Entry of Rickettsia tsutsugamushi into polymorphonuclear leukocytes

Factors involved in the phagocytosis and entry into polymorphonuclear leukocytes (PMNs) of Rickettsia tsutsugamushi were studied by electron microscopy. R. tsutsugamushi propagated in baby hamster kidney cell cultures was incubated with guinea pig peritoneal PMNs in vitro at 35 degrees C. Structurally intact and degenerating rickettsiae were found in phagosomes, but only intact rickettsiae escaped phagosomes and specifically entered the glycogen-rich cytoplasm. The extraphagosomal cytoplasmic rickettsiae were found within 30 min after incubation; continued incubation for 4 h increased the rickettsial entry about fourfold as seen in ultrathin sections. Most rickettsiae in phagosomes were degenerating after 4 h of incubation. When incubated at 25 degrees C, no entry and very few phagocytized rickettsiae were observed. At 40 degrees C, rickettsial entry was greatly reduced, but more rickettsiae were found in phagosomes than at 35 degrees C. Preincubation of rickettsiae at 56 degrees C for 20 min with trypsin or with 2,4-dinitrophenol inhibited entry, but many rickettsiae were in phagosomes. Glutaraldehyde or formaldehyde fixation of rickettsiae and addition of 2-deoxyglucose, iodoacetamide, cytochalasin B, colchicine, or vinblastine inhibited all rickettsial uptake by PMNs. Acid phosphatase cytochemistry of infected PMNs revealed the enzyme activity only in phagosomes with degenerated rickettsiae and not in those with intact rickettsiae. These observations indicated that rickettsiae are passively phagocytized by PMNs, and only those that are intact actively escape from phagosomes, which selectively inhibits lysosomal fusion.