[CANCER RESEARCH 42, 3617-3624,
0008-5472/82/0042-OOOOS02.00
September 1982]
Inhibition of Concanavalin A Response during Osteopetrosis Virus
Infection1
Joseph A. Price2 and Ralph E. Smith3
Department of Microbiology
and Immunology, Ouke University Medical Center, Durham, North Carolina 27710
ABSTRACT
Infection of animals with oncogenic viruses frequently leads
to an immunosuppressed state. We have examined immunosuppression induced by an avian osteopetrosis virus, myeloblastosis-associated
virus of subgroup B inducing osteope
trosis [MAV-2(O)], and our results suggest that this virus in
duces immunosuppression by a novel mechanism. Lymphoid
cells from osteopetrotic chickens did not respond to a wide
dose range of concanavalin A (Con A) over a wide cell density
range. Failure to undergo blastogenesis was not due to a lack
of Con A-binding sites, since 125l-labeled Con A bound to
lymphocytes from infected and uninfected chickens. Infected
lymphocytes failed to respond to sodium metaperiodate stim
ulation, indicating that failure of blastogenesis was not due to
a blockage of Con A receptor sites. MAV-2(O) infection of
chicks 8 days of age resulted in a transient immunosuppression
which appeared 1 to 2 weeks after infection. Cell-mixing ex
periments showed that MAV-2(O)-induced immunosuppression
was not attributable to suppressor cells. In contrast, adherent
cells from normal lymphoid preparations restored mitogenicity
to lymphocytes from MAV-2(O)-infected
animals. Adherent
cells were present in the spleen and peripheral blood lympho
cytes of MAV-2(O)-infected chickens in numbers comparable
to those of the uninfected animal, and both sets of cells
contained Fc-dependent phagocytic activity and nonspecific
esterase. Peritoneal exúdate cells were elicited from osteope
trotic and normal chickens in similar numbers. We conclude
that MAV-2(O) induces immunosuppression by interfering with
an accessory function of macrophage-like adherent cells.
INTRODUCTION
Osteopetrosis is a disease in which excessive bone accu
mulates in a characteristic pattern (19, 44). Children with
osteopetrosis frequently develop anemia (13), thrombocytopenia (24), and an increased susceptibility to infection, possibly
due to monocyte and neutrophil dysfunction (35).
Several animal models are available for the study of osteo
petrosis. The rat and mouse forms of osteopetrosis are re
markably similar to the human disease, in that they appear to
involve a congenital failure in osteoclasis (23, 41). Osteope
trosis in the osteopetrotic mutant rat is accompanied by atrophy
of the thymus and failure of lymphocytes to respond to mito1 This study was supported by Research Grants CA12323 and CA14236 from
gens in vitro (25). Osteopetrosis in chickens is induced by a
number of avian leukosis viruses (18, 20, 40). Excessive bone
growth occurs because of virus-induced osteoblast prolifera
tion (41, 42), rather than the failure of osteoclasis observed in
mammalian osteopetrosis.
Infection of animals with oncogenic viruses frequently leads
to a compromise of the immune system. This immunosuppres
sion is manifested by decreased mitogenic response in vitro
and is mediated in most cases by suppressor cells. For exam
ple, suppressor cells have been found in mice infected with
Moloney (51), Friend (52), and AKR virus (36). Infection of
chickens with reticuloendotheliosis
virus rapidly leads to an
immunosuppressed state (6) attributable to suppressor T-cells
induced by the nondefective helper virus present in stocks of
this virus (39). Suppressor macrophages are found in Marek's
disease virus-infected chickens (22). A defect in macrophage
function has been described recently in spontaneous murine
mammary carcinoma (9) and in leukemic AKR mice (27), both
virus-associated disorders. In fact, low-molecular-weight
polypeptides specified by murine retroviruses have been implicated
in the macrophage defect seen in the former system (10).
Although most strains of avian leukosis virus induce a low
incidence of osteopetrosis (4), several weeks are required for
the appearance of bone lesions (32). A nondefective associ
ated virus derived from avian myeloblastosis virus has been
isolated which induces a high incidence of osteopetrosis within
a short time (47). Injection of this virus, MAV-2(O),4 into 10day-old chick embryos leads to the appearance of osteope
trosis within the first week after hatch and a 100% incidence
by 2 to 3 weeks of age (3). Chicks infected with MAV-2(O)
demonstrate an immunosuppressed state during the develop
ment of massive bone lesions (46, 48). Previous studies using
avian leukosis viruses of subgroup A have reported minor
differences in the antibody response of infected chickens (11,
31, 34) and small changes in cell-mediated immunity (16, 26).
