Noriko Kawata

New flow cytometric method for surface phenotyping basophils from peripheral blood

To clarify the role of basophils in the pathogenesis of allergic disease, we developed a new method for performing surface phenotyping of these cells in centrifugation-enriched mononuclear cell fraction. This method identified basophils on the basic of a negative reactivity with mixed FITC-conjugated monoclonal anti-bodies (mAbs) (anti-CD2, -CD14, -CD16, and -CD19) with analysis performed by flow cytometry. The validity of this approach was confirmed by sorting experiments. Various PE-conjugated mAbs were also used to examine binding to FITC-negative basophils. Basophils from asthmatic patients (n = 14) as well as from normal subjects (n = 6) were shown to express CDw32 (Fc gamma RII), CD25 (IL-2R), but not CD64 (Fc gamma RI). We also detected binding of IgG1 and IgG4 to basophils. This method of phenotyping was very rapid and simple. It thus appears to be useful in the study of allergic disease, as well as of the biology of the basophil.

Successful Treatment of a Patient with Severe Churg-Strauss Syndrome by a Combination of Pulse Corticosteroids, Pulse Cyclophosphamide, and High-Dose Intravenous Immunoglobulin

A 24-year-old woman with a 4-year history of bronchial asthma suffered from bloody sputum, numbness of the extremities, elevated eosinophil count, and hypoxemia. A diagnosis of alveolar hemorrhage was made by bronchoalveolar lavage. Echocardiogram revealed severe hypokinesis of the left ventricular wall. Her respiratory condition deteriorated despite administration of pulse corticosteroids. A second pulse corticosteroids and pulse cyclophosphamide followed by high-dose intravenous immunoglobulin brought about a dramatic improvement of alveolar hemorrhage, cardiac impairment, and peripheral neuropathy. Levels of antimyeloperoxidase-antineutrophil cytoplasmic antibodies, soluble thrombomodulin, soluble interleukin-2 receptor, eosinophil cationic protein were elevated and returned to the normal range in remission. The combination of pulse corticosteroids, pulse cyclophosphamide, and high-dose intravenous immunoglobulin seemed effective for the acute phase of severe Churg-Strauss syndrome.

Purification of human blood basophils using negative selection by flow cytometry

Basophils were purified from peripheral blood of normal donors using Percoll discontinuous gradients and negative selection by flow cytometry. The mean purity of basophils obtained was 84.77±4.1 (s.d.)% (range 77.3–90.0%, n= 13). The overall yield of these procedures was 16.0±2.6% (range 11.0–19.9%, n= 13), and cell viability of purified basophils exceeded 90%.

Signal transduction by IgG receptors induces calcium mobilization, but not histamine release, in the human basophilic cell line KU812F.

Human blood basophils selectively express Fc gamma RII (CDw32) among IgG receptor subtypes, but its functional role in allergic reactions remains unknown. Using the human basophilic leukemia cell line KU812F as a model system, we investigated cellular signaling events mediated through IgG receptor stimulation. KU812F cells express Fc gamma RII on their surface. mRNAs for both Fc gamma RIIA and IIB subtypes were detected by reverse transcriptase-PCR analysis. In this cell line, Fc gamma RII stimulation induced mobilization of free intracellular calcium and actin polymerization. Yet, no significant histamine release was observed, nor did blood basophils stimulated by anti-Fc gamma RII monoclonal antibody IV.3 and by a secondary antibody release histamine. These data indicate that Fc gamma RII stimulation induces cellular signaling events such as calcium mobilization in human basophils. However, these events do not lead to histamine release.