Title

Antibody-Mediated Enhancement of Infection byDengue Virus of the P815 Murine Mastocytoma Cell
LineAuthor(s)Legrando, Luis Fernando Sanchez; Hotta, Hak;
Hotta, Susumu et al.

Citation

Biken journal : journal of Research Institutefor Microbial Diseases. 1986, 29(2), p. 51-55

Version Type VoR
URL

https://doi.org/10.18910/82402

rights
Note

Osaka University Knowledge Archive : OUKA
https://ir.library.osaka-u.ac.jp/Osaka University

BIKEN JOURNAL \101.29.51-55,1986

SHORT COMMUNICATIONANTIBODY-\, IEDIATED ENHANCEMENT OF INFECTION BY DENGUEVIRUS OF THE P815 MURINE MASTOCYTOMA CELL LINE

LUIS FERNANDO SANCHEZ LEGRANDi, 3 }IAI< I-LOTTAi
SUSUMU I{OTTA2 and MORIO HOMMA'.'
Department of IVlicrobiology, 1<0be Uni\'CTSit}. School of A, ledicinc. 7-5-I 1<us unoki~cho,Chuo-ku, 1<0bc, Hyogo, 650 Japan', and Institute for Tropical IVledicine, 1<nnnza\\. a Alledicnl
University, I-I Uchinada-cho, Aza Daigaku, 1<nhoku -gun, ishika\^a, 990-02 Japan*(Received Decembc, 10,1985)

uMMARY Dengue type 2 virus (DEN 2) could replicate only to a limited extent
in a inurine mastocytoma cell line, PSI5. The viral multiplication was en-hanced 10- to 100-fold by mousc anti-DEN 2 antiserum or anti-DEN 2 type-SPCci11c monodonal antibody, diluted beyond their neutralizing titers. Cells incubatedwith virus-antibody, mixtures changed inorphologically developing a mature mastcell-like appacarancc, 4-5 days after infection. The indirect fluorescent antibody,technique showed that the enhancemcnt of infection was caused by an increase inthc number of DEN 2-infected cells. This is the first report that cells of mast celllineage support dengue virus multiplication, and that virus production is enhancedin the presence of anti-dengtic antibodies

Dengue viruses have a predilection for Iymphoid and myeloid tissues including monocytes, macrophages and histiocytes. The
virus permissiveness of mononuclear phagocytes in monkeys and humans is reported tobe enhanced in t}\e presence of subneutralizIng titers of anti-dengue antibodies (Brandt
at a1. , 1982; Daughaday at a1. , 1981; Halstcad
and O'ROUTke, 1977; Halstead at a1. , 1977;
3 Present address : Department of Pediatrics,

Division of Allergy!jinmtinology, Ernofj, Uni\, ersity School of Medicine, 69 Butler Street,
S. E. , AtIanta, GA. 30303, U. S. A+ To \\. horn requests for reprints should be addressed

Hotta at a1. , 1984b). The enhanced infection was due to an incrcasc in the number of

infected cells witlt FC receptors on their surface. The FC receptors apparently acted as
entrance points for the virus after forming
Immunecomplexes with antibodies. Experlmental models in mice have also been usedto study, enhancement of infection, and havedcmonstrated that perltoneal macrophaues
from dengue type I virus-immune inicc show
greater permissiveness than those from non-Immune control mice to dengue type 2 virus(Hotta and Hotta, 1982). Enhanced permisSIveness of mouse macrophage cell lines todengue virus was also observed in the pre-51

sence of anti-dengue antibodies (Halstead ata1. , 1983 ; Hotta at a1. , 1984a).

3

Clinical and laboratory studies have sug-gested that mast cells may be involved in
antibody- and cell-mediated responese todengue virus Infection in man (George andDuraisamy, 1981 ; 1<uberski at a1. , 1977 ;Pavri at a1. , 1979). However, it is still unknown whether mast cells act as a target of
dengue virus replication. In the present
study we investigated the multiplication ofdengue virus and the Infection-enhancing
effect of anti-dengue antibodies in cultures ofa inurine mastocytoma cell line, P815
P815 colts (Dunn and P. tter, 1957), kindly
donated by Dr. Yasuhiro Hosaka (Depart-^2

the mouse antiserum and I : 1,024 for the,,,*.
La.

