Viêm Dập Tắt Nhờ Sự Phân Cắt Monocyte Chemoattractant Protein-3 Bởi Gelatinase A
Tóm tắt
Phân rã mô bởi enzyme matrix metalloproteinase, cụ thể là gelatinase A, đóng vai trò quan trọng trong viêm và di căn. Với việc nhận biết tầm quan trọng của vị trí gắn cơ chất bên ngoài vùng xúc tác, chúng tôi đã sàng lọc các cơ chất ngoại bào bằng cách sử dụng miền hemopexin của gelatinase A làm mồi trong hệ thống hai lai men bia. Protein hóa hướng đơn nhân-3 (MCP-3) được xác định là một cơ chất sinh lý của gelatinase A. MCP-3 sau khi bị phân cắt gắn vào các thụ thể CC-chemokine-1, -2, và -3, nhưng không còn gây ra dòng canxi hoặc thúc đẩy hóa hướng động, mà thay vào đó hoạt động như một chất đối kháng chemokine tổng quát làm giảm viêm. Điều này gợi ý rằng matrix metalloproteinases vừa là tác nhân vừa là điều hòa của phản ứng viêm.
Từ khóa
#gelatinase A #protein hóa hướng đơn nhân #phân rã mô #viêm #matrix metalloproteinaseTài liệu tham khảo
G. A. McQuibban and C. M. Overall unpublished data.
The fluorescence of Fluo-3AM–loaded cells was monitored with a Perkin-Elmer 650-10B spectrofluorimeter (excitation wavelength 506 nm emission wavelength 526 nm). Desensitization assays were performed by sequential addition of MCP-3(5-76) or buffer control followed by the full-length chemokine.
G. A. McQuibban J.-H. Gong I. Clark-Lewis C. M. Overall unpublished data.
125 I-Labeled MCP-3 (4 nM) in the presence of serially diluted unlabeled MCP-3 or MCP-3(5-76) and 0.05% NaN 3 was incubated at 4°C for 30 min with THP-1 cells. Cell-bound and free 125 I–MCP-3 were separated by centrifugation of the cells through a column of dioctyl phthalate: n -butyl phthalate (2:3 v/v). Bound 125 I-labeled MCP-3 in the cell pellet was measured by gamma counting. Nonspecific binding was determined in the presence of a 100-fold concentration of unlabeled ligand and was subtracted from the total.
THP-1 cell migration was assessed in transwell trays (Costar; 6.5-mm diameter 3-μm membrane pore size). Chemokines were added to the lower well and THP-1 cells (1 × 10 7 cells per milliliter) were added to the upper well. After 1.5 hours cells that had migrated to the lower well were counted.
CD-4 mice (five per group) were injected at two subcutaneous sites (500 ng/100 μl of pyrogen-free saline) with chemokine as indicated. In other experiments five mice per group were injected as before but with mixtures of 100 μl of MCP-3 and synthetic MCP-3(5-76). Mice were killed 18 hours after injection and paraffin sections transverse to the skin were analyzed after hematoxylin and eosin (H&E) staining for mononuclear cells.
Peritonitis with an associated 40-fold increase in inflammatory cell numbers was induced in mice by using zymosan A. Fifty micrograms of MCP-3(5-76) or saline was administered to the peritoneal cavity after 24 hours. Infiltrating cells were collected after 2 and 4 hours by saline lavage. Cells were counted on a Coulter Counter gated at 5 to 10 μm. Cytospins (100 μl) were examined by light microscopy after H&E staining.
Peritoneal inflammatory cells were stained for 60 min on ice with rat anti-mouse F4/80 monoclonal antibody or rat IgG2b isotype control (each at 20 μg/ml). After washing cells were stained with fluorescein isothiocyanate–conjugated anti-rat immunoglobulin G for 45 min on ice washed and analyzed by flow cytometry with a FACScan analyzer (Becton Dickinson Oxford UK).
Van Damme J., et al., Chem. Immunol. 72, 42 (1999).
Openakker G., Masure S., Grillet B., Van Damme J., Lymphokine Cytokine Res. 10, 317 (1991).
We thank G. Pelman for synovial fluid samples. Supported by a grant from the National Cancer Institute of Canada (NCIC) with funds provided in part by the Canadian Cancer Society and by Medical Research Council Group grant funding. G.A.M. is supported by a NCIC studentship.