MAV-2(O)-induced immunosuppression is accompanied by a
loss of lymphoid organ mass (3), a decrease in the ability of
infected peripheral and spleen lymphocytes to respond to
stimulation by PHA (48), a decrease in the ability of infected
cells to form IgM-dependent hemolytic plaques (48), and a
depressed response to a variety of T- and B-cell-dependent
mitogens (32). However, little more is known about the nature
of the immunosuppression induced by avian leukosis viruses.
Use of MAV-2(O) offers a unique opportunity to study avian
leukosis virus-induced immunosuppression: (a) MAV-2(O) in
duces proliferative disease more rapidly than any other nondefective avian leukosis virus. MAV-2(O) appears to cause the
the National Cancer Institute, NIH, Bethesda, Mil
2 Postdoctoral trainee supported by NIH Grant CA 09111. Present address:
Department of Biology, Trenton State College, Trenton, N. J. 08625.
3 To whom requests for reprints should be addressed, at Department
Microbiology and Immunology. Box 3020, Duke University
Durham. N. C.
Received November 18, 1981; accepted June 8. 1982.
SEPTEMBER
1982
Medical
of
Center,
* The abbreviations
used are: MAV-2ÕO), myeloblastosis-associated
subgroup B inducing osteopetrosis; PHA, phytohemagglutinin;
hydroxyethyl)-1-piperazineethanesulfonic
acid; PBL, peripheral
cytes; Con A, concanavalin A.
virus of
HEPES. 4-(2blood lympho
3617
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J. A. Price and R. E. Smith
proliferation of osteoblasts or osteoblast stem cells (41, 42);
(o) MAV-2(O) induces lymphoid hypoplasia without the direct
destruction of cells in lymphoid glands and causes a failure of
stem cells to populate the glands or to proliferate in them (17,
46); (c) MAV-2(O) interacts with bone marrow cells in a lytic
fashion, particularly when hatched or bursectomized chickens
are infected (30, 32). The present study investigates the nature
of the depressed blastogenic response observed in MAV-2(O)infected chicks. The results indicate that MAV-2(O) does not
lead to the presence of a suppressor cell, nor does infection
induce a T-cell defect. Instead, lymphocytes from the MAV2(O)-infected chick cannot respond to mitogen because of a
defect in a plastic-adherent cell.
MATERIALS
Viruses.
derivative
of avian myeloblastosis
virus was used in these experiments (47). This virus was designated
MAV-2(O). Serial passages of this virus were made by injection into a
chorioallantoic
membrane vein of 12-day SPAFAS (SPAFAS, Inc.,
Storrs, Conn.) embryos with a 1:20 dilution of viremic serum [10"
plaque-forming
units/0.1
ml, diluted
in phosphate-buffered
saline,
(0.15 M NaCI, 2.7 mM KCI, 1.5 mM KH2PO4, 8 mM Na2HPO4, 0.9 mM
C.-iCI . and 0.5 mM MgCl2-6H2O), pH 7.4, containing 10% calf se
rum]. Sera from birds showing heavy osteopetrosis were pooled and
stored at -70°. MAV-2(O) was assayed by plague formation in chick
embryo fibroblasts (15).
Experimental Animals. Embryonated eggs from the inbred SC line
were obtained from Hyline, Dallas Center, Iowa. Eggs were maintained
in a humidified incubator until 1 day prior to hatching, at which time
infected and control eggs were transferred to separate hatchers. Birds
were maintained in an animal isolation facility designed to prevent
spread of viruses among groups of infected animals and were fed
Purina Growena Chow and water ad libitum. Embryos were infected by
injection into a vein of the chorioallantoic
membrane (2). Virus was
administered to chicks 1 day of age or older by injection into the jugular
vein. Osteopetrosis in diseased animals was essentially as described
previously (33, 40). In most cases, osteopetrosis was observed within
2 weeks, and birds were incapacitated by heavy bone growth within 4
weeks.
Lymphocyte
Proliferation.