~.,

*o01o_I.
^..

merit of Preventive IVledicine, Research Institute for Allcrobial Diseases, Osaka Uni-versity, Osaka, Japan), were cultured in
Eagle's minimum essential medium supplemerited with 107, fetal calf serum withoutantibiotics. Dengue type 2 virus (DEN 2;New Guinea C strain) was prepared as thesupernatant of a 1070 infected suckling mousebrain hornogenate. Anti-DEN 2 antiserum
was raised in eight-week-old male ICR miceby subcutaneous injection of DEN 2 mixedwith I ing of aluminum hydroxide and subsequent booster injections on days 14 and 28.
Control mouse serum was prepared in thesame way except for use of uninfected suckling mouse brain hornogenates. NIOnoclonalanti-DEN 2 antibody, was a product of a hybridoma cell line (3H5), and was characterized as a type-specific neutralizing antibody
and identified as inurine IgGl (Gentry at al. ,1982; Han. hai at a1. , 1982). Th, 507,
PIaque-reduction neutralizing titers (PRNT50)of the antibody, preparations were I : 256 forAE..~,2o345

Days after infection
FIGURE I. Multiplication of DEN 2 in the P815
mastocytoma cell line. DEN 2 \\, as inoculated
into cultures of P815 cells in the presence of mouseantserum diluted 100-fold (A----A) and 1,000-fold (,^A) beyond its PRNTs, , or a monoclonal anti-DEN 2 antibody diluted 100-fold(.----.) and 1,000-fold (.^.) beyond its
PRNT, ,. Open circles (0^0) represent \, iral
growth in the presence of a 1,000-fold dilution ofcontrol

serum

which was obtained from micejinmunized witlT uninfected suckling mouse brain
preparations

washed with Hanks' balanced salt solution toremove uriadsorbed virus, and the complete
medium containing the subneutTalizing antibodies was added to the cultures. Portionsof the culture fluids were taken every d^, after
infection, and the samples were titrated formonoclonal IgGl. DEN 2 was in DCulated
into P815 cell cultures at a multiplicity of inforti. n (M. 0.1. ) of I pinque-forming unit
(PFU) per cell, in the presence or absence

virus infectivity.

of antibodies, which were diluted I : 100 andHotta, 1982; Hotta er a1. , 1984a). whenI : 1,000 beyond their PRNT After virusadsorption at 35 C for 90 min, the cells wereand with tofuidine blue, and observed under52

BIKEN JOURNAL \!o1.29 No. 2 1986

DEN 2 antigen in infected P815 cells wasexamined by the fluorescent antibody (FA)technique as described previously (Hotta andrequired, the cells were stained with Giemsa

TABLE I

Mo, pho/ogi'cal lean, ,'CS of DEN 2-infected P8/5 cells.
inoculum"No. of vacuoles

No. of granules% FA-POSiti\'e

per cell"per cell"cells"

Virus alone

5.0. . 0.3

16. 0 t 3. O

loot2.3

Virus plus antibody

17.0, . 2. O

193.0, = 5 .I

36. 0 t 3.7' DEN 2 was inoculated onto P815 cells at an M. 0.1. of I in the absence (virus alone) or presence (virusplus antibody) of mouse anti-DEN 2 antiserum diltited I : 1,000 from its PRNT, o" Mean value t standard deviation for 300 cells' Mean value ,. standard deviation for three different preparations

a light microscope.

