Scand. J. Immunol.

11, 47-51, 1980

Conditions for Induction of Specific and

Polyclonal Antibody Production by Cowan 1
Bacteria and by Pokeweed Mitogen
O. SJOBERG & J. KURNICK
Blood Centre, University Hospital, and Department of Immunology,
Uppsala University, Uppsala, Sweden

Sjoberg, O. & Kurnick, J. Conditions for Induction of Specific and Polyclonal Antibody
Production by Cowan 1 Bacteria and by Pokeweed Mitogen. Scand. J. Immunol. 11, 47-51,
1980.
Cowan 1 bacteria and pokeweed mitogen (PWM) were used to induce the formation of direct
plaque-forming cells (PFC) against sheep erythrocytes (SRBC) by human peripheral blood
lymphocytes in vitro. It was necessary to absorb the serum supplement with SRBC before
culture to obtain anti-SRBC PFC. Alternatively, sheep serum could be added to the cultures.
The PFC response was specific, and the response was equally high in cultures with a mixture of
absorbed and non-absorbed serum as in cultures with absorbed serum only. Cowan 1 and
PWM could also induce synthesis and secretion of both IgM and IgG polyclonal antibodies.
Absorption with SRBC or addition of sheep serum had no effect on this synthesis. Thus it seems
likely that the induction of anti-SRBC PFC by Cowan 1 or PWM needs the presence of SRBC
antigen and is the result of a synergism between mitogen and antigen. Consequently, the anti-
SRBC PFC response obtained after stimulation with Cowan I or PWM in SRBC-absorbed
serum does not reflect a true polyclonal antibody response.

O. Sjoberg, Blood Centre, University Hospital, S-750 14 Uppsala, Sweden.

Various substances called polyclonal B cell
activators (PBAs) can induce bursa-equivalent
lymphocytes (B cells) from several animal
species to increased DNA synthesis and
polyclonal antibody synthesis [2, 8]. To be
clinically useful, the PBAs should be able to
stimulate B cells present in human peripheral
blood lymphocytes (PBL). Unfortunately, many
of the substances known as PBAs in animal
systems are unable to activate human blood
B cells.
However, some batches of pokeweed mitogen
(PWM) [7, 9, 22], antisera against Pa-micro-
globulin [16], and certain bacterial species [3,
II, 17, 18] have successfully been used to
stimulate human B lymphocytes to increased
DNA synthesis. It has also been known for
some years that PWM can induce polyclonal
Ig synthesis in PBL [13, 20, 24]. Recently, it was
demonstrated that both PWM and the bacteria
mentioned above stimulate a PFC response
against sheep erythrocytes (SRBC) [3, 4, 10,
17, 18].
We here present evidence that both PWM
and Cowan 1 can induce both true polyclonal
secretion of antibodies and anti-SRBC PFC.
In contrast to the polyclonal response, however,
the anti-SRBC PFC response was dependent
on the presence of antigen in the cultures.
MATERIALS AND METHODS
Mitogens. Cowan 1 bacteria were killed by treatment
with 0.5% formaldehyde, stored at 4°C and washed six
times before use. A final concentration of 10-'' was
used in the cultures.
PWM was purchased from Gibco (New York,
USA) and used at a final concentration of I/lOO in
the cu\tures.
Preparation of lymphocytes. PBL were isolated from
blood from healthy donors by centrifugation on a
Ficoll-lsopaque gradient (Ficoll-Paque, Pharmacia
Fine Chemicals, Sweden) [5]. The cells were washed