Cell suspensions were prepared from
the spleen and thymus in Alsever's solution (0.01 M dextrose, 0.03 M
sodium citrate, 0.001 M citric acid, and 0.07 M sodium chloride, pH
6.1). Debris was removed by sedimentation at 1 x g for 4 min. Cells
from the thymus were washed with medium and used without further
processing. Lymphocytes were cultured in the absence of serum in
Roswell Park Memorial Institute Tissue Culture Medium 1640 (Flow
Laboratories, Inc., Rockville, Md.) supplemented with 100 units of
penicillin and 100 jig of streptomycin per ml, 2 mM glutamine, and 10
mM HEPES buffer. Spleen lymphoid cells were separated from erythrocytes by centrifugation at 80 x g for 8 min or at 600 x g for 10 min
over a cushion of Percoli (Pharmacia Fine Chemicals, Inc., Piscataway,
N. J.). The Percoli solution was prepared by mixing 12 parts of Percoli
with 8 parts of 2.1 M NaCI and adding HEPES buffer to a final
concentration of 10 mM, the final pH being adjusted to 7.2 with 1 N
MCI. PBL were obtained by collecting blood into an equal volume of
Alsever's solution, followed by centrifugation over a 14:6 preparation
of Percoli at 900 x g for 20 min. Cells were washed once by centrif
ugation at 450 x g in Alsever's solution, followed by one washing in
medium. Cells were counted, viability was assessed by trypan blue
exclusion, and after adjusting them to the desired concentration, cells
were cultured in microtiter dishes (Microtest II; Falcon Plastics, Oxnard,
Calif.) at 39.5° in humidified CO2 incubator. Labeling was carried out
with 0.05 /iCi of 5-iodo-2'-deoxyuridine
(No. IM 355; Amersham Corp.,
Arlington Heights, III.) overnight, starting 48 hr after initiation of the
cultures. In most experiments, 10 fig of Con A per ml (Miles Laborato
3618
2 to 3 x 106 viable cells.
Periodate activation of lymphocytes was performed by suspending
washed spleen cells in serum-free medium containing sodium metaperiodate (G. Frederick Smith Chemical Co., Columbus, Ohio) at the
indicated concentrations and incubating the cells at room temperature
for 10 min. Samples were then centrifuged, washed in cold medium,
resuspended, and cultured as described above. Cultures were labeled
from 24 to 40 hr and were terminated by washing onto glass fiber filter
paper in a lymphocyte harvester (BélicoGlass, Inc., Vineland, N. J.),
using distilled water. Samples were counted in a Beckman Y counter
with lymphocyte stimulation evaluated by calculating the stimulation
index (cpm of stimulated cultures/cpm
unstimulated cultures) and net
incorporation (Acpm = cpm stimulated - cpm unstimulated).
Binding Assay. Con A was iodinated by the chloramine-T method to
a specific activity of 2.2 x 107 cpm/mg and stored until use at —20°.
Protein concentration was determined by the Bradford method (BioRad Laboratories, Richmond, Calif.). To perform binding assays, 125I-
AND METHODS
An end point-purified
ries, Elkhart, Ind.) were used to stimulate
labeled Con A was diluted in serum-free Roswell Park Memorial Insti
tute Tissue Culture Medium 1640 and mixed with 5 x 106 spleen cells
in 0.2 ml at 39.5° in a U-shaped microtiter dish (Linbro Scientific,
Hamden, Conn.) for the times indicated. These cells were then washed
3 times by centrifugation at 500 x g for 10 min, harvested in phos
phate-buffered saline onto glass fiber filter paper, and counted in a
Beckman y counter. Calculations include a correction for residual label
in wells receiving each concentration of Con A in the absence of cells.
Adherent Cells. Adherent cells were obtained in microtiter wells by
plating washed cell suspensions in adherence medium [Medium 199
containing 8% calf serum (Grand Island Biological Co., Grand Island,
N. Y.), 2% fetal calf serum (Grand Island Biological Co.), 10% tryptose
phosphate broth (Difco Laboratories, Detroit, Mich.), 2 mM glutamine,
100 units penicillin per ml, 100 HQ streptomycin per ml, and 10 mM
HEPES] for 1 hr at 39.5°. Nonadherent cells were removed by washing
gently with culture medium, and adherent cells were counted with an
inverted microscope equipped with a grid in the eyepiece. A similar
procedure was followed for adherent cell depletion. In some experi
ments, Sephadex G10 columns of 10-ml bed volume were equilibrated
with adherence medium at 37°and used with a nonadherent-cell yield
of 50%. Yields of nonadherent cells prepared by gentle washing of
plastic dishes was about 75%. Two sequential platings on plastic
dishes was sufficient to reduce the yield of adherent cells detected by
plating test samples overnight in adherence medium by 98%; a single
plating removed 90 to 95% of the adherent cells.
Packed Cell Volume. Blood samples were collected in heparinized
50-/il microhematocrit tubes (Clay Adams, Parsippany, N. J.). Packed
cell volume was determined for each sample by comparison to a
standard scale. Means ±S.E. were calculated for each determination.
Data Analysis. Analysis was conducted on a Digital PDF 11/10
computer programmed to perform Student's 2-tailed i test for sample
means of unequal variances and Dunnett's multiple range comparison
(49).
RESULTS
Basic Parameters of Lectin Unresponsiveness. To establish
which organ systems of the infected animal were immunologically functional, it was essential to use a method to examine
different tissues for T-cell-dependent function. Responses to
PHA and Con A have been shown to be T-cell dependent in the
chicken (53). Con A was chosen for the present analysis
because it provides a sharp dose response (21). Spleen cells
from normal chicks responded to Con A, while spleen cells
from osteopetrotic chicks were markedly inhibited (Table 1).
