Polyl-histidine

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Abstract
Poly-l-histidine (PHSTD) of molecular weight 26,000 induced the generation of large amounts of Superoxide (O 2 − ) and hydrogen peroxide (H2O2) in human neutrophils (PMNs). Despite its low solubility at neutral pH, PHSTD was bound very rapidly to the PMN surfaces. Maximal generation of O 2 − took place with 4–5 ×10−6 M of PHSTD, starting after a lag of about 25 sec and proceeding for 15–17 min at a rate of 150 nmol/107 PMNs/min, suggesting that this polycation is one of the most potent stimulators of O 2 − generation known, PHSTD was found to be non-toxic for PMNs even at millimolar concentrations. Generation of O 2 − by PHSTD depended on extracellular calcium; it was inhibited by calcium channel blockers and by trifluoperazine, and it triggered a sharp rise in intracellular calcium as determined by the Quin 2 fluorescence technique. The generation of both O 2 − and H2O2 by PHSTD was partially inhibited by cytochalasin B or (CYB, CYE). On the other hand, CYB markedly enhanced the generation of both O 2 − and H2O2 following stimulation of PMNs either by PHSTD, polyarginine, histone, or by antibody-opsonized group A streptococci. Electron microscopic analysis and NBT reduction tests revealed that both PHSTD and PHSTD-opsonized streptococci were avidly phagocytosed by PMNs. Since CYB totally inhibited internalization of both PHSTD and the PHSTD-opsonized streptococci, it was suggested that these agents stimulated oxygen radical generation mainly on the leukocyte surfaces. Complexes (CX) formed between PHSTD and polyanethole sulfonate (a strong polyanion) or between histone and the polyanion mimicked immune CX in their ability to trigger the generation of large amounts of O 2 − which were inhibited by CYB. Generation of O 2 − and chemiluminescence either by PHSTD or by PHSTD-opsonized streptococci were markedly inhibited by poly-l-glutamate, suggesting that PHSTD acted as a cationic agent which interacted via electrostatic forces with some negatively charged sites in the leukocyte membrane. Generation of H2O2 by PHSTD was also markedly inhibited by deoxyglucose, KCN, DASA, as well as by the lipoxygenase inhibitors nordihydroguaiaretic acid, phenidone, and propylgallate. On the other hand, cyclooxygenase inhibitors such as aspirin, indomethacin, and piroxicam were inactive, suggesting that arachidonic acid metabolism via lipoxygenase pathway might have been involved in the activation by PHSTD of the NADPH oxidase in PMNs. PHSTD may mimic the effects of antibodies both as an opsonin and as a potent stimulator of the respiratory burst in PMNs and may thus serve as a model for further study of leukocyte-bacteria interactions in infectious and inflammatory sites and of the pathogenicity of immune complexes.

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