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Characterization of the bovine secondary in vitro antibody response
Veterinary Immunology and lmmunopathology, 7 (1984) 19--32 Elsevier Science Publishers B.V., Amsterdam -- Printed in The Netherlands
CHARACTERIZATION
L.G. Filion 1'2
OF THE BOVINE SECONDARY
H. Bielefeldt
19
IN VITRO ANTIBODY RESPONSE
Ohmann 1'3, P.W. Owen I and L.A. Babiuk 1'3
Iveterinary Infectious Disease Organization, 124 Veterinary Road, Saskatoon, ~askatchewan, S7N OWO, Department of Medical Microbiology, Medical School and 3Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N OWO. Published with the permission (Accepted 6 December
of the Director of VIDO as Journal Series #27.
1983)
ABSTRACT Filion, L.G., Bielefeldt 0hmann, H., Owen, P.W., Babiuk, L.A., Characterization of the bovine secondary in vitro antibody response. Immunol. Immunopathol.,7: 19-32.
1984. Vet.
In the present report, the bovine secondary in vitro antibody response to keyhole limpet hemocyanin is described. The induction of anti-KLH antibody was not dependent upon the presence of mitogen but was antigen specific (KLH vs ~valubumin). Furthermore, this response was dependent upon cell density (i0 v per well), antigen dose (I to i0- ug/culture) and time in culture (5 days). The antibody produced was specific for KLH as measured in several binding assays. An unresponsive state was detected with high concentrations of KLH (more than I0 ug per culture) which was not due to the formation of immune complexes but to the inactivation of B and/or T cells. The induction of the antibody response was dependent on the presence of macrophages (syn~eneic or allogeneic) and their presence could not be replaced with 2-mercaptoethanol.
INTRODUCTION
The
activation
responses
immunobiology. or
T
cell
functions Gershon 1981,
immunoregulation
has
of critical 1979,
Tada
Morimoto
et
indirectly
with
enhanced mitogen cytotoxic
suppression
stimulus
of
a
led
& Okumura
(M~) wide
1981b,
the
1979),
variety
or antigen
of
involved
et been
al, shown
lymphocyte proliferation
induction
elucidations in mice
also in man 1983). to
the
immune
to the study of
in the modulation
and
populations
and recently
has
induced
lymphocyte
cell
in
fundamental
discovery
Morimoto
lineage
of immune responses
0165-2427/84/$03.00
to
immunoregulatory
al,
lymphocytes
is a subject
The study of the mechanisms
reactivity
macrophage/monocyte
vitro
and
to an antigenic
In
of B
of
the
(Cantor
&
(Geha et al, addition,
the
interact
directly
or
subpopulations
leading
to
(Sjoberg
(Yamashita
(Metzgar et al, 1980).
© 1984 Elsevier Science Publishers B.V.
et al,
& Hamaoka
1972), 1979),
in or
20
These interactions which have been identified occur
in
large
conducted domestic
domestic
animals.
to determine whether animals
tory animals.
offers
some unique
few
studies
advantages
have
been
The study of larFe
over studyin F small
the study of the cellular interactions
lymphoid
compartments
performed
(McDermott et al,
or
Recently
the
analysis
populations
has
begun
modulate
organs
labora-
such
as
of
the
interactions Obmann
to understand
man
their
response
where,
respiratory
because
et
between al,
the immune to
tract
of
various
1983a,
may
bovine
b).
response
infectious
studies may have also direct application in
the
in specialized be
1982),
(Bielefeldt
were done in an attempt
infections
very
the case.
The kinetics of the immune response in the same animal may be
followed. Furthermore,
eventually
However
this is indeed
in mice and humans may also
These
of cattle
agents.
cell trials
and
However,
to
such
for controllin~ viral and bacterial
ethics
or
risk,
similar
experiments
could not be performed. In
the
present
peripheral indicate
study,
blood that
lymphocytes.
the
induction
lymphocytes
M~'s This
are
was
levels of soluble
prove
for
useful
studying
for
in
is antigen
by excessive
in
performed.
crucial
response
of
The
vitro
suggestin~
conditions
antibody
results
induction
dose dependent
antigen,
various
vitro
bovine
described of
below
antibody
by
and can be abrogated
that this
required
from
system
should
for immunore~ulation
ranging from induction of a reactive to and/or nonreactive state.
MATERIALS AND METHODS
Lymphocyte Cultures Lymphocytes Hereford
or
were
McGulre & Babiuk 7 days
obtained
from
Holstein-Friesian
after
1983).
peripheral
blood
as d e s c r i b e d
Calves were immunized with
immunization
could not be produced
the
calves
adn
by PBL.
weekly
thereafter
Animals
were
of
4-12
month
previously
old
(Filion,
I mg. of KLH, bled 5 to until
in
reimmunlzed
vitro
at this
antibody time.
No
substantial difference existed between animals in the kinetics of appearance and
disappearance
of
reactive
PBL cells
for
anti-KLH
antibody
production.
Briefly, blood was collected into syringes containing citrated dextrose; huffy coat was collected temperature,
and was
after centrifugation
layered
on Ficoll-Paque
at
1600 g for
(Pharmacia,
the
15 min at room
Uppsala,
Sweden).
The peripheral blood leukocytes
(PBL) present at the interface were washed 3
times
medium
with
containing procedure
minimal
essential
antibiotics yielded
(MEM)
(5 mg amphotericin
greater
than
99%
(GIBCO,
Burlington,
Ontario)
B, 5 mg gentamycin/liter).
mononuclear
cells
with
more
than
This 98%
21
viability.
The
essential volumes
medium into
Fisher
PBL,
resuspended
(FBS-MEM),
96
well
Scientific,
were
(flat
in
10%
aliquoted
bottom)
Edmonton,
bovine
(triplicate
microtltre
Alberta)
Cells and antigen concentration
fetal
and
serum
cultures)
dishes
incubated
-
in 200 ul
(Falcon
at
37°C
varied with the experiment
minimal
test in
IIl,
5%
CO 2.
and are reported
in the appropriate figure or table. Monocyte-Depleted
Cells
To remove monocytes, ed in 10 ml volumes
purified PBL, resuspended in 10% FBS-MEM, were plat-
(1 x 107 cells/ml) onto
ture petri dishes (Corning Glass, C o m i n g , hrs
in
a humidified
5% CO 2 incubator.
100 x 15 mm plastic tissue cul-
NY) and incubated at 37°C for i-3 The nonadherent
removed by washing once with calcium and magnesium-free, saline of
(PBS), centrifuged
and resuspended
I-2.5 x 108 cells/ml.
These
column (Pharmacia, Uppsala,
(Mishell et al,
Sephadex
30 min
for
at
from
50
to
contained
78%
and
less than
gently
buffered
in 10% FBS-MEM at a concentration
cells were passed
through
a Sephadex
G-10
Sweden) containing a nylon wool plug as describ-
ed previously
column with 20-25 mls of
cells were phosphate
1980).
The cells were allowed
37°C before
elution
of nonadherent
10% FBS-MEM at 37°C.
viability
was
>98%.
2 + 1.8% nonspecific
to bind to the cells
from
the
The recovery of cells ranged
The
recovered
esterase
nonadherent
positive
cells
cells
(Koshi et
al, 1976). Monocyte Enrichment The cells adhering to the petri dishes were washed thoroughly with PBS to remove weakly adherent incubating
with
cells.
The cells remaining attached were removed by
3 mM EDTA in PBS were
washed
for
Recovered
cells
three
FBS-MEM.
Analysis of the adherent
15 mins
times
at
with
population
37°C
MEM
and
in a CO 2 incubator. resuspended
in
(designated M~) revealed
10% >90%
nonspecific esterase positive cells with 95% viability. Pulsing of cells with Antigen Various cell populations time as indicated
were pulsed with antigen for various periods of
in the results.
Removal of unbound antigen was performed
by washing the culture three times with 10% FBS-MEM. Antibody Detection The level of anti-KLH antibodies from culture supernatants was determined by an enzyme link immunoassay 1983).
