Preparation of specific polyclonal antibodies to a C-C chemokine receptor, CCR1, and determination of CCR1 expression on various types of leukocytes.
cDNA cloning has revealed the presence of at least three distinct human receptors for macrophage inflammatory protein-1alpha (MIP-1alpha) 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 NH2-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 NH2-terminal portion of CCR1, further indicating that this antibody recognized the NH2-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-1alpha, suggesting that its NH2-terminal portion is critically involved in ligand binding or signaling. Finally, the antibody partially inhibited monocyte chemotactic activities of human rMIP-1alpha, suggesting that CCR1 is a functional receptor for MIP-1alpha on human peripheral blood monocytes.
Su
S B
SB
Department of Pharmacology, Cancer Research Institute, Kanazawa University, Japan.
Mukaida
N
N
Wang
J
J
Nomura
H
H
Matsushima
K
K
D000328:Adult; D000818:Animals; D000906:Antibodies / Q000276:immunology*; D018952:Antigens, CD34 / Q000032:analysis; D001692:Biological Transport / Q000187:drug effects; D001854:Bone Marrow Cells; D002118:Calcium / Q000378:metabolism; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D004797:Enzyme-Linked Immunosorbent Assay; D005312:Fetal Blood / Q000166:cytology; D019084:Fluorescent Antibody Technique, Indirect; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D007231:Infant, Newborn; D017493:Leukocyte Common Antigens / Q000032:analysis; D007962:Leukocytes / Q000187:drug effects / Q000378:metabolism*; D019402:Macrophage Inflammatory Proteins / Q000494:pharmacology; D011817:Rabbits; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine / Q000096:biosynthesis* / Q000235:genetics / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology
D000906:Antibodies; D018952:Antigens, CD34; D054405:Chemokine CCL3; D054407:Chemokine CCL4; D019402:Macrophage Inflammatory Proteins; D019707:Receptors, Chemokine; D018121:Receptors, Cytokine; D011993:Recombinant Fusion Proteins; C080633:macrophage inflammatory protein 1alpha receptor; D017493:Leukocyte Common Antigens; D002118:Calcium