Daigneault D E, Hartshorn K L, Liou L S, Abbruzzi G M, White M R, Oh S K, Tauber A I
Department of Medicine, Boston University School of Medicine, MA 02118.
Blood. 1992 Dec 15;80(12):3227-34.
Although neutrophils are not viewed as a principal defense against influenza A virus (IAV) infection, their interactions are both complex and clinically relevant. Activation of the neutrophil is distinctive from that described for chemoattractants. To more fully characterize the pathway by which IAV stimulates the human neutrophil, we have examined its binding characteristics. First, inhibition studies with various sialic acid-containing and sialic-free sugars showed that IAV binds to sialic acid residues and activates receptors distinct from those used by Concanavalin-A (Con-A) and formyl-methionyl-leucyl-phenylalanine (FMLP) and that overlap those bound by wheat germ agglutinin (WGA). That viral hemagglutinin (HA) mediates viral binding and activation was shown by preincubating neutrophils with purified monovalent bromelain-released HA (BHA) and showing that IAV-induced membrane depolarization and hydrogen peroxide (H2O2) production were inhibited approximately 95%. However, binding inhibition required significantly higher concentrations of purified HA, suggesting that binding and cell activation have different interactive requirements. Desialation of the neutrophil surface membrane by neuraminidase treatment resulted in a 90.6% +/- 4.4% and 53.1% +/- 8.7% inhibition of IAV activation of neutrophils and viral binding, respectively. Resialation with ganglioside GT1b totally restored viral binding, but did not reverse the inhibition of activation. Thus, although HA was shown to mediate binding and neutrophil activation, viral binding per se was insufficient to stimulate the cell. Having demonstrated the functional role of HA, we sought to establish the mechanism of stimulation. HA in three different forms (BHA, HA-rosettes, and HA-liposomes) failed to activate the cell, although H2O2 production evoked by IAV stimulation was reduced in competitive inhibition studies with each preparation. Upon cross-linking with a monoclonal antibody to HA, activation comparable to that of intact virus was observed. The requirement for cross-linking of functional receptors, as opposed to activation through the neutrophil Fc receptor, was confirmed in experiments using staphylococcal A protein. These studies have shown the chemical specificity of IAV binding to the human neutrophil, the character of the receptor(s) stimulated to activate the IAV-evoked response, and the activation requirement for cross-linking those receptors responsible for stimulating functional responses.
尽管中性粒细胞不被视为抵抗甲型流感病毒（IAV）感染的主要防御细胞，但其相互作用既复杂又与临床相关。中性粒细胞的激活与趋化因子所描述的激活方式不同。为了更全面地描述IAV刺激人类中性粒细胞的途径，我们研究了其结合特性。首先，用各种含唾液酸和不含唾液酸的糖类进行抑制研究表明，IAV与唾液酸残基结合并激活与伴刀豆球蛋白A（Con-A）和甲酰甲硫氨酰亮氨酰苯丙氨酸（FMLP）所使用的受体不同的受体，且与麦胚凝集素（WGA）所结合的受体有重叠。用纯化的单价菠萝蛋白酶释放的血凝素（BHA）预孵育中性粒细胞，结果表明病毒血凝素（HA）介导病毒结合和激活，且IAV诱导的膜去极化和过氧化氢（H2O2）产生被抑制了约95%。然而，结合抑制需要显著更高浓度的纯化HA，这表明结合和细胞激活有不同的相互作用要求。用神经氨酸酶处理中性粒细胞表面膜使其去唾液酸化，分别导致IAV对中性粒细胞的激活和病毒结合受到90.6%±4.4%和53.1%±8.7%的抑制。用神经节苷脂GT1b重新唾液酸化完全恢复了病毒结合，但没有逆转激活抑制。因此，尽管已证明HA介导结合和中性粒细胞激活，但病毒结合本身不足以刺激细胞。在证明了HA的功能作用后，我们试图确定刺激机制。三种不同形式的HA（BHA、HA玫瑰花结和HA脂质体）均未能激活细胞，尽管在与每种制剂的竞争性抑制研究中，IAV刺激引起的H2O2产生减少。用抗HA单克隆抗体交联后，观察到与完整病毒相当的激活。在使用葡萄球菌A蛋白的实验中证实了功能性受体交联的要求，而不是通过中性粒细胞Fc受体激活。这些研究表明了IAV与人类中性粒细胞结合的化学特异性、被刺激以激活IAV诱发反应的受体特性，以及交联负责刺激功能性反应的那些受体的激活要求。
