Note: Descriptions are shown in the official language in which they were submitted.
CYTORIWE
This invention relates to cytokines, and in
particular to a polypeptide factor derived from
macrophages.
Background and Prior Art
Publications referred to herein are identified
1~ in full at the end of this specification.
It is well recognized that macrophages play an
essential role in immune and inflammatory processes, both
at a cellular level and via the release of mediators such
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as interleukin-l (IL-l), colony stimulating factor ~CSF)
and tumour necrosis factor (TNF) (Dinarello, 1984;
Burgess and Metcalf, 1980; Le and Vilcek, 1987).
The thymus is a key organ in the generation and
5 maturation of lymphocytes in mammals, particularly during
foetal and neonatal life. Processing by the thymus is
required for the production of T lymphocytes, of which
various subsets are required for specific types of immune
response. These subsets, such as helper T cells,
10 suppressor T cells, cytotoxic T cells etc. may be
identified by the specific antigens present on their cell
surfaces.
Thymus cell suspensions may be divided into
light and dense cell populations by fractionation in
15 density gradients of Percoll (Salisbury et. al., 1979;
Percoll is a trade mark of Pharmacia AB).
In rats, the dense cell fraction comprises
immature thymocytes which are unable to respond to
Concanavalin A, and thymic epithelial cells, which
20 produce keratin.
The viability of dense, immature rat thymocytes
was found by the present inventors to decrease markedly
when a suspension of the cells in complete nutrient
medium was incubated in vitro over a four hour period.
This was attributed to the phenomenon of
programmed cell death, or apoptosis.
It has been estimated that approximately 90% of
the cells which are generated within the thymus die in
situ (Kinnon et. al, 1986). This population of
30 thymocytes is derived from the thymic cortex, and the
cells are characterized by their high density and
susceptibility to cortisone-mediated apoptotic death
(Weissman, 1986). The essential role of thymic
epithelial cells in thymocyte maturation is well
35 documented by studies in nude mice and rats in which the
ectodermally derived epithelial cells have failed to
, :: ~ ' '
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.,. ~
. ~ .
:
develop (Douglas-Jones et. al., 1981). In the cortex of
the normai thymus, the majority of the stromal cellq are
epithelial in nature, and have long processes which
reticulate among the cortical thymoctyes. In addition,
5 the cortex contains a small number of macrophages which
are primarily located in the region adjacent to the
medulla (Adkins et. al., 1981).
The epithelial cells in both the cortex and the
medulla express both class I and class II
10 histocompatibility antigens (Van Ejwik et. al., 1980) and
are thought to induce or select for self class II antigen
recognition by thymo~ytes (Berrih et. al., 1985). Thymic
epithelial cells have also been shown to be the source of
thymic hormones which induce the appearance of maturation
15 markers on the thymocytes (Berrih et. al., 1985). More
recently, the epithelial cells have also been shown to be
a source of IL-l (Le et. al., 1987).
We have now surprisingly found that a factor
released by macrophages in response to stimulation with
20 lipopolysaccharide is able to protect thymocytes from
apoptotic death.
The factor appears to stimulate immature,
non-proliferating thymocytes to differentiate to a stage
at which they are able to survive.
Without wishing to be bound by any proposed
mechanism for the observed beneficial effect, it is
thought that the macrophage-derived factor binds to a
common determinant of the Ia antigen complex on thymic
epithelial cells.
This in turn stimulates the thymic epithelial
cells to release a factor which promotes survival of
thymocytes.
~ The macrophage-derived factor is clearly
different in its biochemical and functional properties
35 from previously known factors such as tumour necrosis
factor and interleukin-l.
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Summary of the Invention
According to one aspect of the present
invention there is provided a monokine released by
macrophages in response to stimulation with
5 lipopolysaccharide, said monokine having the following
properties:
Relative molecular weight 36000 kD;
Isoelectric point 6.3 - 6.4;
Stable at temperatures up to 70C;
Stable at pH 2 to 10;
Activity destroyed by: reduction with
2-mercaptoethanol
treatment with proteinases, or
treatment with urea;
Does not stimulate proliferation of fibroblasts; and
Binds to common determinant of Ia antigen on
thymic epithelial cells.
According to a second aspect of the invention,
there is provided a method of producing said monokine,
20 comprising the steps of incubating macrophages in
nutrient medium in the presence of bacterial
lipopolysaccharide or-muramyl depeptide for 5 minutes to
2 hours, and recovering the monokine.
Preferably lipopolysaccharide is used to
25 stimulate monokine production.
Preferably incubation is for 2 hours.