Briefly,
(OA) (Sigma, pH 9.6.
the antigens, KLH (Calbiochem.,
St. Louis, Mo.) were diluted
Aliquots of i00 ul were added
(Dynatek Laboratories, wells
(ELISA) as described previously
were
washed
Alexandria,
3 times
with
(Filion et al
La Jolla, Ca.) or ovalbumin
to 20 ug/ml with carbonate buffer
to the wells of Immunolon
II plates
VI) and incubated at 4°C overnight.
distilled
water
to
remove
antigen before the addition of i00 ul of culture supernantants
the
The
unabsorbed
to each well.
22
After a ! hr incubation at room
temperature,
the wells were washed
3 times
with distilled water to remove the unbound antibody. Horseradish peroxidaseconjugated
rabbit
anti-bovine
in i00 ul volumes
was
added
IgG
(Cappell
to each well
Laboratories, and
the plates
Cochranville, were
PA)
incubated
a
further hour at room temperature. The ELISA plates were once again washed as described solution allowed
above, before
the addition of
(6.5 mM 5-aminosalicylic
100 ul/well of the enzyme substrate
acid with
0.02% H202).
to proceed at rt for 30 min before reading
The reaction
the optical densities at
490/630 nm in an MR 580 microelisa autoreader
(Dynatek Lab, Alexandra,
Each
controls:
ELISA
plate
contained
two
negative
was
i)
antigen
VI.). plus
substrate, and ii) antigen, horseradish peroxidase conjugate and substrate.
RESULTS The in vitro secondary response of primed PBL to KLH:
Definition of optimal
culture conditions In an attempt of antibody,
to optimize
variations
in cell density,
tion times were investigated.
A
the culture
conditions
for in vitro
antigen concentration,
induction
and incuba-
Cells from 2 different animals were incubated
.2J
o
o~
z a .J
KLH
P n 0
x! i
OA I
no Ag 1
J
I
I
i
I
2.5
5
7.5
10
20
cells x l 0 !
5
culture
Fig. 1 Optimal cell density for in vitro antibody productions. PBL from KLH primed animals were cultured with KLH. Cells ~ere plated at various concentrations and stimulated for 5 days with 10 - ug/culture of KLH ~ - - ~ , 0--0), OA (I--e,A---A) or 10% FBS-MEM (no Ag) before assaying the level of antibody (OD~SD).
23
at
a density
produced
of
106 cells/200
ul with KLH.
after 5 days in culture, whereas
Peak
antibody
cultures containing
ger numbers of cells produced lower antibody levels (Fig.
I).
responses
were
fewer or larThe induction
of the anti-KLH antibody response was found to be antigen specific since in the presence of an unrelated antigen, ie. OA, or in the presence of 10% FBS, anti-KLH antibody was not from animals primed tion with
the maximum
ture (Fig. 2).
formed
to KLH,
(Figure
1,2,3).
In addition,
lymphocytes
responded best at a specific antigen concentra-
response
occurring
between
10 -2 and
10 -5 ug per cul-
Anti-OA antibody was not induced in these cultures
(results
not shown).
Fig. 2 Antigen dose response curve for in vitro antibody induction. PBL's from KLH primed animals were pl~ted at I0 ~ cells/ culture and stimulated for 5 days with various concentrations of KLH
.4_
.3-
A
0 0 0) .2-
>. Iu~ z Lu 0 .J 0
.I
(o--o,e--o),
I
OA ( ~ - - ~ A--&) or 10% FBS-MEM (C). The antibody levels (OD~SD) to KLH were measured employing an ELSIA assay.
,
a. 0
I
c
I
I
10-s
I
I
10
I
-6
I
10
-4
ANTIGEN
I
I
10
-2
DOSE
I
I
I
1
10 2
24
The final parameter
to be tested for optimizing
in vitro antibody synthesis
was the day of the peak response.
As illustrated in Fig. 3, no antibody was
detected
of
2 days
after
initiation
the
culture.
However,
antibody
was
detected 3 days after initiation which peaked by day 5.
.3 A
o
¢0
o
0~ LI
.2 >.
z
LU
a.
0 _x
Z
I
1
I
I
I
2
3
4
5
6
DAYS Fig. 3 Kinetics of induction for in vitro antibody productionm2 PBL (106 cells/culture) were incubated for various time periods with i0 ug/culture of KLH ( O - - O ~ - - ~ ) or OA ( e - - O , A - - A ) . The levels of anti-KLH and anti-OA antibodies were measured by an ELISA assay (OD+SD). Given
the
incubation
above
findings,
a cell
density
of
106
cells/culture
and
an
time of 5 days in the presence of 10 -2 or 10 -3 ug of antigen was
used in subsequent experiments. Specificity of anti-KLH antibody Although
the
above
results
suggest
that
KLH
initiated
a
secondary
in
vitro antibody response,
the specificity of the anti-KLH antibody production
remained
Therefore,
ascertain OA prior
to be
shown.
whether
incubation
to testing
experiments
of the culture
in the ELISA assay could
were
performed
supernatant inhibit
in
order
to
fluids with KLH or
binding
to the ELISA
25
plates
coated
with
antibody
was
fashion,
whereas
results
suggested
KLH.
inhibited the
As by
illustrated
the addition
addition
of
in Fig.
4,
of soluble
soluble
OA
or
the
putative
anti-KLH
KLH in a dose dependent diluent
did
not.
These
that the antibody produced was KLH specific.
o o
.2
z w a
.1 0 0
KLH
l I
I
1
10
I
100
ANTIGEN ADDITION (lxg)
Fig. 4 Inhibition of anti-KLH antibody binding by specific antigen: Supernatant of culture stimulated with KLH, were added to equal volumes of various concentrations of either KLH (O--6), 0A or diluent ( O ~ O ) prior to assaying anti-KLH antibodies by ELISA on KLH coated plates. Antibody levels of culture stimulated with 10% FBS-MEM ( ~ ) or KLH ( A ) without the addition of soluble KLH or 0A are reported also. Values are expressed as the mean 0D+SD/culture of triplicate samples. m
To
further
cultures,
the
confirm
the
supernatants
specificity of KLH,
of
0A or
added to either KLH or 0A coated wells. cultured bound
supernatants
strongly
stimulated
to
culture
bound only minimally KLH
coated
supernatants
wells.
the
antibody
10% FBS
produced
triggered
As shown in Table
in
cultures
OA
were
i, KLH triggered
to 0A coated wells, whereas, Furthermore,
these
or
10%
they
FBS-MEM
bound neither OA nor KLH coated wells.
26
TABLE
1
Lack of binding of culture supernatants
to wells coated with irrelevant
antigen
Stimulus for Ab Production
Exper. No.
KLH
OA
KLH Coated Wells
OA Coated Wells
1 2 3 4 5 6
.270+.010 .1887.015 .432~.032 .246"$.017 .157~.009 .291~.005
.011+.004 .013~.002 .0187.005 .006"$.002 .008~.001 .019~.004
7
.061+.007 .081~.006 .051~.009
.008+.001 .007~.001 .006~.001
.084+.013
.017+.002
8 9 10% FBS
Culture supernatants from KLH, OA or MEM-FBS stimulated cultures were added to either the KLH or OA coated wells and the ELISA was performed. Experiments i-9 were performed at various times. 1F~O KLH or OA stimulated cultures, multiple doses were used, however only ug/well are reported. Pulsing lymphocyte cultures with antigen Previously it was demonstrated ug)
or
low
anti-KLH
(10 -6 , 10 -7
antibody
detectable
ug)
response
response
that cultures with either high
doses (Fig.
to high doses
of
KLH
2).
did
The
not
produce
reason(s)
of antigen
for
a the
(i0, or 100 significant lack
could be attributed
of
either
a to
(i) the formation of the immune complexes during the culture incubation time which
could
suppressor
not
be
detected
pathway mediated
reduced Ab synthesis order were After
or
to differentiate first
pulsed
pulsing
the
with cell
FBS-MEM and the cultures
in
our
assay
system,
(2)
the
induction
of
a
either by M6 or T cells, which would result in
(3) the induction
of B cell
between
these
various
concentrations
free were
antigen
three
was
incubated
unresponsiveness.
possibilities, of KLH for
removed
by
in the absence
cultures i, 4 or
three
washes
of soluble
In
of PBL 24 hrs. in
10%
antigen.
