Polyanionic Agents Inhibit Phagosome‐Lysosome Fusion in Cultured Macrophages: A Reply to the Suggestion of Goren, Vatter, and Fiscus to the Contrary

: The paper and review by Goren et al. (J. Leukocyte Biol. 41, 111, 1987) contain serious objections to the reports from several laboratories on the pattern of fusion of secondary lysosomes with phagosomes (yeasts being predominantly the target) in polyanion-treated macrophages; these reports had concluded that the polyanions were inhibitors of this fusion. The main objection by Goren et al. is to the alleged misuse of electron microscopic (EM) lysosome markers; many instances of phagosome-lysosome (P-L) fusion in the treated cells have therefore been missed. The central argument is that 1) the "hydrocolloid" properties of certain of these polyanions hinder the passage of the enmeshed marker from lysosome to phagosome after their fusion and 2) this hindrance is mistakenly interpreted as indicating that fusion has not taken place, thus giving rise to the belief that the polyanions can inhibit fusion. In reply, we explain that we score as P-L fusion any instance of marker (ferritin) being seen anywhere in a fused phagosome (phagolysosome). For this crucial reason, immobilisation of marker by a hydrocolloid polyanion, e.g., in lysosomal residue of phagolysosomes or just within phagosome membranes (as in Goren's Figs. 5 and 6 [8]), would not seriously threaten the marker distinction between fusion and nonfusion (with consequent underestimation of the former) and therefore would not invalidate the reports of a high incidence of nonfusion in polyanion-treated macrophages. Such inhibition of P-L fusion is supported by using as lysosomal label the nonpermeant fluorescent probe lucifer yellow (accepted by Goren et al. as a reliable indicator of fusion). Further support comes from the correlated inhibition of the saltatory movements of secondary lysosomes previously described; static lysosomes will have their contact with the phagosomes severely restricted. Another criticism is based on the failure of certain salient properties and functions of phagosomes to change significantly after polyanion treatment of the macrophages; these include intraphagosomal digestion, presence of lysosomal enzymes, and acidification. However, this indirect evidence can be accounted for alternatively by the operation of factors (primary lysosomes, endogenous acidification, etc.) not affected by the polyanionic block. We conclude that fusion inhibition by the polyanions is a real phenomenon, as previously reported, notwithstanding the hydrocolloid properties of some of them. Furthermore, an explanation based on hydrocolloid properties is questionable, since one or possibly two of the five main polyanionic agents appear not to be hydrocolloids.