0300-9475/80/0100-0047 |02.00 © 1980 Blackwell Scientific Publications 47

48 O. Sjoherg & J. Kurnick

three times in phosphate-buffered saline and suspended TABLE 1. Effect of absorption of normal human serum
in RPMt 1640 (Flow Laboratories). (NHS) on stimulation of anti-sheep erythrocyte plaque-
Culture conditions. 1 ml portions of a cell suspension forming cells (PFC) by Cowan I and pokeweed
in medium (2.5 X 10" cells) were added to plastic tissue mitogen (PWM)
culture tubes (Falcon Plastics no. 2058) and mitogens
and sera were added as indicated in the Results section.
The serum concentration in the cultures was 5% (v/v) PFC/culture
if not otherwise stated. The tubes were then incubated
for 6 days in a humidified incubator in 5% COj in Serum Mitogen Exp. 1 Exp. 2 Exp. 3
air at 37°C.
NHS 2 7 16
Absorption of normal human serum (NHS) and fetal
Cowan 1 7 19 6
bovine serum (FBS) was performed by incubating 29 10
PWM 21
equal volumes of serum and washed packed SRBC at
4°C for 1 h. Absorbed NHS* 1 6 6
PFC assay. To test for PFC [6, 14] the tubes were Cowan 1 844 3784 208
centrifuged and the supernatants removed. The pellets PWM 514 1772 250
were diluted in balanced salt solution [15] to a suitable
concentration. 100 (JLI of the cell suspension were
mixed with 25 |j.l of a 10% erythrocyte suspension and • NHS was absorbed with an equal volume of washed,
25 (xl of fresh guinea-pig serum diluted 1:4 in balanced packed sheep erythrocytes at 4°C for I h with occasional
salt solution. Thereafter 200 (xl of a 0.5% agarose mixing.
solution kept at 47°C were added. The mixture was
poured into a petri dish and spread out. The dishes were
incubated at 37°C for 2 h. Scanning for PFC was done different experiments. In two of the experiments
in indirect light using a magnifying glass. Values for shown, there was a small induction of PFC by
PFC per culture tube are given as the geometric mean
of three cultures. Cowan 1 and PWM also in cultures without
Assay for Ig syntttesis. Rabbit antisera against human absorbed human serum. Although this occurred
IgM and IgG were kindly provided by Hans Bennich, in approximately two-thirds of the experiments,
Uppsala. They were absorbed with insolubilized IgG it could not be reproduced consistently. The
and IgM, respectively, before use. IgM was prepared use of absorbed serum did not by itself stimulate
from the serum of a myeloma patient by dialysis
against water, followed by separation of the precipitate the PFC response above background values.
on DEAE-Sepharose CL-6B (Pharmacia Fine Chemi-
cals). Human IgG was purchased from Kabi, Sweden.
The Ig preparations were coupled to cyanogen bromide- Antigen is needed for the induction ofanti-SRBC
activated Sepharose 4B (Pharmacia Fine Chemicals) PFC
when used for absorption of antisera.
The necessity to absorb the serum could
Culture supernatants were assayed for contents of
indicate that something—possibly antibody—is
Ig by an inhibition radioimmunoassay as described
by Wide et al. [23]. In essence, cellulose particles to
removed, or alternatively that something is
which the relevant antiserum had been coupled through
added. To determine which alternative might
cyanogen bromide activation were mixed with radio-
be correct, we cultured cells and Cowan t in
active Ig labelled by the chloramine-T method [12] and
known concentrations of Ig or 0.1 ml of culture NHS or absorbed NHS, either alone or together.
supernatant. The binding of radioactivity to theThe response in cultures with a mixture of
particles was measured, and the amount of Ig secreted
NHS and absorbed NHS was not significantly
per culture was calculated from the standard curve.
Three cultures were pooled before assay.
lower than in cultures with absorbed serum
alone (Table II). Thus, it is unlikely that the
absorption removes inhibitory substances.
RESULTS Rather, it appears that something essential
for the induction of PFC is added. The most
likely candidate for this role is soluble antigen.
Absorption of NHS is necessary for the induction To test this, sheep serum (which is known to
ofanti-SRBC PFG contain an antigen, able to stimulate a primary
Direct anti-SRBC PFC could be induced both PFC response against intact SRBC in mouse
with Cowan t and PWM in cultures with PBL. systems[19]) was added to the cultures instead of
However, it was necessary to absorb the NHS absorbed NHS. The results of such experiments
with SRBC to obtain a response (Table J). are shown in Table III. It can be seen that
The values in this table also demonstrate that sheep serum could substitute for the absorption
the response varied considerably between with SRBC. 5 \x\ of sheep serum per culture
 Ig Synthesis Induced by Mitogens 49

TABLE II. Effect of mixing normal human TABLE IV. Specificity of anti-sheep erythrocyte plaque-
serum (NHS) and sheep erythrocyte-absorbed forming cell (PFC) response induced by Cowan 1
NHS on the anti-sheep erythrocyte plaque-
forming cell (PFC) response stimulated by
Cowan 1 PFC/culture
'Antigen' Exp. 1 Exp. 2
PFC/culture
0 1
Serum* Exp. 1 Exp. 2 Sheep serum 479 246
Swine serum 8 3
NHS 0 19 NHS* absorbed with sheep
Absorbed NHS 851 487 erythrocytes 942 272
NHS-1-absorbed NHS 783 328 NHS absorbed with swine
erythrocytes 136 8

• The concentration of each serum was 5%.

* Normal human serum.

TABLE III. Effect of sheep serum on the plaque- were secreted. In contrast to the induction of
forming cell (PFC) response after stimulation with
Cowan 1 and pokeweed mitogen (PWM)*
anti-SRBC PFC the induction of polyclonal Ig
synthesis was not dependent on the presence
of SRBC antigen. Absorption of the FBS with
PFC/culture SRBC did not detectably increase the produc-
Sheep serum tion of Ig (Table VI), nor did addition of sheep
(5 jil/culture) Mitogen Exp. 1 Exp. 2
serum have any effect (Table VII).
Cowan 1 19 4
PWM 54 10
+ Cowan 1 3276 721 TABLE V. Stimulation of polyclonal Ig
+ PWM 6104 600 synthesis by Cowan 1 and pokeweed
mitogen (PWM) in fetal bovine serum-
containing cultures
* 5% normal human serum was used in all
cultures.
Synthesis (ng/culture)
Mitogen IgM IgG
was found to be the optimal dose. The increase 3 181
of anti-SRBC PFC by addition of sheep serum Cowan 1 181 2263
or by absorption of the NHS with SRBC was PWM 189 651
specific for SRBC (Table IV). The results, taken
together, indicate that the antigen must be
present in the cultures for successful induction TABLE VI. Stimulation of polyclonal Ig synthesis by
of anti-SRBC PFC by Cowan 1 or by PWM. Cowan 1 and pokeweed mitogen (PWM) in cultures
Anti-SRBC PFC could also be induced by with absorbed and non-absorbed fetal bovine serum
Cowan 1 in cultures containing FBS instead (FBS)
of NHS. Also in this case it was necessary to
add antigen in some way to the cultures, either Synthesis (ng/culture)
through 'absorption' of the serum or by addition
Exf ). 1 Exp. 2
of sheep serum to the cultures (data not shown).
Serum Mitogen IgM IgG IgM IgG

