Basophil Responses to Chemokines Are Regulated by Both Sequential and Cooperative Receptor Signaling

Abstract

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-3Rα (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-1α > 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.

Targeting of Gr‐1+,CCR2+ monocytes in collagen‐induced arthritis

“…Eotaxin/CCL11, a natural antagonist of CCR2, was recently shown to induce signal transduction via the ERK-1/2 pathway (30). Human basophils also express CCR2 (31) and can be activated via CCR2, resulting in shape changes (31) and the release of cytokines and leukotriene C 4 (32).…”

Identification of Selective Basophil Chemoattractants in Human Nasal Polyps as Insulin-Like Growth Factor-1 and Insulin-Like Growth Factor-2

“…Therefore, it is unclear how basophils might be recruited selectively to sites of allergic inflammation. In a recent study we showed that responses of basophils to certain CC-chemokines were different from those of other leukocytes as a result of their unique expression of multiple chemokine receptors, which can function cooperatively or sequentially (19). However, there may be more specific mechanisms or mediators that bring about the selective recruitment of basophils to sites of allergic inflammation.…”

“…This assay, which has been adapted to measure responses of human basophils (19), in addition to eosinophils, neutrophils, and monocytes (20), is highly sensitive, detecting responses to Ͻ100 pM eotaxin-1/CCL11, and is therefore a useful tool for identifying leukocyte-stimulating activity in extracts of inflamed tissue. We have used this assay to investigate nasal polyp tissue, using a similar strategy to that used previously to identify eotaxin-1/CCL11 (21).…”

“…Leukocytes from citrated whole blood were separated into a polymorphonuclear (PMN) cell population (containing eosinophils and neutrophils) and a PBMC population (containing basophils, monocytes, and lymphocytes) by dextran sedimentation and centrifugation on Histopaque 1077 as described previously (19,20,30,31). For chemotaxis experiments, basophils and eosinophils were further purified from PBMC or PMN preparations, respectively, by negative magnetic selection using Ab mixtures from StemCell Technologies, according to the manufacturer's protocol.…”

“…Eosinophil, neutrophil, basophil, and monocyte shape change was assayed as described previously (19,20). PMN or PBMC preparations were resuspended in assay buffer (PBS with Ca 2ϩ /Mg 2ϩ , supplemented with 0.1% BSA, 10 mM HEPES, and 10 mM glucose, pH 7.4) at 1 ϫ 10 7 cells/ml, and 25-l aliquots were mixed with 25 l of agonists and incubated for 4 min at 37°C.…”