Control cultures were not washed in order to assure that the induction of in vitro antibody was successfully accomplished. 5 suggest
that exposure
production of Ab.
The results outlined
in Fig.
to antigen for 1 hr was sufficient to stimulate the
27
~ r .
~ r .
24 hr.
c~ 0 m
OA
.5 zm
.4-
.3 "2 /
0
.1o ~o2,o
~
1G'~-2
0
KLH
10210
0
10~10
0 102 10
1 1()1 1t~2
DOSEOFANTIGEN Fig. 5 Pulsin~ of cultures with various doses of KLH for 1,4 and 24^hours: Various concentrations of antigen, KLH (bottom panel) or OA (0 ~o 102 ug) (~]) (top panel) or diluent ( ~ ) were added to PBL cultures (i0 v cells/culture). After 1,4 or 24 hrs these cultures were washed 3 times and incubated in fresh MEM +10% FBS for a total of 5 days. Antigen was not readded to these cultures. Control included cultures where the KLH or OA was not removed are presented in the left most panels. Furthermore, antibody
removal
synthesis
of
when
excess high
Ag
by
antigen
washing,
prevented
concentrations
were
suppression used.
of
Cultures
left in contact with the antigen for 4 hrs produced lower levels of antibody with the higher concentrations longer that
incubation the
antigen
lack was
periods
of
not
before
detectable
due
to
of antigen.
the
antigen
This was even more evident with removal.
These with
results
antibody
in
cultures
10
formation
of
izamune complexes
or but
suggest
100 due
ug to
of the
inactivation of B cells by antigen or by the induction of a suppresssor cell pathway. The role of macrophages
in the induction of a s e c o n d a r y
in v i t r o
antibody
response The
M4
plays
interactions
a
crucial
(reviewed
been shown either
central
in Unanue
role
& Rosenthal
to enhance or suppress
1980, Morimoto et al, 1980).
in
the
regulation
1980).
of
Furthermore,
the immune response
cell-cell M6 have
(Metzgar et al
In an attempt to determine the role of M6's in
the in vitro induction of antibody and in the suppression of these responses with
high
doses
of
antigen,
depletion
and
reconstitution
experiments
were
employed. Lymphocyte populations with few M6 (nonadherent cells (NA)) were obtained by sequential
plastic
adherence
and passage
through a Sephadex G-10 column
28 of PBL.
This procedure
routinely
produced
relatively
lations with very few M6 (2 - .5% nonspecific the M~ population ed in vitro with
to a change
of KLH
experiments
employing
Furthermore,
contaminating
of
from PBL completely abrogated the antibody response induc-2 ug of KLH (Fig. 6 and 7). The effect of M6-depletion
revealed shown).
popu-
The elimination
10
was not due by
pure lymphocyte
esterase).
concentration
enriched
lymphocytes
induce an antibody response
a wide M~
since
optimum
antigen
population
5 x
was
105/culture
for
dose
the NA cells
range
(results
sufficiently
of
these
free
cells
could
as not of not
to KLH (Fig. 6).
z
0
1l J PBL NA+2ME N A ÷ M(~
Fig. 6 Reconstitut~on of the in ~ antibodY5response with syngeneic M6: Cultures of PBL ~i0 ), M~ (5 ~-'~0 ), NA (5 x 10 ) cells/culture, with 2-ME (range: 2 x IO-JM to 2 x i0- J"~)" or without 2-ME and mixtures of M6 + NA were incubated with either 10- ug of KLH (Fl) or 10% FBS-MEM ( ~ ) . Supernantants of the culture were harvested 5 days after culture initiation and the antibody levels were measured by ELISA. Values (OD) expressed are maans of triplicate determinations. However, various
repopulating numbers
cells/culture) (Fig. 6). Furthermore,
of
syngeneic
resulted
of M6
NA
cells
(ranging
in a significant
(5 x from
the reducing
1978) was unable
agent
105 5
production
No effect was seen with M6 numbers
has M~ substituting
populations
cultures
x
cells/culture) 105
to
x
105
antibody
below 1.25xi05 cells/culture.
2 mercaptoethanol
(2-ME),
which
activity in some in vitro in~nune responses
to reconstitute
1.25
of KLH specific
with
the antibody
response
reportedly
(Opitm et al,
of nonadherent
cell
(Fig. 6).
Pulsing of macrophages Macrophages
play
(Fi~. 6), however
with different
a crucial
role
concentrations in
the
they may also be involved
in vitro
of KLH induction
in suppression
of antibody
of the response
29
under conditions ty,
lymphocytes
such as high antigen dose. were
added
with
various
concentrations
were
pulsed
with
antigen,
high
or
to M6 cultures of KLH. low
To investigate
which had been pulsed
Regardless
doses
of
this possibili-
KLH,
of whether
following
for 24 hr
the macrophages
removal
of
soluble
an antibody response was produced in cultures of M6 and lymphocytes
(Fig. 7).
o ¢ej 0 .15
T
l
I-
Z w
0 Io. 0
+ +
.05
PBL
NA
NA + MO 0
NA + M ~
NA+M~ .01
NA + M ~ 1
10
Fig. 7 Induction of in vitro antibody with pulsed M~: M~ (105 cells/culture) were pulsed for 24 hours with 0, .01, i or 10 ug of KLH prior to_removing the excess unbound antigen by washing 3 times._2NA cells (5 x 10 D cells/culture) were added to the pulsed M6 and KLH (10 ug/culture) ( ~ ) or 10% FBS-MEM ( ~ ) was added to the wells. NA and PBL cultures were included to ascertain the levels of antibody produced by these cells. After • o 5 days in a 5% COp incubator at 37 C, the supernatants were harvested and the antibody levels were determined by ELISA. Thus high concentrations turn could reduce pulsed
cells
immunogenic KLH
being
of KLH did
activate
suppressor
the level of in vitro antibody synthesis.
efficiently
form, the
not
with
most
presented
those M~'s efficient
antigen
pulsed
to
the
cells.
which
in
In fact, these
lymphocytes
with the highest
presenting
M6's
dose
in
an
(I0 ug) of
Therefore,
under
the
experimental conditions employed, M6 did not act as suppressor cells.
DISCUSSION The antibody of
KLH
present response only
if
study
demonstrates
could be induced these
cells
came
that
a
specific
secondary
in
vitro
by culturing bovine PBL in the presence from
animals
primed
to
this
antigen.
30
Additional was
evidence
for
from experiments
the
which
specificity demonstrated
antibody to KLH coated ELISA plates. was dependent
on repeated
of
the
in vitro
that KLH
antibody
inbibited
response
bindin~
of
the
The induction of an in vitro response
immunization
of animals with KLH.
Regardless
of
the conditions for antibody induction, maximum antibody levels were produced 5 days
post-stimulation.
body induction. vitro
Finally,
M~ were
found
to be necessary
for anti-
These results are similar to those recently reported for in
antibody
induction
in man
(Morimoto
et
al,
]981b,
Gerrard
& Fsuci,
1982 and Volkman et al, ]982). The
cost
performing
of
large
detailed
in the development animals
offer
numbers
of
domestic
analysis
animals
of the immune
considerable
specific
cell
has
generally
of the interactions response
advantages populations
cannulation of various lymphocytic
of these
since
been
3 deterrant
between various species.
it is possible
by alveolar
However, to obtain
or mammary
gl~nd
tissue or by repeated bleeding.
more, experiments with infectious agents can be performed wherein fic cellular
mechanism
involved
in protection
can be
for
cell types such large
lava~e, Further-
the speci-
determined.