The optimal dose of Con A for normal lymphocytes was con
sistently 5 to 10 jig/ml. Varying the concentration of Con A
from 1 to 100 jug/ml did not restore the response of cells from
infected chicks assayed individually or as pools. If there was a
CANCER
RESEARCH
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VOL. 42
Immunosuppression
change in the relative proportion of T- and B-cells in osteopetrotic chickens, response might be corrected by altering the
number of cells per culture. However, varying the number of
cells per culture between 1 and 8 x 106 cells/well did not
Induced by Avian Osteopetrosis
Virus
although additional receptors might be present on infected
cells. However, further studies are needed to precisely mea
sure the affinity of binding, to establish the number of receptors
on normal and infected cells, and to determine whether the 2
cell populations differ in the rate of internalization of labeled
Con A. One explanation for an increased Con A binding by
infected cells is that it binds to viral glycoproteins (50). An
excess of Con A sites due to the presence of viral glycoprotein
might also interfere with blastogenesis. To examine this pos
sibility, a stimulus was chosen for mitogenesis which did not
require specific receptors. Oxidative stimulation by sodium
metaperiodate triggers a single round of cell division and is
receptor independent, due to the general nature of the oxida
tion of exposed surface components (28). Sodium metaperio
date oxidation caused normal chick spleen cells to proliferate,
but the spleen cells from osteopetrotic chicks failed to respond
over a wide dose range (Chart 1). This result suggests that the
lack of blastogenic response in osteopetrotic lymphocytes was
not due to a block in Con A receptors.
Immunosuppression
Accompanies
MAV-2(O)-mduced
Anemia. To determine when the lack of blastogenic respon
siveness first appears in infected animals, chicks were infected
with MAV-2(O) 8 days after hatch and examined for immunosuppression. Chicks infected at this time develop a transient
anemia without osteopetrosis, with antiviral antibody appearing
during convalescence (30, 32). The blastogenic response of
restore response in osteopetrotic populations (Table 1). The
procedure used to purify the lymphocytes influenced the blastogenic response obtained. Normal lymphocytes purified by
differential centrifugation responded over a cell density range
between 3 and 7 x 106 cells/well, while lymphocytes purified
by a Percoli gradient had a narrow cell density optimum at 2
x 106 cells/well. Lymphocytes from MAV-2(O)-infected chick
ens failed to respond to Con A regardless of the purification
technique used.
It is known that subgroup B viruses are cytopathic in certain
cells, hence the basis for the plaque assay which is used to
quantitate MAV-2(O) (15). The possibility that MAV-2(O) was
simply killing the cultured spleen cells was ruled out by exam
ining their viability and observing that there was no significant
difference in the viability of cultured normal and osteopetrotic
spleen cells. Furthermore, there was no morphological evi
dence for blast cells in cultures of osteopetrotic lymphocytes,
although blast cells were abundant in cultures of normal cells,
therefore arguing against an artifact in labeling as the expla
nation for the results described above.
An inhibition of response might be encountered if Con A
failed to bind to infected cells, perhaps due to interference by
viral proteins at the cell surface (26). This is unlikely to be the
explanation for reduced blastogenesis of osteopetrotic cells,
because iodinated Con A bound to infected cells better than to
control cells (Table 2). These experiments indicated a similar
affinity for Con A by normal and osteopetrotic spleen cells,
o NORMAL
•OSTEOPETROTIC
Table 1
Effect of spleen cell number on Con A response
Spleen lymphocytes were obtained from 4 to 6 birds, pooled, and purified
using a Percoli cushion. Con A was added at 10 fig/ml to the concentration of
lymphocytes indicated. Assays were performed in triplicate. Cells were cultured
for 48 hr in serum-free Roswell Park Memorial Institute Tissue Culture Medium
1640, and 0.05 /iCi [125IJ-5-iodo-2'-deoxyuridine was added overnight. Cells were
harvested onto glass fiber filters, and radioactivity
spectroscopy.
was measured by scintillation
I.O
Lymphocyte stimulation
Normal
Chart 1. Sodium metaperiodate-induced
blastogenesis. Washed spleen cells
of normal and osteopetrotic chicks were stimulated with the concentrations of
sodium metaperiodate shown and then labeled from 24 to 40 hr. Net cpm = cpm
in stimulated cultures —cpm in unstimulated cultures.
Osteopetrotic
Minion
cells/well1
15,250
90
2
1.745
11
4
1Acpm0
0Sla7
8Acpm478
a SI, stimulation index (cpm of mitogen-stimulated
313
936
19SI1
cultures/cpm
6
16
2
of unstimu
'00
•
SPLEEN
•THIMI io
80|-
lated cultures).
