours of thioglycollate-induced peritonitis were double-stained for surface expression of L-selectin and neutrophil marker Ly-6G. Relative staining levels were determined by flow cytometry. For all histogram plots, the dashed line indicates staining by an isotype-matched negative control mAb. The y-axis indicates cell number and the x-axis indicates log-10 fluorescence. Results are representative of 3 or more independent experiments.

Figure 2.

Basal L-selectin expression levels by neutrophils from ADAM17^ΔZn/ΔZn and control mice. Resting peripheral-blood leukocytes from the ADAM17^ΔZn/ΔZn- and ADAM17^+/+-chimeric mice, and wild-type C57BL /6 mice (WT) were double-stained for surface expression of L-selectin (left panel) or Mac-1 (right panel) and neutrophil marker Ly-6G. Relative staining levels were determined by flow cytometry. The dashed line indicates staining by an isotype-matched negative control mAb. The y-axis indicates cell number and the x-axis indicates log-10 fluorescence. Results are representative of 3 or more independent experiments.

Figure 3.

L-selectin down-regulation by neutrophils from ADAM17^ΔZn/ΔZn- and ADAM17^+/+-chimeric mice following spontaneous apoptosis. (A) Bone marrow neutrophils from the indicated mice were cultured for approximately 24 hours, and then triple-stained for surface expression of L-selectin, neutrophil marker Ly-6G, and annexin V–FITC. Relative staining levels were determined by flow cytometry. The contour plots represent gated neutrophils, and the x- and y-axes indicate log-10 fluorescence. (B) Bone marrow neutrophils from the ADAM17^ΔZn/ΔZn (ΔZn/ΔZn)– and ADAM17^+/+ (+/+)–chimeric mice were cultured for approximately 24 hours in the presence or absence of the metalloprotease inhibitor TAPI-0, as indicated. Media supernatant was then collected and passed through a syringe filter to remove membrane fragments larger than 0.22 μm. The presence of soluble L-selectin in the media was determined by ELISA analysis, as described in “Materials and Methods.” Neutrophils from the ADAM17^ΔZn/ΔZn- and ADAM17^+/+-chimeric mice were plated in triplicate wells, and the supernatant from each well was analyzed in duplicate. Each bar represents the mean ± SD. *P < .01 versus ADAM17^+/+ plus TAPI-0; #P < .01 versus ADAM17^ΔZn/ΔZn plus TAPI-0. Data are representative of 3 independent experiments.

Figure 4.

Immunoprecipitation and immunoblot blot analysis of plasma L-selectin levels from the ADAM17^ΔZn/ΔZn- and ADAM17^+/+-chimeric mice. Plasma (approximately 40 μL) from 2 ADAM17^ΔZn/ΔZn- and 2 ADAM17^+/+-chimeric mice were subjected to immunoprecipitation using the L-selectin mAb MEL-14 and then to nonreducing SDS-PAGE and immunoblotting, as described in “Materials and Methods.”