Thus by
employing in vitro models to discern the cellular interactions durin~ development
and
determine but
expression
of
better methods
also
in man.
the
immune
response,
of infectious
In fact,
it has
disease
been
it
should
be
possible
to
control not only in animals
suggested
that
the delineation
of
immune mechanisms which regulate lung immunity may be best analyzed in large domestic animals because
many
(McDermott et al 1982).
important
diseases
occur
initiated a series of comparative
It is for these reasons as well as in
humans
and
bovines
that
studies employing a bovine model
we
have
to study
cellular immune mechanisms. The cellular mechanisms for the bovine valin-A
(Con A) suppressor
et
1981,
al,
Bielefeldt
generated
in
adherence
and passage
dependant
upon
Ohmann results
et
al
is
or cell mediated
immune
the
Ohmann
of
These
demonstrate
similar
et al fraction
1983a).
These
(M~ depleted
to
M~
previous
that
those
(Tadakuma
findings
the immune
of
and
humans
(Smith
cells were
sequential
plastic
but the ~eneration was
Pierce coupled
response and
suppressor by
through Sephadex G-10 column),
presence
1983b).
response Concona-
cells have been induced and characterized
the non-adherent
clearly
animals
of the humoral
system has only begun to emerge in the literature.
of
small
1976, with large
animal
Bielefeldt the
present
domesticated species
thus
providing a possible model for analyzing immune responses of other species. The antibody adherence addition
response
and passage of
2-ME
after
through
suggesting
the elimination Sephadex that
M~
G-10, are
of M~ by sequential
could
required
not as
be replaced antigen
plastic by
the
presenting
81
cells.
Further
evidence
cell was obtained induced (Fig.
7),
or M~
the
role
of
from the experiments
to produce
addition,
for
antibody
not
wherein unprimed
if they were
recombining did
the M~ as an antigen
nonadherent
seem
incubated cells
to mediate
the
immunogenic
rather
reports
(1981),
but
can
attributed
be
through
to
G-10
from PBL.
yields
a considerable
cells,
even
ing
of
results).
cell
necessary
a
These (1972),
efficacy
of
only was
not
Replating
third
These results in
order
Further
experiments.
characterization of
specific After
of
involved
with
Geha et al
The
discrepancy
procedures.
adherence
In
or passage
accessory
blood monocytes
esterase
attempted
assure
cell
positive
the
to plastic
monocyte-like
(Bielefeldt
Ohmann,
un-
that strict monitor-
purity
of
these
cells
is
for in vitro induction of antibody. bovine
These
this accomplishment,
the cellular mechanisms
M~
antigen in an
in agreement
to eliminate
clearly demonstrate
the
antiserum.
In
since
is by no means a definite marker for
is
to
M~
antigen.
(1981),
(1978).
plastic
of peripheral
and that the M~ is essential
production
are
could be pulsed
activity
deprivation
sufficient
replating
and
et al
et al
number of nonspecific
population
results
the M6
adherence
M6
in presenting
Inaba
from this laboratory,
Moreover,
bovine blood monocytes.
after
et al
to those of Opltz
the
experiments
Sephadex
published
tolerogenic.
Sjoberg
are contrary
preliminary
function
than
of
antigen
suppressive
pulsed with all doses of antigen were effective
previous
lymphocytes
with
with
presenting
immune
will
response
facilitate
trials may be devised
in the pathogenesis
awaits
cell
the
separation
to determine
of bovine diseases.
Acknowledgements The authors would like to acknowledge the expert technical assistance of Ms. Jeanette Heise, Mr. Barry Carroll and Richard Monseler and also Mrs. Bonnie Neufeld for typing the manuscript. Dr. Bielefeldt Ohmann is a recipient of post doctoral fellowship grants from the NATO Science Fellowship program (No. 23-03 51/81 and No 23-03 38/82). Funding for the project was provided through a research contract with Agriculture Canada and the Department of Supply and Services, and by a grant from the Max Bell Foundation of Toronto. REFERENCES Bielefeldt Ohmann, H., Filion, L.G. & Babluk, L.A., 1983a. Bovine monocytes and macrophages: An accessory role in suppressor cell generation by Con A and in lectin induced proliferations. (Immunology in press) Bielefeldt Ohmann, H., Filion, L.G. & Babiuk, L.A., 1983b. Cellular interactions in the generation of bovine Con A-suppressor cells and in mitogenlc proliferative response. (Veterinary Immunology and Immunopathology in press) Cantor, H. & Gershon, R.K., 1979. Immunological circuits: cellular composition. Fed. Proc. 38:2058. Filion, L.G., M c G u i r e , R.L. & Babiuk, L.A., 1983. The n o n s p e c l f l c suppressive effect of BHV-I on bovine leukocyte functions (Infection and Immunity in press).
32 Geha, R.S., Jansen, M.E., Ault, B.H., Yunis, E. & Braff, M.O., 1981. Macrophage T cell interaction in man: Binding of antigen specific human proliferating and helper T cells to antigen pulsed macrophages. J. Immunol 126:781. Gerrard, T.L., & Fauci, A.S., 1982. Activation and immunoregulation of antigen-specific human B lymphocyte response: multlfacet role of the monocyte. J. Immunol. 128:2367. Inaba, K., Nakano, K. & Muramatsu, S., 1981. Cellular synergy in the manifestation of accessory cell activity for in vitro antibody response. J. Immunol. 127:452. Koshi, I.R., Poplack, D.G. & Blaise, R.M., 1976. A nonspecific esterase stain for the identification of monocytes and macrophages: In vitro methods of cell mediated and tumor immunology, p. 399. Bloom, B.R. & David, S.R. (eds.) Academic Press, N.Y. McDermott, M.R., Befus, A.D. & Bienenstock, J., 1982. The structural basis for immunity in the respiratory tract. Int. Rev. of Exp. Pathol. 23:47. Mishell, B.B., Mishell, R.I. & Sbigi, J.M., 1980. In selected methods in cellular immunology. Mishell, B.B. and Shigi, S.M. eds. N.H. Frieman and Company, San Francisco. Metzgar, Z., Hoffeld, J.T. & Oppenheim, J.J., 1980. Macrophage mediated suppression: I Evidence for participation of both hydrogen peroxide and prostaglandins in supression of murine lymphocyte proliferation. J. Immunol. 124:982. Morimoto, C., Todd III, R.F., Distaso, J.A. & Schlossman, S.F., 1981. The role of macrophage in in vitro primary anti-DNP antibody production in man. J. Immunol. 127:1139. Morimoto, C., Reinherz, E.L. & Schlossman, S.F., 1981b. Regulation of in vitro anti-DNP antibody production by functional subsets of T lymphocytes in man. J. Innnunol. 127:69. Morimoto, C., Reinherz, E.L., Borel,Y. & Schlossman, S.F., 1983. Direct demonstration of the human suppressor inducer subset by anti-T cell antibodies. J. Immunol. 130:157. Opitz, H.G., Lemke, H. & Hewlett, G., 1978. Activation of T cells by a macrophage or 2-mercaptoethanol activated serum factors is essential for induction of a primary immune response to heterologous red cells in vitro. Immunol. Rev. 40:53. Sjoberg, 0., Anderson, J. & M o l l e r , G., 1972. Requirements for adherent cells in the primary and secondary immune response in vitro. Eur. J. Immunol. 2:123. Smith, W.G.,~singer, W.R. & Splitter, G.A., 1981. Bovine Con-A induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action. Immunol. 43:91. Tada, T. & Okumura, K., 1979. The role of antigen specific T cell factors in the immune response. In "Advances in Immunology". Ed. F.J. Dixon and H.G. Kunkel. Academic Press, N.Y. p.1. Tadakuma, T. & Pierce, C.W., 1976. Site of action of a soluble immune response suppressor (SIRS) produced by Con-A activated spleen cells. J. Immunol. 117:967. Unanue, E.R.---~ Rosenthal, A.S. (eds), 1980. Macrophage regulation of immunity. Acad. Press. N.Y. Volkman, D.J., Allyn, S.P. & Fauei, A.S., 1982. Antigen induced in vitro antibody production in humans: Tetanus toxoid-specific antibody synthesis. J. Immunol. 129:107. Yamashita, U. & Hamaoka, T., 1979. The requirement of Ia-positive accessory cells for the induction of hapten-reaetive cytotoxic T lymphocytes in vitro. J. Immunol. 123:2367.