Preferably recovering the monokine comprises
the steps of:
recovering medium from macrophage cultures,
removing lipopolysaccharide or muramyl
dipeptide,
removing material of Mr less than about 20,000,
subjecting the remainder to sequential steps of gel
filtration, ion exchange chromatography, and hydrophobic
35 interaction chromatography, and
recovering fractions having monokine activity.
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. ~: ' ,.
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., ~ .
- 5 -
Further purification may be effected by methods
such as preparative gel electrophoresis and/or high
performance liquid chromatography, which are well known
to persons skilled in the art.
According to a third aspect of the invention
there is provided a factor produced by thymic epithelial
cells in response to stimulation with said monokine, said
factor having the following properties:
Relative molecular weight 320,000;
Activity destroyed by trypsin treatment or by
boiling;
Does not require active protein synthesis for
production;
Binds to immature thymocytes;
Protects immature thymocytes from apoptotic
death; and
Binding to thymocytes inhibited by
preincubation with antibody to CD4 antigen.
According to a fourth aspect of the invention,
20 there is provided a method of bioassay of the monokine
comprising the steps of:
(a) adding a sample of fluid containing or
suspec~ed of containing monokine to either dense thymic
cells, or whole thymus suspension depleted of adherent
25 cells, in nutrient medium in the absence of serum and
indicator dye for a period of 3 to 16 hours, preferably 3
to 7 hours, ;
(b) adding MTT (3-(4,5-dimethylthiazol-2-yl)- -~
2,5-diphenyl tetrazolium bromide)
(c) incubating for 1 hour at 37C,
(d) removing untransformed MTT,
(e) adding isopropanol to the samples,
(f) estimating extinction at 540 or 560 nm,
and
(g) calculating the amount of monokine in the
sample.
~, ....... . ..
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:.: .~,
' ~ - ' , ~ -
... ~ .... : . . . .
-- 6 --
Preferably the assay is carried out in 96 well
microtitre piates, and each well contain~ 0.5 to 5 x 106,
more preferably 2 x 106, dense thymic cells in 100 ~1
medium, suitably RPMl 1640 medium containing no phenol
5 red or serum.
Preferably 100 ,ul of sample to be assayed is
used per 2 x 107 cells.
The preferred incubation time before adding MTT
is 3 hours if adherent cells are not first depleted, and
10 7 hours if adherent cells have been depleted.
Detailed Description of the Invention
The invention will now be described in detail
by way of reference only to the following non-limiting
examples, and to the accompanying drawings, in which:
Fig. 1 shows viability of the dense thymic
cells, alone or in the presence of stimulated macrophage
supernatant. Percentage cell viability is shown as a
function of time. Results are the mean + s.e.m. of 25
experiments. By Student's t-test, *P c0.05 and
20 **P~ 0.01;
Fig. 2 shows results of molecular weight and
isoelectric point measurement of the monokine (a)
Molecular weight determination. Sephacryl S200 column
run. A column (100 cm x 1~6 cm) calibrated with
25 Combithek II proteins in DPBS with 0.1 mol/l NaCl was run
with a 5 ml sample under identical total conditions.
Flow rate 10.8 ml/h, sensitivity x 0.1, chart speed 2
cm/h., run time 17 hours. All of the fractions were
tested and MF activity was detected in only one (O.8 ml)
30 fraction. Hatched area indicates activity. (b)
Iso-electric point. LKB broad range ampholine gel (pH
3-9). Initial run, activity localized between 6 and 7
with reference to a standard curve. Second run, samples
applied as a 10 cm band with standard proteins at either
35 end. Standard curves were identical and 2 mm strips of
t
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':
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: ', ` .
gel were taken between pH 6 and 7 and the activity was
localized to one strip: pH 6.3 - 6.4. Hatched area
indicates activity;
Fig. 3 shows the production of the macrophage
5 factor in response to varying levels of two different
stimuli. Production was determined by the titre
resulting in 100% protection in the thymocyte viability
assay. No protection refers to anything less than 100%
protection. The supernatant was collected at three
10 different times as shown. Results are the mean of four
experiments;
Fig. 4 shows the time course for the production
of the macrophage factor. Supernatants from macrophages
stimulated with 20 ,ug/ml LPS were collected at various
15 time intervals after stimulation as shown. Production
was expressed as the titre of macrophage factor resulting
in 100% protection in the thymocyte viability assay. Any
protection less than total was referred to as no
protection. Representative experiment of two
20 experiments;
Fig. 5 shows the effect of protein synthesis
inhibitors on the production of the macrophage factor.
