Phagocytosis of herpes simplex virus by human granulocytes and monocytes

Abstract

Polymorphonuclear leukocytes (PMN) can mediate cytotoxic reactions against virus infected targets cells. We observed very efficient binding of PMN to HSV-infected fibroblasts when loaded with HSV-specific antibodies. Using electron microscopy, infected fibroblasts were found to be totally surrounded by PMN and the phagocytosis of virions and fragments of infected cells was demonstrated. To quantify and study this phenomenon, and to compare PMN with monocytes, we developed radiometric and fluorometric phagocytosis assays. Leukocytes were mixed with [3H]glucosamine- or FITC-labeled virus and incubated at 37 degrees C. PMN associated radioactivity or fluorescence per cell as measured by flow cytometry was determined. PMN phagocytosis was dependent on the presence of specific anti-HSV antibodies and could be enhanced by addition of complement. Monocytes were also able to phagocytize virions; however, the rate of uptake was less than that for PMN. Under optimal conditions the total amount of herpes simplex particles that could be associated with one PMN or monocyte was about 10,000. PMN and monocytes are capable of phagocytosis of HSV. This may be an important factor in preventing the spread of infection in vivo.

Flow cytometric quantitative determination of ingestion by phagocytes needs the distinguishing of overlapping populations of binding and ingesting cells

“…The most frequently used particles in phagocytotic measurements are zymosan (1)(2)(3)(4)(5) and Staphylococcus aureus (1,2,6-9), labeled almost without exception with fluorescein isothiocyanate (FITC). FITC-labeled Streptococcus pneumoniae (10,11), Escherichia coli (12), Neisseria meningitis (13), latex particles (5,12,14,15), herpes simplex virus (16,17), and entire yeasts such as Saccharomyces cerevisiae (18)(19)(20), and Candida albicans (8,21,22) have also been used. In this study, zymosan was used as a phagocytosable particle.…”

Degradation of Herpes Simplex Virions by Human Polymorphonuclear Leukocytes and Monocytes

“…There are several reasons to assume that degradation of virions occurs. After phagocytosis and phagosomelysosome fusion (Van Strijp et al, 1989a) it is likely that virus particles are inactivated and eventually degraded, due to the antiviral effects of lysosome constituents, such as defensins, cationic proteins (Rouse et al, 1980;Selsted & Harwig, 1987;Thorne et aL, 1984;Zerial et al, 1987;Daher et al, 1986) and the reactive oxygen species liberated by the PMN. It has been shown that toxic oxygen products are able to inactivate virus infectivity (Belding et al, 1970).…”

“…HSV particles was observed, but spontaneous uptake of virions by MN reached a level of 25% after a 2 h incubation period. Addition of opsonins to this assay enhanced the uptake of virions for the phagocytes, but for MN the uptake process proceeded over a longer time (Van Strijp et al, 1989a). On the basis of the above mentioned measurements, for the following experiments, in which the degradation of HSV glycoproteins was studied, we used a 30rain phagocytosis time with radiolabelled virus.…”

“…Purity was checked by electron microscopy and it was shown that over 95 % of the particles were enveloped viruses. To determine phagocytosis of the virus PMN or MN were mixed with purified fluorescein-labelled virus and human serum, incubated for various time periods and measured on a per cell basis in a FACStar flow cytometer as described (Van Strijp et al, 1989a). In the absence of opsonins no uptake by PMN of.…”

“…No difference was observed between phagocytes of donors that were seropositive or seronegative for HSV. Virus was radiolabelled using 20 rtCi [3H]thymidine per ml or methionine-free medium supplemented with 35 gCi [3SS]methionine per ml, and purified as described (Van Strijp et al, 1989a). Virus was stored at -70 °C in small volumes at a standard concentration of 350 I.tg/ml.…”