CHARACTERIZATION
L.G. Filion 1'2
OF THE BOVINE SECONDARY
H. Bielefeldt
19
IN VITRO ANTIBODY RESPONSE
Ohmann 1'3, P.W. Owen I and L.A. Babiuk 1'3
Iveterinary Infectious Disease Organization, 124 Veterinary Road, Saskatoon, ~askatchewan, S7N OWO, Department of Medical Microbiology, Medical School and 3Department of Veterinary Microbiology, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada, S7N OWO. Published with the permission (Accepted 6 December
of the Director of VIDO as Journal Series #27.
1983)
ABSTRACT Filion, L.G., Bielefeldt 0hmann, H., Owen, P.W., Babiuk, L.A., Characterization of the bovine secondary in vitro antibody response. Immunol. Immunopathol.,7: 19-32.
1984. Vet.
In the present report, the bovine secondary in vitro antibody response to keyhole limpet hemocyanin is described. The induction of anti-KLH antibody was not dependent upon the presence of mitogen but was antigen specific (KLH vs ~valubumin). Furthermore, this response was dependent upon cell density (i0 v per well), antigen dose (I to i0- ug/culture) and time in culture (5 days). The antibody produced was specific for KLH as measured in several binding assays. An unresponsive state was detected with high concentrations of KLH (more than I0 ug per culture) which was not due to the formation of immune complexes but to the inactivation of B and/or T cells. The induction of the antibody response was dependent on the presence of macrophages (syn~eneic or allogeneic) and their presence could not be replaced with 2-mercaptoethanol.
INTRODUCTION
The
activation
responses
immunobiology. or
T
cell
functions Gershon 1981,
immunoregulation
has
of critical 1979,
Tada
Morimoto
et
indirectly
with
enhanced mitogen cytotoxic
suppression
stimulus
of
a
led
& Okumura
(M~) wide
1981b,
the
1979),
variety
or antigen
of
involved
et been
al, shown
lymphocyte proliferation
induction
elucidations in mice
also in man 1983). to
the
immune
to the study of
in the modulation
and
populations
and recently
has
induced
lymphocyte
cell
in
fundamental
discovery
Morimoto
lineage
of immune responses
0165-2427/84/$03.00
to
immunoregulatory
al,
lymphocytes
is a subject
The study of the mechanisms
reactivity
macrophage/monocyte
vitro
and
to an antigenic
In
of B
of
the
(Cantor
&
(Geha et al, addition,
the
interact
directly
or
subpopulations
leading
to
(Sjoberg
(Yamashita
(Metzgar et al, 1980).
© 1984 Elsevier Science Publishers B.V.
et al,
& Hamaoka
1972), 1979),
in or
20
These interactions which have been identified occur
in
large
conducted domestic
domestic
animals.
to determine whether animals
tory animals.
offers
some unique
few
studies
advantages
have
been
The study of larFe
over studyin F small
the study of the cellular interactions
lymphoid
compartments
performed
(McDermott et al,
or
Recently
the
analysis
populations
has
begun
modulate
organs
labora-
such
as
of
the
interactions Obmann
to understand
man
their
response
where,
respiratory
because
et
between al,
the immune to
tract
of
various
1983a,
may
bovine
b).
response
infectious
studies may have also direct application in
the
in specialized be
1982),
(Bielefeldt
were done in an attempt
infections
very
the case.
The kinetics of the immune response in the same animal may be
followed. Furthermore,
eventually
However
this is indeed
in mice and humans may also
These
of cattle
agents.
cell trials
and
However,
to
such
for controllin~ viral and bacterial
ethics
or
risk,
similar
experiments
could not be performed. In
the
present
peripheral indicate
study,
blood that
lymphocytes.
the
induction
lymphocytes
M~'s This
are
was
levels of soluble
prove
for
useful
studying
for
in
is antigen
by excessive
in
performed.
crucial
response
of
The
vitro
suggestin~
conditions
antibody
results
induction
dose dependent
antigen,
various
vitro
bovine
described of
below
antibody
by
and can be abrogated
that this
required
from
system
should
for immunore~ulation
ranging from induction of a reactive to and/or nonreactive state.
MATERIALS AND METHODS
Lymphocyte Cultures Lymphocytes Hereford
or
were
McGulre & Babiuk 7 days
obtained
from
Holstein-Friesian
after
1983).
peripheral
blood
as d e s c r i b e d
Calves were immunized with
immunization
could not be produced
the
calves
adn
by PBL.
weekly
thereafter
Animals
were
of
4-12
month
previously
old
(Filion,
I mg. of KLH, bled 5 to until
in
reimmunlzed
vitro
at this
antibody time.
No
substantial difference existed between animals in the kinetics of appearance and
disappearance
of
reactive
PBL cells
for
anti-KLH
antibody
production.
Briefly, blood was collected into syringes containing citrated dextrose; huffy coat was collected temperature,
and was
after centrifugation
layered
on Ficoll-Paque
at
1600 g for
(Pharmacia,
the
15 min at room
Uppsala,
Sweden).
The peripheral blood leukocytes
(PBL) present at the interface were washed 3
times
medium
with
containing procedure
minimal
essential
antibiotics yielded
(MEM)
(5 mg amphotericin
greater
than
99%
(GIBCO,
Burlington,
Ontario)
B, 5 mg gentamycin/liter).
mononuclear
cells
with
more
than
This 98%
21
viability.
The
essential volumes
medium into
Fisher
PBL,
resuspended
(FBS-MEM),
96
well
Scientific,
were
(flat
in
10%
aliquoted
bottom)
Edmonton,
bovine
(triplicate
microtltre
Alberta)
Cells and antigen concentration
fetal
and
serum
cultures)
dishes
incubated
-
in 200 ul
(Falcon
at
37°C
varied with the experiment
minimal
test in
IIl,
5%
CO 2.
and are reported
in the appropriate figure or table. Monocyte-Depleted
Cells
To remove monocytes, ed in 10 ml volumes
purified PBL, resuspended in 10% FBS-MEM, were plat-
(1 x 107 cells/ml) onto
ture petri dishes (Corning Glass, C o m i n g , hrs
in
a humidified
5% CO 2 incubator.
100 x 15 mm plastic tissue cul-
NY) and incubated at 37°C for i-3 The nonadherent
removed by washing once with calcium and magnesium-free, saline of
(PBS), centrifuged
and resuspended
I-2.5 x 108 cells/ml.
These
column (Pharmacia, Uppsala,
(Mishell et al,
Sephadex
30 min
for
at
from
50
to
contained
78%
and
less than
gently
buffered
in 10% FBS-MEM at a concentration
cells were passed
through
a Sephadex
G-10
Sweden) containing a nylon wool plug as describ-
ed previously
column with 20-25 mls of
cells were phosphate
1980).
The cells were allowed
37°C before
elution
of nonadherent
10% FBS-MEM at 37°C.
viability
was
>98%.
2 + 1.8% nonspecific
to bind to the cells
from
the
The recovery of cells ranged
The
recovered
esterase
nonadherent
positive
cells
cells
(Koshi et
al, 1976). Monocyte Enrichment The cells adhering to the petri dishes were washed thoroughly with PBS to remove weakly adherent incubating
with
cells.
The cells remaining attached were removed by
3 mM EDTA in PBS were
washed
for
Recovered
cells
three
FBS-MEM.
Analysis of the adherent
15 mins
times
at
with
population
37°C
MEM
and
in a CO 2 incubator. resuspended
in
(designated M~) revealed
10% >90%
nonspecific esterase positive cells with 95% viability. Pulsing of cells with Antigen Various cell populations time as indicated
were pulsed with antigen for various periods of
in the results.
Removal of unbound antigen was performed
by washing the culture three times with 10% FBS-MEM. Antibody Detection The level of anti-KLH antibodies from culture supernatants was determined by an enzyme link immunoassay 1983).