Table 2
Binding of'2Bl-Con A to spleen cells of normal and osteopetrotic
chicks
Spleen cells were obtained from a pool of 5 normal 4-week-old chicks ami 7
osteopetrotic chicks of the same age. Numbers shown are the average of
triplicate determinations. The standard error was 10% or less.
A2
cellsNormal
Spleen
time(hr)1.5
40
125l-ConA bound following
addition of labeled Con
jig/ml8,011ng/ml10.656
/ig/ml21,752
fig/ml29,716
7
DAYS POST
OsteopetroticNormal1.54
14,6298.725
21,49510,909
31.00118,235
38,28021,650
51,802NDa
OsteopetroticBinding4cpm
a ND, not done.
15,7345
SEPTEMBER
100
10
SODIUM METAPERIODATE (mM)
1982
23.84110ng/ml12,707
44,42820 53.85450
ND
14
21
INFECTION
Chart 2. Onset of depressed blastogenic response to Con A by cells from
infected chicks. Eight-day-old chicks were infected with 2 x 104 plaque-forming
units of MAV-2(O) by the i.v. route. At the times indicated, spleens and thymuses
were removed from infected and age-matched controls, and the responses to
Con A were measured.
3619
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J. A. Price and R. E. Smith
Table 3
Pathology of MAV-2(O) infection of 8-day-old chicks
At indicated times after infection of 8-day-old chicks, animals were exsanguinated and weighed. Organ mass data were adjusted to mass of organ/body
before statistical analysis. None of the infected chickens was osteopetrotic at the time of sacrifice.
postin-fection
at6X3miri3-
massBody
mass
mass as % of body
vINO. i >
GX-amined554488Days
Experi
ment
Virus1
ControlMAV-2(O)2
cell vol
tion161617172828Packed
ume29
1a22±
±3p
0.05e32
<
ControlMAV-2(O)3
(g)143
mass
790±
8p ±
0.01182
<
0.0310.201
±
0.044p
±
0.050.145
<
131±
5123±
1NSNDNDOrgan
±
19p ±
0.01297
<
ControlMAV-2(O)MII
0.0520.495
±
0.061p
±
0.050.607
<
0.0180.267
±
0.0560.458
±
0.059p
±
0.084p<
±
<0.01NONDNDBursa0.651
0.050.573
361 ±
34p
34 ±
< 0.01Spleen0.150
0.0140.209
±
0.067p
±
0.050.497
<
0.0780.374
±
0.0770.224
±
0.096p
±
0.059p
±
< 0.01ThymusND6ND0.307
< 0.01
" Mean ±S.E.
'' ND, not done; NS, no significant difference.
c Level of significance as measured by Student's 2-tailed f test.
both spleen and thymus cells from infected chicks declined
rather quickly after infection (Chart 2). For example, 7 days
after infection, the blastogenic response of thymus cells from
infected chicks was 50% of uninfected cells, and spleen cells
showed a similar level of inhibition 14 days postinfection. The
response of both spleen and thymus cells from infected chicks
was reproducibly depressed to less than 10% of control values
by 16 to 18 days postinfection. The depression of blastogenic
responsiveness was transient, since chicks recovered the abil
ity to respond to Con A. Preliminary observations indicate that
individual chickens vary in the speed of recovery, an observa
tion consistent with a heterogeneity in recovery from anemia
(30). The response of PBL from osteopetrotic chicks to Con A
was not significantly depressed, similar to results obtained
earlier with PHA stimulations (48). These results indicate that
a depression of blastogenic response was reproducibly ob
served in thymus and spleen cells but not in circulating cells.
To further examine the interaction of MAV-2(O) with the
lymphoid cells of chicks infected after hatching, infected and
control chickens were sacrificed at 16, 17, and 28 days after
infection, and the packed cell volume, body mass, and spleen,
bursa, and thymus weights were measured. The results show
that MAV-2(O) induced a depression in body weight and a
hypoplasia of the bursa and thymus (Table 3). A hyperplasia of
the spleen was observed (Table 3), which has been found
previously in osteopetrotic chicks shortly after hatch (46).
Given the fact that chicks infected after hatch do not develop
osteopetrosis despite the development of a depressed blasto
genic response to Con A, the possibility that the substantial
bone hyperplasia present in osteopetrotic animals at the time
of sacrifice results in the observed immunosuppression seems
to be eliminated.
