RANTES and related chemokines activate human basophil granulocytes through different G protein‐coupled receptors

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.

Section: Discussionmentioning

confidence: 93%

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

Heinemann

Hartnell

Stubbs

et al. 2000

“…Among the chemokines detected in airways of patients with allergic asthma is monocyte chemotactic protein (MCP)-1 (16). Various roles have been attributed to MCP-1 in promoting allergic responses, including recruitment of basophils (17) and clumping of mast cells (18), IgE-independent induction of basophil (17) and mast cell (18) histamine release, chemotaxis of T cells (19), and enhancement of polarization of naive T cells to IL-4-producing Th2 cells (20,21). Further evidence that MCP-1 may play a role in allergic asthma can be found in studies that observe that MCP-1 immunoneutralization resulted in reduction in AHR, pulmonary inflammation, and production of lymphocyte-derived inflammatory mediators in allergen-challenged animals (22)(23)(24).…”

mentioning

confidence: 99%

Enhanced Airway Th2 Response After Allergen Challenge in Mice Deficient in CC Chemokine Receptor-2 (CCR2)

Kim

Sung

Kuziel

et al. 2001

To evaluate the role of CCR2 in allergic asthma, mutant mice deficient in CCR2 (CCR2−/−) and intact mice were sensitized with i.p. OVA with alum on days 0 and 7, and challenged by inhalation with nebulization of either OVA or saline. Airway hyperreactivity, measured by the methacholine-provoked increase in enhanced pause, was significantly increased (p < 0.05) in OVA-challenged CCR2−/− mutant mice, compared with comparably challenged CCR2+/+ mice. OVA-challenged CCR2−/− mutants also were also found to have enhanced bronchoalveolar lavage fluid eosinophilia, peribronchiolar cellular cuffing, and Ig subclass switching, with increase in OVA-specific IgG1 and IgE. In addition, RNase protection assay revealed increased whole lung expression of IL-13 in OVA-challenged CCR2−/− mutants. Unexpectedly, serum monocyte chemotactic protein-1 levels were 8-fold higher in CCR2−/− mutants than in CCR2+/+ mice sensitized to OVA, but OVA challenge had no additional effect on circulating monocyte chemotactic protein-1 in either genotype. Ag stimulation of lymphocytes isolated from OVA-sensitized CCR2 mutants revealed a significant increase (p < 0.05) in IL-5 production, which differed from OVA-stimulated lymphocytes from sensitized CCR2+/+ mice. These experiments demonstrate an enhanced response in airway reactivity and in lung inflammation in CCR2−/− mutant mice compared with comparably sensitized and challenged CCR2+/+ mice. These observations suggest that CC chemokines and their receptors are involved in immunomodulation of atopic asthma.

“…Most of the cellular responses following exposure of the cells to chemotactic agonists and all the signaling events known to date are induced very rapidly and only transiently generally returning to baseline within a few minutes. At sufficiently high concentrations of the agonist, there is also a complete homologous receptor desensitization of the corresponding chemoattractant receptor, preventing further signal transduction and a second cellular response upon repeated stimulation (4,5,7,8). Despite the desensitization for these rapid and transient responses, basophils exposed to C5a and IL-3 produce the cytokines IL-4 and IL-13 and continuously release leukotriene C4 (LTC4) during a culture period of 18 h (9, 10), indicating that some components of the signaling pathway may be long-lasting and/or that the C5aR can be continuously engaged by the ligand activating a pathway that is not desensitized.…”

mentioning

confidence: 99%

Requirements for C5a Receptor-Mediated IL-4 and IL-13 Production and Leukotriene C4 Generation in Human Basophils

Eglite

Plüss

Dahinden

2000

Self Cite

Anaphylatoxin derived from the fifth complement component (C5a) in the presence of IL-3 induces continuous leukotriene C4 generation and IL-4 and IL-13 expression in human basophils for a period of 16–18 h. This indicates that the G protein-coupled C5a receptor (C5aR) can induce long-lasting cellular responses. Using anti-N-terminal C5aR Abs, C-terminal C5a hexapeptide analogs, and pertussis toxin, we demonstrate that the putative activation site of the C5aR is both necessary and sufficient for these late cellular responses. Furthermore, continuous pertussis toxin-sensitive G protein-coupled receptor activation and receptor-ligand interaction is ongoing and required during the entire period of product release. However, the late basophil responses have a more stringent requirement for optimal receptor activation. Leukotriene C4 generation appears to be influenced mostly by the way the receptor is activated, because the most active hexapeptide is a superagonist for this response. By contrast, C5adesarg, lacking the C-terminal arginine, induces minimal lipid mediator formation but is fully active to induce IL-4 production and is even a superagonist for IL-13 release. Nevertheless, IL-4/IL-13 synthesis in response to C5adesarg could be blocked by both C-terminal antagonistic peptide as well as anti-N-terminal C5aR Abs, indicating only minor differences of ligand-receptor interactions between C5a and C5adesarg. Taken together, our data demonstrate that long-lasting and continuous signaling occurs through a limited activation domain of the C5aR, which can differentially promote separate basophil functions.