Three protein synthesis inhibitors, actinomycin D (1
~g/ml), cycloheximide (10 ,ug/ml) or puromycin (100 ~ug/ml)
25 were added to macrophage cultures simultaneously with LPS
(20 ~g/ml) and supernatants were collected at six
intervals between five minutes and two hours for testing
in the thymocyte viability assay. Protective activity of
the macrophage factor at the various times is indicated
30 by the percentage viability of the thymyocytes at three
hours. Results are the mean of three experiments;
Fig. 6 shows the effect of control supernatants
on the production of the macrophage factor. Three
control supernatants, lysed macrophage supernatant,
35 trypsin treated lysed macrophages and unstimulated
macrophage supernatants were compared to macrophage
- - ~., . .~ . .
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- ~ . , . . - : . -
... , . . ~. :: . :: :
supernatant from macrophages stimulated with 20 ~g/ml
LPS. The percèntage viability of the thymocytes over
four hours is shown. Results are the mean of five
experiments;
Fig. 7 shows the effect of restimulation of
macrophage cultures with LPS on the production of the
macrophage factor. Supernatants were collected at two
hourly intervals and cultures were restimulated with
either LPS only (20 ~g/ml) or LPS (20 pg/ml) and
10 indomethacin (10 5M) in new medium. Production of the
factor was expressed as the titre resulting in 100
protection in the thymocyte viability assay for
supernatants collected at two, four, six and eight hours
after initial stimulation;
Fig. 8 shows the role of the Ia molecule as a
receptor for the monokine (representative experiments are
shown). In every experiment, the unprotected populations
were less than 70% of the control population by 4 hours
incubation.
(a) Stimulated macrophage supernatant can be
mimicked by a monoclonal antibody to Ia (MRC OX6). This
effect is also mediated via an epithelial cell. Control
DCF; DCF + 2.5 ~ug/ml MRC OX6 added (reproduced in four
experiments): Ia-negative DCF with 2.5 ~g/ml MRC OX6
25 added (reproduced in three experiments); Ia-negative DCF
with supernatant from thymic epithelial cells stimulated
with 2.5 ,ug/ml MRC OX6; and DCF + 2.5,ug/ml MRC OX3, the
monoclonal antibody to rat Ia which is strain-specific in
its recognition (reproduced in three experiments), are
30 compared.
(b) Removal of MF activity by its absorption
on to Ia-positive cells. MF absorbed on to thymic
epithelial cells prior to addition to DCF (reproduced in
four experiments); MF absorbed on to rat spleen cells
35 (reproduced in three experiments); MF absorbed on to rat
spleen cells depleted of Ia-positive cells (using MRC
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,~"~-"."" ~ ", , - , ~ ~ ,' ~ ' ,~ ,,, ,' ' . ~
.: . ., , - , - ~ . :
- 9 -
OX6/complement lysis) (reproduced in three e~periments);
and MF absorbed on to rat RBC (reproduced in two
experiments) were compared.
(c) The binding site of the MF on spleen
5 cells is blocked by an antibody to Ia (MRC OX6). MF were
absorbed on to spleen cells (as in Fig. 3b) but spleen
cells were previously incubated with 2.5 ~g/ml of MRC
OX6. The possibility of carry over of MRC OX6 into the
assay system was eliminated by labelling the antibody
10 with 125I (reproduced in three experiments). MF absorbed
on to spleen cells previously incubated with rat-specific
antibody to Ia (MRC OX3) (reproduced in three
experiments);
Fig. 9 shows the role of the Fab portion of MRC
15 OX6 in stimulating protective activity and in blocking
the binding of MF. Populations compared were
DCF + MRC OX6; DCF + Fab MRC OX6;
DCF previously incubated with Fab MRC OX 6 +
MF;
Fig. 10 shows the role of thymic epithelial
cells in thymocyte viability. A representative ~-
experiment of percentage cell viability as a function-of - -
time is shown. Note that in every experiment the
unprotected populations were less than 70% of the control
25 population by 4 hours incubation. Populations compared
were
Control, DCF depleted of Ia-positive cells
(using MRC OX6 and complement in a lysis technique);
Ia-negative DCF (i.e. DCF after the depletion
30 of Ia-positive cells) with the addition of MF (reproduced
over four experiments);
Ia-negative DCF with l hour supernatant from
unstimulated thymic epithelial cells (reproduced in two
experiments);
-- 10 --
Ia-negative DCF with supernatant from thymic
epithelial cèlls incubated for 1 hour at 37C with MF
(reproduced in two experiments);
Fig. 11 shows results of molecular weight