Stimulation of polyctonat Ig synthesis by FBS — 60 130 280 1790

Cowan 1 and PWM Absorbed FBS 80 150 170 1630
FBS Cowan I 410 670 950 3260
Certain batches of PWM are known to Absorbed FBS Cowan 1 430 740 1750 2340
induce the production of polyclonal antibodies. FBS PWM 300 230 3290 8090
The results in Table V show that Cowan 1 has Absorbed FBS PWM 320 290 3040 8020
the same effect. Both IgM and IgG antibodies
50 O. Sjoberg & J. Kurnick
TABLE VII. Stimulation of polyclonal Ig synthesis by
Cowan 1 in cultures with or without sheep serum
Synthesis (ng/culture)
Sheep serum Mitogen IgM IgG
32 163
Cowan 1 747 1834
+ Cowan 1 675 1107
DISCUSSION
It is well known that PWM can induce secretion
of polyclonal Ig in cultures with human PBL.
Fauci & Pratt [tO] showed that stimulation with
PWM can also give rise to an anti-SRBC PFC
response. They found it necessary to use
absorbed serum and claimed that the anli-
SRBC PFC was a reflection of polyclonal
antibody synthesis. Recently, several other
studies have demonstrated that certain bacteria
can induce anti-SRBC PFC responses in cultures
of human PBL [3, 4, 17, 18]. In all these studies
SRBC-absorbed NHS was used to obtain
PFC responses, and the authors considered the
PFC responses to be the result of a direct
polyclonal activation of B cells.
The results presented in this paper make it
unlikely that such an assumption is correct.
Thus, it was necessary to use SRBC-absorbed
serum to obtain an anti-SRBC PFC response.
Alternatively, sheep serum could be used
instead of absorbed NHS. The same would
probably also be true for stimulation with the
other bacteria used in the studies mentioned
above.
Fauci & Pratt suggested that the etfect of the
absorption was to remove inhibitory factors—
presumably antibodies. Our results make this
assumption unlikely for the following reasons:
(1) Cultures with mixtures of SRBC-absorbed
and unabsorbed serum gave as good responses
as cultures with absorbed serum alone. (2)
Addition of sheep serum could substitute for
the absorption. (3) Addition of swine serum or
of NHS absorbed with swine erythrocytes
together with mitogen did not result in pro-
duction of anti-SRBC PFC. (4) No increase in
polyclonal Ig secretion could be detected when
absorbed serum or sheep serum was used
instead of only normal FBS.
In contradistinction, our results favour the
view that the anti-SRBC PFC response obtained
after stimulation with PWM or various bacteria
is the result of a synergism between mitogen and
antigen. The anti-SRBC PFC response therefore
does not seem to be the result of a true poly-
clonal stimulation of B cells by mitogen, and
other factors, such as the patient's immune
status against SRBC, may influence the results.
This conclusion is also supported by the results
of Allan et at. [1], who found that absorption
of equine IgG with SRBC resulted in the release
of SRBC antigen into the IgG preparation and
that the antigen could induce a primary immune
response against SRBC if the IgG was injected
into normal rats. Thus, the anti-SRBC PFC
response seems a less suitable indicator of B
cell function in general.
The importance of thymus-derived lympho-
cytes (T cells) in this system is not clear. Our
results show, however, that it is impossible to
draw any conclusions about the T cell depend-
ency of the anti-SRBC PFC response, if
rosetting of T cells with SRBC is used as a
method of depleting a PBL population of T
cells, since antigen-specific B cells would be
removed at the same time.
As shown in the second part of this study.
Cowan 1 was an equally potent inducer of
secretion of polyclonal Ig as PWM. The response
was independent of the presence of SRBC
antigen in the cultures, and both IgM and IgG
were produced. The variability in the capacity
of different batches of PWM to induce Ig
production [9, 21] and the clear T cell depen-
dency [13] make it a somewhat less desirable
agent for studies of human B cells. The avail-
ability of Cowan 1 suggests that this mitogen
may have more widespread applicability in such
investigations.
ACKNOWLEDGMENTS
We are grateful to Hans Bennich for supplying
antisera and to Leif Wide for valuable advice
and for supplying cyanogen bromide-activated
cellulose particles. This study was supported
by grants from the Swedish Cancer Society,
the Swedish Medical Research Council, NIH
contract NCl-CB-74129-3], and from Svenska
Livforsakringsbolags namnd for medicinsk
forskning.

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Received 21 May 1979
Received in revised form 27 August 1979