Different proteolytic mechanisms involved in Fc gamma RIIIb shedding from human neutrophils - PubMed (original) (raw)
Different proteolytic mechanisms involved in Fc gamma RIIIb shedding from human neutrophils
P J Middelhoven et al. Clin Exp Immunol. 2001 Jul.
Abstract
The Fc gamma receptor type IIIb (CD16) is highly expressed on human neutrophils and is found in a soluble form in plasma and in other body fluids. Upon activation of neutrophils in vitro, Fc gamma RIIIb is shed from the cell surface by proteolytic cleavage. We have now investigated the effect of metalloproteinase inhibitors and a serine proteinase inhibitor on the shedding of Fc gamma RIIIb induced by phorbol 12-myristate 13-acetate (PMA) or cytochalasin B (cyto B) + N-formyl-methionyl-leucyl-phenylalanine (fMLP). Metalloproteinase inhibitors blocked to a large extent PMA-induced, but not cyto B + fMLP-induced shedding of Fc gamma RIIIb. Inhibition of members of the ADAM (a disintegrin and metalloproteinase) family appeared most efficient. In contrast, the serine protease inhibitor N-methoxysuccinyl-alanine-alanine-proline-valine-chloromethylketone (MeOsuc-AAPV-CMK) largely blocked cyto B + fMLP-induced, but not PMA-induced shedding of Fc gamma RIIIb. Metalloproteinase inhibitors in combination with the serine proteinase inhibitor resulted in full inhibition of Fc gamma RIIIb shedding induced by either PMA or cyto B + fMLP. The shedding of Fc gamma RIIIb that accompanied apoptosis was inhibited by 60% in the presence of inhibitors of metalloproteinases but was insensitive to inhibition of serine proteinases. These results show that distinct types of proteolytic enzyme are involved in the stimulus-induced shedding of Fc gamma RIIIb from human neutrophils and suggest that these proteinases may become differentially activated under various physiological or pathological conditions.
Figures
Fig. 1
Inhibition of PMA-induced FcγRIIIb shedding by metalloprotease inhibitors measured by ELISA. Human neutrophils were preincubated with various concentrations of Ro32–7066 (a), Ro32–3580 (b) or Ro32–1541 (c) for 10 min at 37°C. After 10 min of PMA (200 ng/ml) stimulation, supernatants were collected and soluble FcγRIIIb was measured by ELISA. The concentration of soluble FcγRIIIb ±SD in the absence of inhibitors (closed squares) was taken as 100%. This concentration amounted to 0·9 ± 0·4 pmol/ml (n = 4) in (a), 1·2 ± 0·5 pmol/ml (n = 4) in (b) and 2·0 ± 0·9 pmol/ml (n = 5) in (c). The percentage of soluble FcγRIIIb ±SD in the absence of PMA and without inhibitors (open squares) is also indicated. The results shown were obtained in (n) independent experiments.
Fig. 2
PMA-induced and cyto B + fMLP-induced FcγRIIIb release from neutrophils. Human neutrophils (107/ml) were preincubated with vehicle (DMSO), Ro32–7066 (50 µ
m
), MeOSuc-AAPV-CMK (CMK; 200 µ
m
) or the combination of Ro32–7066 and CMK for 10 min at 37°C. After stimulation of the neutrophils with PMA (200 ng/ml) for 10 min at 37°C (filled bars) or with a combination of cyto B (0·5 µg/ml) for 5 min + fMLP (1 µ
m
) for 10 min at 37°C (hatched bars), supernatants were collected and soluble FcγRIII was measured by ELISA. As a negative control, unstimulated neutrophils are shown (open bars). The concentration ±SD of FcγRIIIb in the supernatants of the PMA incubations [2·0 ± 0·9 pmol/ml (n = 4)] was taken as 100% and compared with the concentration of FcγRIIIb when protease inhibitors were also present. The concentration ±SD of FcγRIIIb in the cyto B + fMLP incubations [3·4 ± 1·4 pmol/ml (n = 4)] was taken as 100% and compared with the concentration of FcγRIIIb when protease inhibitors were also present. The results shown were obtained in four independent experiments.
Fig. 3
PMA-induced and cyto B + fMLP-induced
l
-selectin down-regulation from neutrophils. Human neutrophils (107/ml) were preincubated with vehicle (DMSO), Ro32–7066 (50 µ
m
), MeOSuc-AAPV-CMK (CMK; 200 µ
m
), or a combination of inhibitors for 10 min at 37°C. After stimulation with PMA (200 ng/ml) for 10 min at 37°C (filled bars), or with a combination of cyto B (0·5 µg/ml) for 5 min + fMLP (1 µ
m
) for 10 min at 37°C (hatched bars),
l
-selectin expression was determined by FACS analysis. Unstimulated neutrophils are shown as open bars. The expression of
l
-selectin ±SD on DMSO-incubated cells without inhibitors [MFI 346 ± 59, n = 5] was taken as 100% and compared with the expression of
l
-selectin on cells incubated with activators ± inhibitors.
Fig. 4
Elastase release upon neutrophil activation with different stimuli. Human neutrophils (107/ml) were stimulated with PMA (200 ng/ml) for 10 min at 37°C, or with a combination of cyto B (0·5 µg/ml) for 5 min + fMLP (1 µ
m
) for 10 min at 37°C. Supernatants were collected and elastase was measured by ELISA. The results shown represent the concentration of elastase ±SD of six independent experiments.
Fig. 5
Inhibition of FcγRIIIb release by serine and metalloprotease inhibitors during apoptosis. Human neutrophils were incubated with vehicle (DMSO), MeOSuc-AAPV-CMK (CMK; 200 µ
m
), Ro32–7066 (10 µ
m
), Ro32–3580 (20 µ
m
) or Ro32–1541 (50 µ
m
) for 24 h at 37°C (
). The amount of soluble FcγRIIIb was determined by ELISA. The concentration ±SD of soluble FcγRIIIb in the 24-h supernatants without inhibitors [1·9 ± 0·6 pmol/ml (n = 4)] was taken as 100%. The percentage of soluble FcγRIIIb at t = 0 is also shown (□). The results shown were obtained in four independent experiments.
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