Briefly,
(OA) (Sigma, pH 9.6.
the antigens, KLH (Calbiochem.,
St. Louis, Mo.) were diluted
Aliquots of i00 ul were added
(Dynatek Laboratories, wells
(ELISA) as described previously
were
washed
Alexandria,
3 times
with
(Filion et al
La Jolla, Ca.) or ovalbumin
to 20 ug/ml with carbonate buffer
to the wells of Immunolon
II plates
VI) and incubated at 4°C overnight.
distilled
water
to
remove
antigen before the addition of i00 ul of culture supernantants
the
The
unabsorbed
to each well.
22
After a ! hr incubation at room
temperature,
the wells were washed
3 times
with distilled water to remove the unbound antibody. Horseradish peroxidaseconjugated
rabbit
anti-bovine
in i00 ul volumes
was
added
IgG
(Cappell
to each well
Laboratories, and
the plates
Cochranville, were
PA)
incubated
a
further hour at room temperature. The ELISA plates were once again washed as described solution allowed
above, before
the addition of
(6.5 mM 5-aminosalicylic
100 ul/well of the enzyme substrate
acid with
0.02% H202).
to proceed at rt for 30 min before reading
The reaction
the optical densities at
490/630 nm in an MR 580 microelisa autoreader
(Dynatek Lab, Alexandra,
Each
controls:
ELISA
plate
contained
two
negative
was
i)
antigen
VI.). plus
substrate, and ii) antigen, horseradish peroxidase conjugate and substrate.
RESULTS The in vitro secondary response of primed PBL to KLH:
Definition of optimal
culture conditions In an attempt of antibody,
to optimize
variations
in cell density,
tion times were investigated.
A
the culture
conditions
for in vitro
antigen concentration,
induction
and incuba-
Cells from 2 different animals were incubated
.2J
o
o~
z a .J
KLH
P n 0
x! i
OA I
no Ag 1
J
I
I
i
I
2.5
5
7.5
10
20
cells x l 0 !
5
culture
Fig. 1 Optimal cell density for in vitro antibody productions. PBL from KLH primed animals were cultured with KLH. Cells ~ere plated at various concentrations and stimulated for 5 days with 10 - ug/culture of KLH ~ - - ~ , 0--0), OA (I--e,A---A) or 10% FBS-MEM (no Ag) before assaying the level of antibody (OD~SD).
23
at
a density
produced
of
106 cells/200
ul with KLH.
after 5 days in culture, whereas
Peak
antibody
cultures containing
ger numbers of cells produced lower antibody levels (Fig.
I).
responses
were
fewer or larThe induction
of the anti-KLH antibody response was found to be antigen specific since in the presence of an unrelated antigen, ie. OA, or in the presence of 10% FBS, anti-KLH antibody was not from animals primed tion with
the maximum
ture (Fig. 2).
formed
to KLH,
(Figure
1,2,3).
In addition,
lymphocytes
responded best at a specific antigen concentra-
response
occurring
between
10 -2 and
10 -5 ug per cul-
Anti-OA antibody was not induced in these cultures
(results
not shown).
Fig. 2 Antigen dose response curve for in vitro antibody induction. PBL's from KLH primed animals were pl~ted at I0 ~ cells/ culture and stimulated for 5 days with various concentrations of KLH
.4_
.3-
A
0 0 0) .2-
>. Iu~ z Lu 0 .J 0
.I
(o--o,e--o),
I
OA ( ~ - - ~ A--&) or 10% FBS-MEM (C). The antibody levels (OD~SD) to KLH were measured employing an ELSIA assay.
,
a. 0
I
c
I
I
10-s
I
I
10
I
-6
I
10
-4
ANTIGEN
I
I
10
-2
DOSE
I
I
I
1
10 2
24
The final parameter
to be tested for optimizing
in vitro antibody synthesis
was the day of the peak response.
As illustrated in Fig. 3, no antibody was
detected
of
2 days
after
initiation
the
culture.
However,
antibody
was
detected 3 days after initiation which peaked by day 5.
.3 A
o
¢0
o
0~ LI
.2 >.
z
LU
a.
0 _x
Z
I
1
I
I
I
2
3
4
5
6
DAYS Fig. 3 Kinetics of induction for in vitro antibody productionm2 PBL (106 cells/culture) were incubated for various time periods with i0 ug/culture of KLH ( O - - O ~ - - ~ ) or OA ( e - - O , A - - A ) . The levels of anti-KLH and anti-OA antibodies were measured by an ELISA assay (OD+SD). Given
the
incubation
above
findings,
a cell
density
of
106
cells/culture
and
an
time of 5 days in the presence of 10 -2 or 10 -3 ug of antigen was
used in subsequent experiments. Specificity of anti-KLH antibody Although
the
above
results
suggest
that
KLH
initiated
a
secondary
in
vitro antibody response,
the specificity of the anti-KLH antibody production
remained
Therefore,
ascertain OA prior
to be
shown.
whether
incubation
to testing
experiments
of the culture
in the ELISA assay could
were
performed
supernatant inhibit
in
order
to
fluids with KLH or
binding
to the ELISA
25
plates
coated
with
antibody
was
fashion,
whereas
results
suggested
KLH.
inhibited the
As by
illustrated
the addition
addition
of
in Fig.
4,
of soluble
soluble
OA
or
the
putative
anti-KLH
KLH in a dose dependent diluent
did
not.
These
that the antibody produced was KLH specific.
o o
.2
z w a
.1 0 0
KLH
l I
I
1
10
I
100
ANTIGEN ADDITION (lxg)
Fig. 4 Inhibition of anti-KLH antibody binding by specific antigen: Supernatant of culture stimulated with KLH, were added to equal volumes of various concentrations of either KLH (O--6), 0A or diluent ( O ~ O ) prior to assaying anti-KLH antibodies by ELISA on KLH coated plates. Antibody levels of culture stimulated with 10% FBS-MEM ( ~ ) or KLH ( A ) without the addition of soluble KLH or 0A are reported also. Values are expressed as the mean 0D+SD/culture of triplicate samples. m
To
further
cultures,
the
confirm
the
supernatants
specificity of KLH,
of
0A or
added to either KLH or 0A coated wells. cultured bound
supernatants
strongly
stimulated
to
culture
bound only minimally KLH
coated
supernatants
wells.
the
antibody
10% FBS
produced
triggered
As shown in Table
in
cultures
OA
were
i, KLH triggered
to 0A coated wells, whereas, Furthermore,
these
or
10%
they
FBS-MEM
bound neither OA nor KLH coated wells.
26
TABLE
1
Lack of binding of culture supernatants
to wells coated with irrelevant
antigen
Stimulus for Ab Production
Exper. No.
KLH
OA
KLH Coated Wells
OA Coated Wells
1 2 3 4 5 6
.270+.010 .1887.015 .432~.032 .246"$.017 .157~.009 .291~.005
.011+.004 .013~.002 .0187.005 .006"$.002 .008~.001 .019~.004
7
.061+.007 .081~.006 .051~.009
.008+.001 .007~.001 .006~.001
.084+.013
.017+.002
8 9 10% FBS
Culture supernatants from KLH, OA or MEM-FBS stimulated cultures were added to either the KLH or OA coated wells and the ELISA was performed. Experiments i-9 were performed at various times. 1F~O KLH or OA stimulated cultures, multiple doses were used, however only ug/well are reported. Pulsing lymphocyte cultures with antigen Previously it was demonstrated ug)
or
low
anti-KLH
(10 -6 , 10 -7
antibody
detectable
ug)
response
response
that cultures with either high
doses (Fig.
to high doses
of
KLH
2).
did
The
not
produce
reason(s)
of antigen
for
a the
(i0, or 100 significant lack
could be attributed
of
either
a to
(i) the formation of the immune complexes during the culture incubation time which
could
suppressor
not
be
detected
pathway mediated
reduced Ab synthesis order were After
or
to differentiate first
pulsed
pulsing
the
with cell
FBS-MEM and the cultures
in
our
assay
system,
(2)
the
induction
of
a
either by M6 or T cells, which would result in
(3) the induction
of B cell
between
these
various
concentrations
free were
antigen
three
was
incubated
unresponsiveness.
possibilities, of KLH for
removed
by
in the absence
cultures i, 4 or
three
washes
of soluble
In
of PBL 24 hrs. in
10%
antigen.