5 determination of the thymic epithelial cell factor using
Sepharose Cl-6B chromatography;
Fig. 12 shows the time course for the
production of the thymic epithelial cell factor;
Fig. 13 shows the enhanced proliferation of
10 dense immature thymocytes in response to either whole
- macrophage supernatant or an antibody to rat Ia (2.5
~g/ml of clone MRC OX6 or clone MRC OX3). Representative
experiment of eight experiments showing the mean +
standard deviation for quadruplicate cultures;
Fig. 14 shows the enhanced proliferation of
immature thymocytes in response to partially purified
macrophage supernatant. The addition of partially
purified macrophage supernatant to dense immature
thymocytes results in an increase in proliferation over
20 that of a control population. Pooled data from three
preparations of purified Mr 36 000 monokine showing means
values + standard error of the mean;
Fig. 15 shows the effect of purified macrophage
factor on thymocyte proliferation. Addition of macrophage
25 supernatants which have been treated in one of three
different ways restores the proliferative response of the
adherent cell depleted population to that of a control
population. Representative of two experiments showing
mean ~ standard deviations; and
-
Fig. 16 shows the characteristics of the
thymocyte response. The response of divided, recombined
and whole thymus to varying levels of Con-A in the
presence of 0.5 ~g of LPS is shown. Representative
experiment of five experiments showing the mean +
35 standard deviations of quadruplicate cultures.
.
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~, ~ ' : '
;!~:, `, ,,. ~ .
:~` ~ . .' . ' "'
~.~'1,," " . "
Abbreviations used hereina~ter are deflned as
follows:
Con-A Concanavalin-A
DCF Dense cell fraction of thymus
DPBS Dulbecco's phosphate-buffered
saline
DTEC Dense thymic epithelial cells
FCS Foetal calf serum
IL-I Interleukin-l
LPS Lipolysaccharide
MDP Muramyl dipeptide
MF Macrophage factor (monokine)
Mr Relative molecular weight
RBC Red blood cells
TECF Macrophage-induced thymic
- epithelial cell factor
TNF TumouF necrosis factor
Example 1 Preparation of macrophage supernatant
Macrophage cultures were prepared from the
20 peritoneal washings of 6-8 week old Wistar-Furth rats.
The cells in RPMI-1640 medium containing 10% FCS were
seeded into tissue culture flasks at a density of 1 x 106
cells/ml (5 x 106 per flask), and allowed to adhere for 1
hour at 37C. The monolayers were then washed five times
25 with DPBS, 5 ml serum-free medium was added per flask,
and the cells were stimulated with 20 ~ug/ml
lipopolysaccharide from Salmonella enteritidis (Sigma).
The supernatant was harvested at 2 hours, filtered
through an XM300 filter (relative molecular weight (Mr)
30 300,000 cut off) to remove high Mr components, dialysed
against DPBS and concentrated over a PM10 membrane filter
(Mr 10,000 cut off). Both XM300 and PM10
ultra-filtration membranes were from Amicon Ltd. By
titration it was shown that the described MF activity was
35 increased at least 500 times over that in control,
~ .
- ~
~, " ,~ ' ' - ' ' ' '
!;. - -
unstimulated macrophage cultures. The culturedperitoneal cèlls were identi~ied as macrophages hy the
criteria of being adherent cells, with 99.6% showing a
positive reaction for lysozyme.
5 Example 2 Effect of Macrophage Supernatant on the
Viability of Thymus Cells
Thymus glands were removed from 4-8 week old
male and female rats, taking care to dissect the thymus
free from surrounding tissues including lymph nodes.
10 Thymuses were washed in RPMI-1640 containing lO~ FCS,
sliced and pressed through a 0.5 mm pore size stainless
steel wire mesh. The resulting thymic suspension was
fractionated on a gradient of Percoll according to a
described method (Salisbury et. al., 1979) The dense
15 fraction was then washed three times in RPMI-1640 with
10% FCS.
Purified dense thymic epithelial cells and
dense thymocytes were prepared by treating whole thymus
suspensions with antibody and complement prior to
20 fractionation on Percoll. Thymus cell suspensions
containing 1 x 108 cells in 5 ml RPMI-1640 were treated
; with monoclonal antibody (MRC OX52, a rat pan T cell and
thymocyte marker; Robinson et. al., 1986 for thymocytes,
or MRC OX6, directed against a common determinant on rat
25 Ia, obtained from Sera Labs, for Ia-positive cells) at
2.5 pg/ml final concentration, together with complement
at 2% final concentration. The preparations were
incubated for 1 hour at 37C and then layered on to
Percoll.