Absence of Suppressor Cells. Evidence for suppressor cells
was sought by mixing cells from normal and nonresponding
animals and observing for a depression of response in the
normal population. Osteopetrotic lymphocytes did not inhibit
the mitogenic response of normal lymphocytes in mixed cul
tures (Table 4), despite the inability of osteopetrotic cells to
respond to Con A. In contrast, addition of very few normal cells
to a majority of osteopetrotic spleen cells resulted in a normal
blastogenic response (Table 4, Experiment 2, Line 5). In a
3620
Table 4
Effect of mixing osteopetrotic and normal spleen cells on Con A mitogenicity
Spleen cells from normal and osteopetrotic chicks were prepared as described
in "Materials and Methods," using a low-speed centrifugation separation pro
cedure to remove RBC. Viability of each population was 70% by trypan blue
exclusion. Cells were mixed in varying proportions, stimulated with 10 fig Con A
per ml, and labeled between 24 and 40 hr.
cells/wellExperi
Million
AAbsent52840242837421418336710379264171225119Present9,7177
normal
ment12Osteo
petrotic00.20.51.01.52.04.001.51.872.252.623.0Normal4.03.83.53.02.52.003.01.51.120.750.
Acpm10082100981
SI. stimulation index (cpm of mitogen-stimulated
lated cultures).
b
cultures/cpm
¿cpmtest cell mixture
Acpm normal cells without osteopetrotic
cells
of unstimu-
x 100
second experiment, splenic lymphocytes were obtained from
chicks during an anemic phase which occurs 14 days after
infection of 8-day-old hatched chickens, from osteopetrotic
chicks 4 weeks of age which had been infected as 15-day-old
embryos, and from normal 4-week-old chicks. When these
lymphocytes were mixed, a normal response was obtained at
a normal celhinfected cell ratio of 1:9.
Irradiation of the normal PBL or spleen cells did not prevent
blastogenesis of the mixture (Table 5), indicating that normal
T-cells were not responsible for the mitogen reactivity observed
in the mixed-cell population. The doses of X-irradiation used
were sufficient to prevent division of T-cells but may have been
insufficient to prevent replication of the more radioresistantadherent macrophage-like cells. Division of these adherent
macrophage-like cells is unlikely to influence the incorporation
of label in mixed populations.
CANCER
RESEARCH
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VOL. 42
Immunosuppression
The above experiments using mixtures of normal and in
fected lymphocytes showed no evidence for suppressor cells.
Furthermore, it is clear that the T-cells from MAV-2(O)-infected
chicks were able to respond to mitogen when normal cells were
added to the infected population. We therefore attempted to
identify the cell present in normal populations which was re
sponsible for restoring blastogenic activity of infected lympho
cytes.
Adherent Cells from Normal Chickens That Restore Mito
gen Responsiveness to Infected Lymphocytes. The first ap
proach was to perform cell separations which selected adher
ent and nonadherent populations. In the first experiment, nor
mal lymphocyte populations were added to microtiter wells,
and nonadherent cells were carefully removed by washing.
Microscopic inspection of cells present on the surface of the
well after washing indicated that the only cells present were
macrophage-like cells. The adherent cells were counted and
nonresponding cells were added to the cultures. Both osteopetrotic and anemic chick spleen cell responses were aug
mented substantially by additional adherent cells from normal
spleen or blood (Table 6). Since chick spleen cells adhere
poorly peripheral blood cells were also used to provide more
homogeneous populations of adherent cells. Lymphoid cells
from both osteopetrotic and anemic chickens were stimulated
Induced by Avian Osteopetrosis
Virus
by the addition of normal adherent cells (Table 6). In the second
experiment, the number of unfractionated PBL which restored
response was roughly 6 times higher than an adherent popu
lation, while a nonadherent cell population was 10 times less
effective (Chart 3). The presumptive identification of the ad
herent cells which reconstitute mitogen responsiveness as
macrophage-like was strengthened by showing that these cells
possessed Fc receptors, were phagocytic, and had nonspecific
esterase activity.
The above results show clearly that normal adherent cells
restore blastogenic responsiveness to MAV-2(O)-infected lym
phocyte populations. The following experiment was performed
to determine whether the failure in blastogenic response was
due to the absence of adherent cells in the infected chicken.
Spleen cells from normal and osteopetrotic chickens were
observed for adherent cells, and no difference was observed
between the 2 populations. In addition, quantitation of the
peritoneal exúdate cells from both normal and osteopetrotic
chickens revealed no difference in the yield. These results
show clearly that MAV-2(OHnfected chickens possess normal
numbers of adherent cells which, while normal in several re
spects, fail to function as accessory cells for mitogen-induced
blastogenic responses.
Table 5
Addition of untreated or X-irradiated normal cells to infected spleen cells
io -
Irradiation of PEL was at 1600 rads and of spleen cells at 2000 rads; each
treatment produced at least 99% reduction in blastogenic response. Each cell
type was added as 10% of the infected cell population.
o
of
normalcellsadded(X
Expericellment Infected
source1
X
o.
cellsourceNonePBLIrradiatedPBLNoneSpleenIrradiatedspleenNo.
