Basophil responses to chemokines are regulated by both sequential and cooperative receptor signaling.
To investigate human basophil responses to chemokines, we have developed a sensitive assay that uses flow cytometry to measure leukocyte shape change as a marker of cell responsiveness. PBMC were isolated from the blood of volunteers. Basophils were identified as a single population of cells that stained positive for IL-3Ralpha (CDw123) and negative for HLA-DR, and their increase in forward scatter (as a result of cell shape change) in response to chemokines was measured. Shape change responses of basophils to chemokines were highly reproducible, with a rank order of potency: monocyte chemoattractant protein (MCP) 4 (peak at <1 nM) >/= eotaxin-2 = eotaxin-3 >/= eotaxin > MCP-1 = MCP-3 > macrophage-inflammatory protein-1alpha > RANTES = MCP-2 = IL-8. The CCR4-selective ligand macrophage-derived chemokine did not elicit a response at concentrations up to 10 nM. Blocking mAbs to CCR2 and CCR3 demonstrated that responses to higher concentrations (>10 nM) of MCP-1 were mediated by CCR3 rather than CCR2, whereas MCP-4 exhibited a biphasic response consistent with sequential activation of CCR3 at lower concentrations and CCR2 at 10 nM MCP-4 and above. In contrast, responses to MCP-3 were blocked only in the presence of both mAbs, but not after pretreatment with either anti-CCR2 or anti-CCR3 mAb alone. These patterns of receptor usage were different from those seen for eosinophils and monocytes. We suggest that cooperation between CCRs might be a mechanism for preferential recruitment of basophils, as occurs in tissue hypersensitivity responses in vivo.
Heinemann
A
A
Leukocyte Biology Section, Biomedical Sciences Division, Imperial College School of Medicine, South Kensington, London, United Kingdom.
Hartnell
A
A
Stubbs
V E
VE
Murakami
K
K
Soler
D
D
LaRosa
G
G
Askenase
P W
PW
Williams
T J
TJ
Sabroe
I
I
D001491:Basophils / Q000166:cytology / Q000276:immunology* / Q000378:metabolism*; D048429:Cell Size / Q000276:immunology; D018932:Chemokine CCL2 / Q000378:metabolism / Q000494:pharmacology; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D018925:Chemokines / Q000097:blood / Q000494:pharmacology*; D016207:Cytokines; D004804:Eosinophils / Q000166:cytology / Q000378:metabolism; D005434:Flow Cytometry; D006801:Humans; D015640:Ion Channel Gating; D007963:Leukocytes, Mononuclear / Q000166:cytology; D018945:Monocyte Chemoattractant Proteins / Q000494:pharmacology; D009000:Monocytes / Q000166:cytology / Q000378:metabolism; D054390:Receptors, CCR2; D054397:Receptors, CCR3; D019707:Receptors, Chemokine / Q000096:biosynthesis / Q000097:blood / Q000502:physiology*; D012542:Scattering, Radiation; D015398:Signal Transduction / Q000276:immunology*
C099677:CCL13 protein, human; C515450:CCL7 protein, human; C515453:CCL8 protein, human; C515334:CCR2 protein, human; C515350:CCR3 protein, human; D018932:Chemokine CCL2; D054410:Chemokine CCL7; D054412:Chemokine CCL8; D018925:Chemokines; D016207:Cytokines; D018945:Monocyte Chemoattractant Proteins; D054390:Receptors, CCR2; D054397:Receptors, CCR3; D019707:Receptors, Chemokine