Coiling phagocytosis discriminates between different spirochetes and is enhanced by phorbol myristate acetate and granulocyte-macrophage colony-stimulating factor

Abstract

The mechanisms involved in coiling phagocytosis are not yet known, and it is not even clear whether this phenomenon is either an incidental event or a specific response. Therefore, the phagocytic uptake of Borrelia burgdorferi and other spirochetes by human monocytes in vitro was used to investigate the involvement of both sides--microbes and phagocytes--in coiling phagocytosis. As seen with electron microscopy, morphologically similar Borrelia, Leptospira and Treponema strains induced markedly different frequencies of coiling phagocytosis. The monocytes used coiling phagocytosis for both live (motile) and killed (nonmotile) B. burgdorferi, but pseudopod coils were observed neither with fragmented B. burgdorferi nor with cell-free supernatant from B. burgdorferi cultures. Investigation of the relationship of coiling phagocytosis with other pseudopod-based cellular mechanisms revealed that the use of bioreagents that inhibit conventional phagocytosis also inhibited coiling phagocytis but did not affect membrane ruffling. Bioreagents that increase membrane ruffling did not affect phagocytosis of B. burgdorferi, except for granulocyte-macrophage colony-stimulating factor and phorbol myristate acetate, which increased coiling phagocytosis selectively. These results demonstrate that coiling phagocytosis is not induced by microbial motility, viability, or a certain morphology and that it is not a random event. Rather, it is a selective uptake mechanism actively driven by the phagocytes. However, whether coiling phagocytosis represents an independent alternative to conventional phagocytosis or, alternatively, a fault in conventional phagocytosis remains to be determined.

FIG. 1

Schematic drawing of the morphological characteristics of macropinocytosis and of conventional and coiling phagocytosis. Single membrane folds which bend back toward the cell surface constitutes macropinocytosis; large droplets of pericellular fluid (which may contain some particles) are randomly trapped during this process. In conventional phagocytosis, particles are specifically engulfed by circumferential pseudopods which are guided by direct receptor-ligand interactions with the particle. Characteristic of coiling phagocytosis are unilateral pseudopods repetitively rotating around particles, giving rise to largely self-apposed pseudopod whorls. Depending on the microbes studied, pseudopod coils will transform either to ribosome-studded replicative vacuoles (23) or to organelle exclusion zones (34). The microbe-apposed membranes of the engulfing pseudopods are drawn bold.

FIG. 2

Dendrogram showing the phylogenetic relationship of the different spirochetes used in this study.

FIG. 3

Frequencies of coiling and conventional phagocytosis observed with different spirochetes. Human monocytes (2 × 10⁶) were incubated with different spirochetes (2 × 10⁷) for 45 min, and the frequencies of phagocytosis were determined by electron microscopy as described in Materials and Methods. The normalized results are expressed as means ± standard errors of the means of results from three separate experiments using monocytes from different donors, but the value given for B. burgdorferi sensu lato summarizes the mean values for the eight different species tested (Table 1). For the differences between B. burgdorferi sensu lato and the other spirochetes, a P of <0.05 was considered to be significant (*). Although they are quite similar in morphology, the different spirochetes induced totally different frequencies of coiling phagocytosis which show no correlation with their phylogenetic relationship. L.i., L. interrogans.

FIG. 4

Electron micrographs showing the uptake of various spirochetes by human monocytes. Human monocytes (2 × 10⁶) were incubated with various spirochetes (2 × 10⁷; arrows) for 45 min. Pseudopod coils are shown for B. afzelii (A; bar = 0.1 μm), T. phagedenis (B; bar = 0.4 μm), L. interrogans SV icterohaemorrhagiae Bücker (C; bar = 0.5 μm), and B. parkeri (D; bar = 0.5 μm); in panels A, C, and D, the pseudopod coils are partly covered by contrarotating surplus pseudopods (arrowheads). In panel E, a pseudopod coil enwrapping an L. interrogans SV pomona organism has already been transformed to an organelle exclusion zone (asterisk) within the cytoplasm of the monocyte. The remnants of the fused pseudopod membranes are clearly visible (bar = 2.0 μm). The pseudopod whorls are easily distinguished from the funnel-like, symmetrical surface extensions of conventional phagocytosis, shown in panel F for a B. burgdorferi sensu stricto organism (bar = 0.2 μm). The asterisk in panel D indicates a spacious phagosome.

FIG. 5

Electron micrographs showing the effects of various bioreagents on the phagocytosis of B. burgdorferi by human monocytes. Human monocytes (2 × 10⁶) were pretreated for 10 min and then incubated with B. burgdorferi cells (2 × 10⁷) for another 45 min. (A) Borrelia (arrows) are not found inside monocytes which had been poisoned with 0.1% NaN₃ (bar = 1.0 μm). (B) Upon treatment with 10 μM bafilomycin A1, monocytes still extend membrane ruffles (asterisks) but do not phagocytose Borrelia (bar = 2.0 μm). (C) Although 100 nM wortmannin almost completely inhibits phagocytosis, Borrelia (arrows) are occasionally internalized in spacious vacuoles (asterisk) reminiscent of macropinosomes (bar = 0.4 μm). (D) GM-CSF at 100 ng/ml induces a dramatic increase in the rate of coiling phagocytosis (bar = 0.3 μm). (E) Even abundant membrane ruffles of monocytes, as seen in the presence of 10% serum enriched with activated platelets, do not spontaneously form whorls (bar = 0.6 μm). (F) Empty pseudopod coils possibly indicating spontaneously coiled pseudopods, such as this one (asterisk) displayed by an untreated monocyte, are an extremely rare occurrence (bar = 0.6 μm).

FIG. 6

Effects of various stimulatory and inhibitory bioreagents on the frequency of coiling and conventional phagocytosis by human monocytes. Human monocytes (2 × 10⁶) were pretreated with substances which either inhibit conventional phagocytosis or stimulate membrane ruffling. After 10 min, B. burgdorferi cells (2 × 10⁷) were added for another 45 min; only the incubation medium containing NaN₃ was replaced by normal medium. The frequencies of phagocytosis were determined by electron microscopy as described in Materials and Methods; untreated monocytes gave the spontaneous phagocytic activity, set at 100%. The normalized results are expressed as means ± standard errors of the means of results for usually three (five for control, PMA, and GM-CSF) separate experiments using monocytes from different donors. All inhibitors of phagocytosis reduced coiling and conventional phagocytosis equally, regardless how strong their inhibitory effect was. The ruffling-stimulating or activating substances did not affect the uptake of Borrelia except for PMA and particularly GM-CSF, which increased the frequency of coiling phagocytosis selectively (*, P = 0.05 by the χ² test) compared with the spontaneous uptake.

FIG. 7

Effects of GM-CSF on the frequency of coiling phagocytosis by different human phagocytes. Human monocytes, neutrophils, and eosinophils (2 × 10⁶) isolated from the same individual (different from those represented in Fig. 6), were incubated with B. burgdorferi cells (2 × 10⁷) for 45 min in the presence or absence of GM-CSF (100 ng/ml) added 10 min before. The bars represent the relative frequencies of coiling and conventional phagocytosis with GM-CSF and without, determined by electron microscopy as described in Materials and Methods, given as means ± standard errors of the means of triplicate determinations. Essentially the same results were observed in two additional identically performed experiments with phagocytes from different donors, and the results shown are therefore considered to be representative. PMA and especially GM-CSF increase the frequency of coiling phagocytosis selectively. The stimulated frequencies of the different phagocyte populations peak at about the same level, although their spontaneous frequencies are quite different.