105>022033SI"2101362219Acpm852847170351019371497
Osteopetrotic2
O
AnemicNormal
LOG,,,
SI, stimulation index (cpm of mitogen-stimulated cultures/cpm of unstimulated cultures).
i
2345
NORMAL
CELLS
ADDED PER WELL
Chart 3. Enhanced Con A response in spleen cells from osteopetrotic chicks
following addition of normal PBL. Increasing numbers of unfractionated, adher
ent, or nonadherent normal PBL were added to cultures of 2 x 106 cells from
infected chicks, and responses were measured after culture. •,
adherent cells;
A. unfractionated cells; •nonadherent cells.
Table 6
Effect of normal adherent cells on the mitogen response of spleen cells from infected chicks
Adherent cells were obtained by plating normal PBL or spleen cells in microtiter wells in adherence medium (see "Materials and Methods")
overnight and then removing the nonadherent cells by gentle washing the next day. Lymphocytes from osteopetrotic or anemic chicks were
added at 2 x i o1 cells/well, cultured, labeled, and harvested as indicated in Table 1. The Acpm of infected cells in the absence of normal
adherent cells was 5% of age-matched uninfected controls.
ad-added/well010210310403X1013
of normal
of nor
adher-added/well0102103104010110210310"SI10604317412193627SpleenAcpm7
mal
ResponderOsteopetroticAnemicNo.
1023
X
IO33X10"SI"6.530.353.739.549162183PBLAcpm5103285500935163701312264945968195Rl1.06.49.86.91.03.57.212.422.0No.
X
a SI, stimulation index (cpm of mitogen-stimulated cultures/cpm of unstimulated cultures); Rl, relative increase in response (Acpm infected
with added normal adherent cells/Acpm infected without normal cells).
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3621
J. A. Price and R. E. Smith
DISCUSSION
Results with chickens infected with subgroup A avian leukosis virus indicated that a slight increase in the optimal dose
of PHA was required for maximal response when infected cells
were tested (26). However, no evidence was obtained that
mitogen responsiveness could be restored in lymphocyte pop
ulations from MAV-2(OHnfected chickens, despite varying the
Con A concentration over a wide dose range or manipulating
the cell numbers (Table 1). Furthermore, if anything, ¡odinated
Con A bound to infected cells better than to uninfected cells
(Table 2). Although the increasing binding of Con A to infected
cells was probably due to the presence of viral glycoprotein on
the surface of the cell (26, 50), use of a chemical stimulus,
sodium metaperiodate, did not result in blastogenesis of in
fected lymphocytes (Chart 1). These results may indicate that
avian leukosis viruses of subgroup B have a different effect on
lymphoid function than do viruses of subgroup A. Support for
this hypothesis has been obtained recently. It was found that
spleen cells from chickens infected with avian leukosis viruses
of subgroup A responded to Con A, PHA, and pokeweed
mitogen, while spleen cells from chickens infected with viruses
of subgroup B did not respond (38).
Several viruses induce suppressor cell activity in chick lym
phocytes. Marek's disease virus induces an adherent suppres
sor cell which reduces mitogen response (22), and reticuloendotheliosis virus induces a suppressor T-cell (7, 8). In the
present study, there was no suppressor cell activity detectable
in mixtures of normal and osteopetrotic chick cells (Table 4).
This result clearly indicates that MAV-2(O) induces immunosuppression by a mechanism that is different from that reported
previously for other avian tumor viruses. These cell-mixing
experiments also demonstrated that normal cells restored mi
togen responsiveness to infected populations (Table 4). Fractionation of the normal lymphocyte preparations showed that
adherent cells were the most active in restoring mitogenic
response (Table 6; Chart 3). Furthermore, the ability to restore
blastogenic function in infected lymphocytes was retained
when normal T-cells were prevented from dividing by irradiation
(Table 5). These results show clearly that the defect in the
infected cell population was present in the adherent cells.
However, the exact nature of the defect present in the adherent
cell from the MAV-2(O)-infected
animal is not established.
Infected chicks were found to possess adherent cells in both
spleen and PBL populations in numbers similar to those of
uninfected chickens, and these cells demonstrated Fc recep
tors, Fc-dependent phagocytosis, and nonspecific esterase. It
must therefore be concluded that adherent cells from infected
chicks must be relatively differentiated and that the effect of
MAV-2(O) is to inhibit only their accessory cell function for
blastogenesis.
There are several possible explanations for the apparent
selective inhibition of cell accessory function by MAV-2(O).
