Premium
Preparation of specific polyclonal antibodies to a C‐C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes
Author(s) -
Su Shaobo,
Mukaida Naofumi,
Wang Jianbin,
Nomura Hideki,
Matsushima Kouji
Publication year - 1996
Publication title -
journal of leukocyte biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.819
H-Index - 191
eISSN - 1938-3673
pISSN - 0741-5400
DOI - 10.1002/jlb.60.5.658
Subject(s) - ccr1 , microbiology and biotechnology , biology , chemokine receptor , polyclonal antibodies , cc chemokine receptors , antibody , transfection , receptor , monocyte , chemokine , immunology , cell culture , biochemistry , genetics
cDNA cloning has revealed the presence of at least three distinct human receptors for macrophage inflammatory protein‐1α (MIP‐1α) and RANTES: C‐C chemokine receptor (CCR) 1, 4, and 5. To clarify the physiological role of CCR1, we prepared specific antibodies to CCR1 by immunizing rabbits with recombinant glutathione‐ S ‐transferase (GST) fused with its NH 2 ‐terminal portion. The resultant antibodies stained positively 293 cells transfected with CCR1 cDNA but neither parental cells nor cells transfected with CXCR1 [interleukin‐8 (IL‐8) receptor type A] cDNA, confirming its specificity. Immunofluorescence analysis revealed that peripheral blood lymphocytes and monocytes but not neutrophils express CCR1. Positive staining of transfectants, monocytes, and lymphocytes was inhibited by the GST protein fused with the NH 2 ‐terminal portion of CCR1, further indicating that this antibody recognized the NH 2 ‐terminal portion of CC CKR1. A majority of CD3 + , CD4 + , CD8 + , or CD16 + peripheral blood lymphocytes but not CD19 + lymphocytes expressed CCR1. Among CD4 + peripheral blood lymphocytes, CD45RO + cells expressed a larger number of CCR1 compared with CD45RO ‐ . Moreover, CD34 + cells in human bone marrow as well as cord blood were uniformly stained with this antibody. Furthermore, the antibody inhibited calcium mobilization in CCR1 transfectants stimulated with human rMIP‐1α, suggesting that its NH 2 ‐terminal portion is critically involved in ligand binding or signaling. Finally, the antibody partially inhibited monocyte chemotactic activities of human rMIP‐1α, suggesting that CCR1 is a functional receptor for MIP‐1α on human peripheral blood monocytes. J. Leukoc. Biol . 60: 658–666; 1996.