PSI5 cells supported DEN 2 multiplication to some extent even in the absence of theantibodies and dose-dependent virus production was observed in the range of 0.01 to I
NIO. I. (data not shown). Viral production

The secretion of chemical nlediators frommast cells and the role of these products in
immediate and delayed type hypersensitivity
have been studied extensively (Askenasc,1977; AUSten at a1. , 1976) but there havebeen few studies on the role of mast cells as

In the cultures was enhanced 10- or 100-folda host of microbial replication (Padawer,

in the presence of subneutralizing concentrations of mouse anti-DEN 2 antiserum (dilutedtion indicated that mast cells arc derived

I: 100 or I : 1,000 from its PRNT , respectively) (Fig. I). The monoclonal neutralIzIng antibody caused the same extent of enhancement of infection as the polyclonal antiserum (Fig. I).
At 5 days after infection, the number ofvacuoles in the infected P815 cells had increased almost threefold and the number ofmetachromatic granules had increased 100-fold in the presence of subneutralizing concentrations of anti-DEN 2 antiserum (TableI). Cell viability decreased slowly to 6014 ofthat of the uninfected cell cultures. Byphase contrast microscopy, the cells infectedwith virus plus antibody were seen to be1971). Previous studies on cell differentia-

from mononudcar phagocytes (Czarnetzki ata1. , 1979; 1981; 1982; 1983). Thus mast
cells or their precursor cells may act as thetarget for dengue virus infection, as do mononuclear 11hagocytes (Brandt et a1. , 1979; 1982;
Daughaday, at a1. , 1981; Halstead and O'Rourke, 1977; Halstead at a1. , 1977; }lotta andHotta, 1982; Hotta at a1. , 1984a; 1984b)
The present study showed that DEN 2 rePIicated in the PSI5 mastocytoma cell line toa limited extent. A more interesting finding
was that 10- to 100-fold enhancement ofviral production was observed when PSI5cells were infected with DEN 2 in the prcsenceof subneutralizing concentrations of mouselarge mononuclear cells with spreading cyto-anti-DEN 2 antiserunT or a monoclonal anti-

PIasmic processes and Increased cytoplasmic
granulation (data not shown). These changes

DEN 2 antib, dy (IgGl). Th" onhan. ,montof infection is probably mediated by antiDEN 2 IgG antibody through the interactionof virus-antibody complexes with IgG receptors on the surface of mast cells (Ovary, 1971)
Although P815 cells show considerable dedifferentiation, as evidenced by marked re-were not observed in cells infected with virus

alone. On FA staining of DEN 2 antigen,positive fluorescence was seen in the cytoPIasm but not the nudeus. The percentageof FA~positive cells was 36% in the presenceof the enhancing antibody, and 107, in cells
Infected with virus alone on day 4 after inforti. n (T, bl, I).

duction in the histamine content and cellularmetachromasia, we consider that they

areuseful for studies on enhancement of infoc-SHORT COMAiu>:ICATioN53

tion in cells of mast cell lineage, since theimmunoglobulin receptors on their surface

appear to have specificity for all classes ofmouse IgG molecules and show many SImiIanties to those of macrophages and normal
mast cells (Cline and warner, 1972; Ovary,1971).

presence of the same helminth antigen (Ishizaka et a1. , 1977), and also that a mast cell
growth factor present in antiserum preparations could promote differentiation of precursor cells (Tertian at a1. , 1981; Yung at al. ,1981). \\;hether the in orphological alter ations shown by DEN 2-infected PSI5 cells

P815 cells infected with DEN 2 in the pres-were due to differentiation of the cells during

ence of anti-DEN 2 antiserum underwent

the course of virus infection or merely tocytopathic effects remains to be clarified.

in orphological alterations, I. e. , they spreadon plastic surfaces and developed the ap-

pearance of mature mast cells. In this connection, it Is interesting that precursors ofmast cells from Schi'$1030",@ I"""sowi'-immune

animals were found to show a tendency todifferentiate when they were cultured in theACKNOWLEDGiVIENT

One of the authors (L. F. SL. ) was recipient of ascholarship from the Japanese Government (NIOnbusho) from April 1979 to March 1984

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SHORT COM xiuNicATioN35