Evidence from the murine system indicates a functional heter
ogeneity for macrophages from different tissues, a result we
see reflected in an inhibition of Con A responses in spleen and
thymus lymphocytes but not in those from the blood. Cell
populations from these sources may be nonuniform with re
spect to the types of macrophages present, with those mac
rophages especially active in lectin responses being most
affected by MAV-2(O) infection. Since very few such cells are
3622
required to trigger a response (less than 1%), they might easily
be missed in enzyme or phagocytosis assays. A more likely
possibility is that viral infection of these cells causes a block in
the expression of certain differentiated functions which occur
in mature cells, such as presenting lectin, or elaborating a
factor needed for T-cell proliferation. It remains to be estab
lished whether adherent cells from the osteopetrotic chick are
capable of binding iodinated Con A to the same extent as do
adherent cells from uninfected animals. It is also not known
whether the infected adherent spleen population is able to
produce macrophage-specific
factors, such as interleukin I,
which participate in the mitogen response. In fact, little is
known about the functions of the normal chicken macrophage.
It is interesting to note that thymocytes from the osteopetrotic
mutant rat (op/op) do not respond to mitogens (25) but pro
duce a normal chemotactic lymphokine.5 In addition, peritoneal
macrophages from the op/op rat produce normal levels of
lymphocyte-activating factor but do not migrate in response to
a chemotactic lymphokine.
A variety of events may inhibit a lectin response in infected
cell populations. Loss of viability due to cytopathic infection of
the responding population is one possibility. In the MAV-2(O)
system, where the virus is cytopathic to chick embryo fibroblasts, a loss of viability in responder cells was ruled out by
direct examination by dye exclusion (Table 2). Adherent cells
were present at the same frequency in infected populations as
in normal ones. These cells therefore were present and adher
ent but not functional in the blastogenic assay. Furthermore,
examination of the macrophage-associated
properties of the
adherent cells present in infected populations showed that they
had normal levels of activity. The present results with adherent
macrophage-like cells from MAV-2(0)-infected
chickens may
be similar to a defect observed recently in mice infected with
Trypanosoma rhodesiense (1). la+ macrophages, which have
been shown to be essential for processing and presentation of
antigen for proliferation of primed T-cells (5, 14), were first
decreased in spleens of infected mice and later in lymph nodes
(1). The present results suggest that, in the chicken, which
lacks a lymph node system, la+-like macrophages possibly
responsible for lectin processing are decreased after MAV2(O) infection first in the spleen, and la+-like macrophages of
the PBL are either unaffected or altered at very late stages of
the disease. It is of interest to speculate whether addition of
adherent cells from the PBL of an osteopetrotic animal will
restore mitogen responsiveness to the spleen population of the
same animal. Although this experiment was not performed in
the present investigation, we predict that the outcome is likely
to be positive, that is, that the adherent osteopetrotic PBL will
restore mitogen responsiveness to the spleen population of the
same animal. If this result is obtained, it would offer further
proof for the compartmentalization of the 2 macrophage pop
ulations.
The immunosuppression which accompanies MAV-2(O) in
fection was shown to have an unusual manifestation in cultured
lymphocytes. In vitro, lymphocytes from MAV-2(O)-infected
chickens revealed that a profound suppression of mitogen
responsiveness was present. Mitogen-stimulated
lymphocyte
blastogenesis is a useful system for studying the physiological
state of certain cells of the immune system. In several species,
5 N. Hochman, L. M. Wahl, and A. L. Sandberg, personal communication.
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Immunosuppression
including chickens, both PHA and Con A are T-cell-dependent
stimuli (53), and it is generally accepted that T-cell lectin
responses are dependent upon macrophage-like accessory
cells (12, 37). The role of macrophages in the control of
hematopoietic cell differentiation and bone résorption is not
clear. The present work describing MAV-2(O) functional abro
gation of adherent macrophage-like cells is especially intrigu
ing since this virus produces anemia, lymphoid involution, and
bone hyperplasia. It is also of considerable interest that a
macrophage-like cell has been identified as the precursor for
the osteoclast (23). It is of interest to point out that osteopetrosis in the rat and mouse is accompanied by a failure of
mitogen responsiveness (25, 29), and natural killing by mouse
lymphocytes is decreased in osteopetrotic mice (43). Natural
killing assays, developed recently for avian cells (45, 55), have
not been applied to avian osteopetrosis. However, it is likely
that natural killing will be depressed in MAV-2(O)-infected
chicks, consistent with the generalized immune paralysis that
has been reported (Refs. 17 and 48; this paper). In any event,
the infection of chicks by MAV-2(O) offers a useful probe in
differentiation in the lymphoid series and of classes of functions
expressed by macrophages. It remains for further studies to
elucidate the full spectrum by alterations in macrophage func
tions which are induced by MAV-2(O) infection.
ACKNOWLEDGMENTS
We thank Jeffrey Collins and Brice Weinberg for critically reviewing the
manuscript, Ted Wheeler for diligent technical support, and Ann Sandberg for
communicating results prior to publication.
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Inhibition of Concanavalin A Response during Osteopetrosis
Virus Infection 1
Joseph A. Price and Ralph E. Smith
Cancer Res 1982;42:3617-3624.
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