Control cultures were not washed in order to assure that the induction of in vitro antibody was successfully accomplished. 5 suggest
that exposure
production of Ab.
The results outlined
in Fig.
to antigen for 1 hr was sufficient to stimulate the
27
~ r .
~ r .
24 hr.
c~ 0 m
OA
.5 zm
.4-
.3 "2 /
0
.1o ~o2,o
~
1G'~-2
0
KLH
10210
0
10~10
0 102 10
1 1()1 1t~2
DOSEOFANTIGEN Fig. 5 Pulsin~ of cultures with various doses of KLH for 1,4 and 24^hours: Various concentrations of antigen, KLH (bottom panel) or OA (0 ~o 102 ug) (~]) (top panel) or diluent ( ~ ) were added to PBL cultures (i0 v cells/culture). After 1,4 or 24 hrs these cultures were washed 3 times and incubated in fresh MEM +10% FBS for a total of 5 days. Antigen was not readded to these cultures. Control included cultures where the KLH or OA was not removed are presented in the left most panels. Furthermore, antibody
removal
synthesis
of
when
excess high
Ag
by
antigen
washing,
prevented
concentrations
were
suppression used.
of
Cultures
left in contact with the antigen for 4 hrs produced lower levels of antibody with the higher concentrations longer that
incubation the
antigen
lack was
periods
of
not
before
detectable
due
to
of antigen.
the
antigen
This was even more evident with removal.
These with
results
antibody
in
cultures
10
formation
of
izamune complexes
or but
suggest
100 due
ug to
of the
inactivation of B cells by antigen or by the induction of a suppresssor cell pathway. The role of macrophages
in the induction of a s e c o n d a r y
in v i t r o
antibody
response The
M4
plays
interactions
a
crucial
(reviewed
been shown either
central
in Unanue
role
& Rosenthal
to enhance or suppress
1980, Morimoto et al, 1980).
in
the
regulation
1980).
of
Furthermore,
the immune response
cell-cell M6 have
(Metzgar et al
In an attempt to determine the role of M6's in
the in vitro induction of antibody and in the suppression of these responses with
high
doses
of
antigen,
depletion
and
reconstitution
experiments
were
employed. Lymphocyte populations with few M6 (nonadherent cells (NA)) were obtained by sequential
plastic
adherence
and passage
through a Sephadex G-10 column
28 of PBL.
This procedure
routinely
produced
relatively
lations with very few M6 (2 - .5% nonspecific the M~ population ed in vitro with
to a change
of KLH
experiments
employing
Furthermore,
contaminating
of
from PBL completely abrogated the antibody response induc-2 ug of KLH (Fig. 6 and 7). The effect of M6-depletion
revealed shown).
popu-
The elimination
10
was not due by
pure lymphocyte
esterase).
concentration
enriched
lymphocytes
induce an antibody response
a wide M~
since
optimum
antigen
population
5 x
was
105/culture
for
dose
the NA cells
range
(results
sufficiently
of
these
free
cells
could
as not of not
to KLH (Fig. 6).
z
0
1l J PBL NA+2ME N A ÷ M(~
Fig. 6 Reconstitut~on of the in ~ antibodY5response with syngeneic M6: Cultures of PBL ~i0 ), M~ (5 ~-'~0 ), NA (5 x 10 ) cells/culture, with 2-ME (range: 2 x IO-JM to 2 x i0- J"~)" or without 2-ME and mixtures of M6 + NA were incubated with either 10- ug of KLH (Fl) or 10% FBS-MEM ( ~ ) . Supernantants of the culture were harvested 5 days after culture initiation and the antibody levels were measured by ELISA. Values (OD) expressed are maans of triplicate determinations. However, various
repopulating numbers
cells/culture) (Fig. 6). Furthermore,
of
syngeneic
resulted
of M6
NA
cells
(ranging
in a significant
(5 x from
the reducing
1978) was unable
agent
105 5
production
No effect was seen with M6 numbers
has M~ substituting
populations
cultures
x
cells/culture) 105
to
x
105
antibody
below 1.25xi05 cells/culture.
2 mercaptoethanol
(2-ME),
which
activity in some in vitro in~nune responses
to reconstitute
1.25
of KLH specific
with
the antibody
response
reportedly
(Opitm et al,
of nonadherent
cell
(Fig. 6).
Pulsing of macrophages Macrophages
play
(Fi~. 6), however
with different
a crucial
role
concentrations in
the
they may also be involved
in vitro
of KLH induction
in suppression
of antibody
of the response
29
under conditions ty,
lymphocytes
such as high antigen dose. were
added
with
various
concentrations
were
pulsed
with
antigen,
high
or
to M6 cultures of KLH. low
To investigate
which had been pulsed
Regardless
doses
of
this possibili-
KLH,
of whether
following
for 24 hr
the macrophages
removal
of
soluble
an antibody response was produced in cultures of M6 and lymphocytes
(Fig. 7).
o ¢ej 0 .15
T
l
I-
Z w
0 Io. 0
+ +
.05
PBL
NA
NA + MO 0
NA + M ~
NA+M~ .01
NA + M ~ 1
10
Fig. 7 Induction of in vitro antibody with pulsed M~: M~ (105 cells/culture) were pulsed for 24 hours with 0, .01, i or 10 ug of KLH prior to_removing the excess unbound antigen by washing 3 times._2NA cells (5 x 10 D cells/culture) were added to the pulsed M6 and KLH (10 ug/culture) ( ~ ) or 10% FBS-MEM ( ~ ) was added to the wells. NA and PBL cultures were included to ascertain the levels of antibody produced by these cells. After • o 5 days in a 5% COp incubator at 37 C, the supernatants were harvested and the antibody levels were determined by ELISA. Thus high concentrations turn could reduce pulsed
cells
immunogenic KLH
being
of KLH did
activate
suppressor
the level of in vitro antibody synthesis.
efficiently
form, the
not
with
most
presented
those M~'s efficient
antigen
pulsed
to
the
cells.
which
in
In fact, these
lymphocytes
with the highest
presenting
M6's
dose
in
an
(I0 ug) of
Therefore,
under
the
experimental conditions employed, M6 did not act as suppressor cells.
DISCUSSION The antibody of
KLH
present response only
if
study
demonstrates
could be induced these
cells
came
that
a
specific
secondary
in
vitro
by culturing bovine PBL in the presence from
animals
primed
to
this
antigen.
30
Additional was
evidence
for
from experiments
the
which
specificity demonstrated
antibody to KLH coated ELISA plates. was dependent
on repeated
of
the
in vitro
that KLH
antibody
inbibited
response
bindin~
of
the
The induction of an in vitro response
immunization
of animals with KLH.
Regardless
of
the conditions for antibody induction, maximum antibody levels were produced 5 days
post-stimulation.
body induction. vitro
Finally,
M~ were
found
to be necessary
for anti-
These results are similar to those recently reported for in
antibody
induction
in man
(Morimoto
et
al,
]981b,
Gerrard
& Fsuci,
1982 and Volkman et al, ]982). The
cost
performing
of
large
detailed
in the development animals
offer
numbers
of
domestic
analysis
animals
of the immune
considerable
specific
cell
has
generally
of the interactions response
advantages populations
cannulation of various lymphocytic
of these
since
been
3 deterrant
between various species.
it is possible
by alveolar
However, to obtain
or mammary
gl~nd
tissue or by repeated bleeding.
more, experiments with infectious agents can be performed wherein fic cellular
mechanism
involved
in protection
can be
for
cell types such large
lava~e, Further-
the speci-
determined.
