Characterization of Siglec-5 (CD170) expression and functional activity of anti-Siglec-5 antibodies on human phagocytes.
OBJECTIVE
The Siglec family of proteins consists of at least 10 members with immunoglobulin and lectin domains and with similar sialic acid-binding properties. Many Siglec family members are expressed on hematopoietic cells and are involved in cell/cell interactions. Some family members are suspected of regulating cellular processes through specific signaling pathways. Monoclonal antibodies were generated against specific epitopes of Siglec-5 (CD170) and were used to determine expression of Siglec-5 on normal blood and marrow cells and cell lines. The antibodies also were used to elucidate functional activity for Siglec-5 on blood neutrophils.


METHODS
Flow cytometry and ELISA were used to determine the specificity of the monoclonal antibodies for Siglec-5 and to determine expression patterns. Chemiluminescence assays were employed to measure the oxidative burst activity of whole blood or purified neutrophils following treatment with the anti-Siglec-5 antibodies.


RESULTS
Cell surface expression analysis demonstrated that the protein was expressed on gated human neutrophil and monocyte populations, both in the blood and bone marrow. Expression on neutrophils was enhanced by one-hour treatment with fMLP or TNF-alpha. Epitope-specific anti-Siglec-5 monoclonal antibodies did not directly activate human neutrophils; however, antibody binding augmented neutrophil oxidative burst activity as determined by fMLP-induced luminol-dependent chemiluminescence.


CONCLUSIONS
Data demonstrating expression of Siglec-5 on cells of the myelomonocytic lineage and alteration of its expression by inflammatory stimuli suggest a role for this protein in cell/cell interactions following microbial exposure.
Erickson-Miller
Connie L
CL
Department of Molecular Virology and Host Defense, SmithKline Beecham Pharmaceuticals, Collegeville, PA, USA. Connie_L_Erickson-Miller@GSK.com
Freeman
Sylvie D
SD
Hopson
Christopher B
CB
D'Alessio
Karla J
KJ
Fischer
Elizabeth I
EI
Kikly
Kristine K
KK
Abrahamson
Julie A
JA
Holmes
Stephen D
SD
King
Andrew G
AG
D000818:Animals; D000911:Antibodies, Monoclonal / Q000494:pharmacology*; D015703:Antigens, CD / Q000032:analysis / Q000502:physiology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000032:analysis / Q000502:physiology*; D002450:Cell Communication; D002460:Cell Line; D006801:Humans; D037102:Lectins / Q000032:analysis / Q000502:physiology*; D008163:Luminescent Measurements; D051379:Mice; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000502:physiology; D010586:Phagocytes / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D015214:Antigens, Differentiation, Myelomonocytic; D037102:Lectins; C114531:SIGLEC5 protein, human; D014409:Tumor Necrosis Factor-alpha; D009240:N-Formylmethionine Leucyl-Phenylalanine; D013755:Tetradecanoylphorbol Acetate