Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


Role of the Metalloproteinase ADAM-8 in the Regulation of the Inflammatory Response. PSGL-1 Shedding by ADAM-8 in Human Neutrophils.

Dominguez-Luis4,  Maria Jesus, Urzainqui-Mayayo1,  Ana, Herrera-Garcia4,  Ada Maria, Diaz-Martin5,  Ana, Arce-Franco5,  Maria Teresa, Mollinedo2,  Faustino, Sanchez-Madrid1,  Francisco

Immunology Service, Hospital de La Princesa, Madrid, Spain
Instituto Superior del Cancer, Salamanca, Spain
Rheumatology Service, Hospital Universitario de Canarias, La Laguna, Tenerife, Spain
Rheumatology Service, Hospital Universitario de Canarias, La Laguna, Spain
Rheumatology Service, Hospital Universitario de Canarias, Spain

ADAM (A disintegrin and metalloproteinase domain) proteins are a family of transmembrane proteases with heterogeneous functions and expression profile. One of its members, ADAM-8 is constitutively present at both cell surface and intracellular granules levels in human neutrophil, and it has been implicated in the L-selectin shedding. However, the potential role of this metalloproteinase in the regulation of other "sheddable" proteins involved in the initial phase of the inflammatory response such as PSGL (P-selectin glycoprotein ligand)-1, had not been investigated.

Objective:

To study the capability of ADAM-8 to process the ectodomain of PSGL-1 in human neutrophils.

Methods:

In this study have been used human peripheral blood neutrophils from healthy donors, HL-60 cells (human promyelocytic leucemia cells) and CEM (T-lymphoblastic leucemia cell line). The association PSGL-1/ADAM-8 was studied by co-immunoprecipitation assays in HL60 cells. The membrane and intracellular expression of these two proteins in neutrophils was assessed by flow cytometry and confocal immunofluorescence microscopy. Supernatant concentration of PSGL-1 was determined by ELISA. Native and catalytically inactive ADAM-8 was expressed in CEM line (not possess endogenous ADAM-8) by nucleofection. PSGL-1 function was evaluated in a flow chamber by the dynamic behavior of CEM cells preincubated with termolisyn-activated recombinant ADAM-8 over a P-selectin-coated surface.

Results:

Both confocal microscopy studies and co-immunoprecipitation assays showed the association ADAM-8/PSGL-1 in human neutrophils and HL60, respectively. Pull-down assays established that the binding capacity of ADAM-8 to PSGL-1 was lost if cytoplasmic tail of PSGL-1 was truncated before amino acid 18. Incubation of human neutrophils with thermolysin-activated recombinant soluble ADAM-8 resulted in PSGL-1 shedding assessed by ELISA respect to cells incubated with inactive soluble ADAM-8 (t-student, p<0.05). In addition, CEM cells transfected with native ADAM-8 showed a reduction in PSGL-1 surface expression (by flow cytometry) and a decrease in the number of cells that roll on recombinant P-selectin (in flow chamber) respect to cells transfected with a catalytic-inactive ADAM-8 (t-student, p<0.05).

Conclusions:

Both the membrane and soluble form of ADAM-8 are able to regulate the PSGL-1 and L-selectin surface expression in human neutrophils. All these data support a potential relevant role for ADAM-8 in the recruintment of neutrophils into tissues during the inflammatory response.

To cite this abstract, please use the following information:
Dominguez-Luis, Maria Jesus, Urzainqui-Mayayo, Ana, Herrera-Garcia, Ada Maria, Diaz-Martin, Ana, Arce-Franco, Maria Teresa, Mollinedo, Faustino, et al; Role of the Metalloproteinase ADAM-8 in the Regulation of the Inflammatory Response. PSGL-1 Shedding by ADAM-8 in Human Neutrophils. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1509
DOI: 10.1002/art.29275

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