Thus by
employing in vitro models to discern the cellular interactions durin~ development
and
determine but
expression
of
better methods
also
in man.
the
immune
response,
of infectious
In fact,
it has
disease
been
it
should
be
possible
to
control not only in animals
suggested
that
the delineation
of
immune mechanisms which regulate lung immunity may be best analyzed in large domestic animals because
many
(McDermott et al 1982).
important
diseases
occur
initiated a series of comparative
It is for these reasons as well as in
humans
and
bovines
that
studies employing a bovine model
we
have
to study
cellular immune mechanisms. The cellular mechanisms for the bovine valin-A
(Con A) suppressor
et
1981,
al,
Bielefeldt
generated
in
adherence
and passage
dependant
upon
Ohmann results
et
al
is
or cell mediated
immune
the
Ohmann
of
These
demonstrate
similar
et al fraction
1983a).
These
(M~ depleted
to
M~
previous
that
those
(Tadakuma
findings
the immune
of
and
humans
(Smith
cells were
sequential
plastic
but the ~eneration was
Pierce coupled
response and
suppressor by
through Sephadex G-10 column),
presence
1983b).
response Concona-
cells have been induced and characterized
the non-adherent
clearly
animals
of the humoral
system has only begun to emerge in the literature.
of
small
1976, with large
animal
Bielefeldt the
present
domesticated species
thus
providing a possible model for analyzing immune responses of other species. The antibody adherence addition
response
and passage of
2-ME
after
through
suggesting
the elimination Sephadex that
M~
G-10, are
of M~ by sequential
could
required
not as
be replaced antigen
plastic by
the
presenting
81
cells.
Further
evidence
cell was obtained induced (Fig.
7),
or M~
the
role
of
from the experiments
to produce
addition,
for
antibody
not
wherein unprimed
if they were
recombining did
the M~ as an antigen
nonadherent
seem
incubated cells
to mediate
the
immunogenic
rather
reports
(1981),
but
can
attributed
be
through
to
G-10
from PBL.
yields
a considerable
cells,
even
ing
of
results).
cell
necessary
a
These (1972),
efficacy
of
only was
not
Replating
third
These results in
order
Further
experiments.
characterization of
specific After
of
involved
with
Geha et al
The
discrepancy
procedures.
adherence
In
or passage
accessory
blood monocytes
esterase
attempted
assure
cell
positive
the
to plastic
monocyte-like
(Bielefeldt
Ohmann,
un-
that strict monitor-
purity
of
these
cells
is
for in vitro induction of antibody. bovine
These
this accomplishment,
the cellular mechanisms
M~
antigen in an
in agreement
to eliminate
clearly demonstrate
the
antiserum.
In
since
is by no means a definite marker for
is
to
M~
antigen.
(1981),
(1978).
plastic
of peripheral
and that the M~ is essential
production
are
could be pulsed
activity
deprivation
sufficient
replating
and
et al
et al
number of nonspecific
population
results
the M6
adherence
M6
in presenting
Inaba
from this laboratory,
Moreover,
bovine blood monocytes.
after
et al
to those of Opltz
the
experiments
Sephadex
published
tolerogenic.
Sjoberg
are contrary
preliminary
function
than
of
antigen
suppressive
pulsed with all doses of antigen were effective
previous
lymphocytes
with
with
presenting
immune
will
response
facilitate
trials may be devised
in the pathogenesis
awaits
cell
the
separation
to determine
of bovine diseases.
Acknowledgements The authors would like to acknowledge the expert technical assistance of Ms. Jeanette Heise, Mr. Barry Carroll and Richard Monseler and also Mrs. Bonnie Neufeld for typing the manuscript. Dr. Bielefeldt Ohmann is a recipient of post doctoral fellowship grants from the NATO Science Fellowship program (No. 23-03 51/81 and No 23-03 38/82). Funding for the project was provided through a research contract with Agriculture Canada and the Department of Supply and Services, and by a grant from the Max Bell Foundation of Toronto. REFERENCES Bielefeldt Ohmann, H., Filion, L.G. & Babluk, L.A., 1983a. Bovine monocytes and macrophages: An accessory role in suppressor cell generation by Con A and in lectin induced proliferations. (Immunology in press) Bielefeldt Ohmann, H., Filion, L.G. & Babiuk, L.A., 1983b. Cellular interactions in the generation of bovine Con A-suppressor cells and in mitogenlc proliferative response. (Veterinary Immunology and Immunopathology in press) Cantor, H. & Gershon, R.K., 1979. Immunological circuits: cellular composition. Fed. Proc. 38:2058. Filion, L.G., M c G u i r e , R.L. & Babiuk, L.A., 1983. The n o n s p e c l f l c suppressive effect of BHV-I on bovine leukocyte functions (Infection and Immunity in press).
32 Geha, R.S., Jansen, M.E., Ault, B.H., Yunis, E. & Braff, M.O., 1981. Macrophage T cell interaction in man: Binding of antigen specific human proliferating and helper T cells to antigen pulsed macrophages. J. Immunol 126:781. Gerrard, T.L., & Fauci, A.S., 1982. Activation and immunoregulation of antigen-specific human B lymphocyte response: multlfacet role of the monocyte. J. Immunol. 128:2367. Inaba, K., Nakano, K. & Muramatsu, S., 1981. Cellular synergy in the manifestation of accessory cell activity for in vitro antibody response. J. Immunol. 127:452. Koshi, I.R., Poplack, D.G. & Blaise, R.M., 1976. A nonspecific esterase stain for the identification of monocytes and macrophages: In vitro methods of cell mediated and tumor immunology, p. 399. Bloom, B.R. & David, S.R. (eds.) Academic Press, N.Y. McDermott, M.R., Befus, A.D. & Bienenstock, J., 1982. The structural basis for immunity in the respiratory tract. Int. Rev. of Exp. Pathol. 23:47. Mishell, B.B., Mishell, R.I. & Sbigi, J.M., 1980. In selected methods in cellular immunology. Mishell, B.B. and Shigi, S.M. eds. N.H. Frieman and Company, San Francisco. Metzgar, Z., Hoffeld, J.T. & Oppenheim, J.J., 1980. Macrophage mediated suppression: I Evidence for participation of both hydrogen peroxide and prostaglandins in supression of murine lymphocyte proliferation. J. Immunol. 124:982. Morimoto, C., Todd III, R.F., Distaso, J.A. & Schlossman, S.F., 1981. The role of macrophage in in vitro primary anti-DNP antibody production in man. J. Immunol. 127:1139. Morimoto, C., Reinherz, E.L. & Schlossman, S.F., 1981b. Regulation of in vitro anti-DNP antibody production by functional subsets of T lymphocytes in man. J. Innnunol. 127:69. Morimoto, C., Reinherz, E.L., Borel,Y. & Schlossman, S.F., 1983. Direct demonstration of the human suppressor inducer subset by anti-T cell antibodies. J. Immunol. 130:157. Opitz, H.G., Lemke, H. & Hewlett, G., 1978. Activation of T cells by a macrophage or 2-mercaptoethanol activated serum factors is essential for induction of a primary immune response to heterologous red cells in vitro. Immunol. Rev. 40:53. Sjoberg, 0., Anderson, J. & M o l l e r , G., 1972. Requirements for adherent cells in the primary and secondary immune response in vitro. Eur. J. Immunol. 2:123. Smith, W.G.,~singer, W.R. & Splitter, G.A., 1981. Bovine Con-A induced suppressor cells: generation, macrophage requirements and possible mechanisms of regulatory action. Immunol. 43:91. Tada, T. & Okumura, K., 1979. The role of antigen specific T cell factors in the immune response. In "Advances in Immunology". Ed. F.J. Dixon and H.G. Kunkel. Academic Press, N.Y. p.1. Tadakuma, T. & Pierce, C.W., 1976. Site of action of a soluble immune response suppressor (SIRS) produced by Con-A activated spleen cells. J. Immunol. 117:967. Unanue, E.R.---~ Rosenthal, A.S. (eds), 1980. Macrophage regulation of immunity. Acad. Press. N.Y. Volkman, D.J., Allyn, S.P. & Fauei, A.S., 1982. Antigen induced in vitro antibody production in humans: Tetanus toxoid-specific antibody synthesis. J. Immunol. 129:107. Yamashita, U. & Hamaoka, T., 1979. The requirement of Ia-positive accessory cells for the induction of hapten-reaetive cytotoxic T lymphocytes in vitro. J. Immunol. 123:2367.