The isolated populations were washed and
characterized by indirect immunofluorescence and by
en~yme histochemistry. Results for 10 thymus
preparations are summarized in Table 1.
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~:: ~ ~ v~ 8 + + + ~ ~
c ,~e~ O O O I i ~ a~ > ~ O ~ o
_. ~ C o. V C~ ~ ~ C ~
+~ +I X _ . a ~ X +l +l +I J o ~ o o ~aP
_~ ~~ ~~
~V7 ~, C
o ~.~ _ ~ u ~ a
~ ~ , c aJ ~, C
n i _ ~ oO ~ o s o
_ - CJ _ X ~ X ~ o 1~
0 _ o.~ ~.~ ~ ~ V
a LL ^ ca ~_ o c
~1 0 3 ~ -
~ 'J_ -O E~Xnl
~ j o--C~ ~ . ~ V V
~ ~ O
C C ~',> C~ .
_ CJ ~O'_ ~ (J .. -.
:~ C~ ~CJ .~
~",~ ' .
Aliquots of DCF were counted, and the number of
viable cells (excluding trypan ~lue) was expressed as a
percentage o~ the starting population. The results shown
in Fig. 1 indicate a rapid loss of viability. This was
5 typically in the range of 40-70% of the starting
population after 4 hours incubation.
The addition of 50 ~l/ml of supernatant from
macrophage cultures which had been stimulated with 20 ~g
lipopolysaccharide/ml 2 hours previously protected the
10 dense cell fraction from loss of viability. Neither
lipopolysaccharide (from 1-20 ~g/ml) alone, nor
supernatant from unstimulated macrophages protected the
DCF. The supernatant from stimulated macrophages was
titrated, and was found to be protective at an end-point
15 dilution of 1/500. Approximately half of the peritoneal
cells which were seeded into culture flasks became
adherent macrophages. This gave 2.5 x 106 cells per
flask in 5 ml medium. Therefore 50 ~1 of supernatant
represents the release from 2.5 x 104 macrophages. This
20 equates to this number of macrophages releasing enough
activity to protect 500 x 107 dense thymic cells, or one
macrophage protecting 2 x 105 dense thymic cells.
The loss of viability in vitro was confined to
the dense, immature population and did not affect the
25 buoyant fraction or the thymic epithelial cells alone.
The protective effect was found in whole
supernatant, and in the fraction retained above the PM10
membrane. Although these membranes have a nominal cut
off of Mr 10,000, it was found that under the conditions
30 used, soybean trypsin inhibitor (Mr 23,000) was filtered
out while bovine serum albumin (Mr 67,000) was retained.
Repeated assessment of macrophage cultures by
examining at least 200 cells per culture showed that 99.6
+ 0.6% of the adherent cells were positive for lysozyme
35 by indirect immunofluorescence staining. The specificity
of the anti-rat lysozyme serum was demonstrated by a
.:
... ~ . . . .
.. . . - ~. - .
-: . .. . . . .
.. ,.;... . . . . .
- 15 -
single arc in immunoelectrophoresis against a rat
neutrophil lysate and again~t the purified protein used
for immunization. A control or second stage antibody
alone was negative. The cells in the cultures were 100%
5 positive for acid phosphatase, the reaction being
characterized by diffuse cytoplasmic staining of variable
intensity. For further studies a batch of fractionated
macrophage supernatant was prepared from the peritoneal
washings of 30 Wistar-Furth rats. This preparation (MF)
10 was used at a concentration equivalent to a 1/250
dilution of whole supernatant in subsequent protection
studies.
Example 3 Characterization of Monokine (MF)
(a) Physical properties.
The Mr of the monokine was determined by gel
filtration fractionation on Sephacryl S200. The results
are shown in Fig. 2a. MF eluted at Mr 36,000.
The isoelectric point of the MF was determined
by isoelectric focusing. Standard proteins and 100 ~ul of
20 MF were loaded on to a broad range (pH 3-9) LKB ampholine
gel and run to equilibrium. A standard curve was
prepared from the reference proteins as shown in Fig. 2b
and strips of the gel were cut at known intervals in
relation to this. The strips were homogenized, dialysed
25 against DPBS and tested. A second run was used to narrow
the range further. The activity was localized between pH
6.3 and 6.4.
The effect of heating was assessed by treating
aliquots of MF for 30 minutes at 40, 56, 60, 70 and 80C.
30 Activity was destroyed at 80C, but was stable at lower
temperatures. The effect of pH on the stability was
tested by adding MF to appropriate 0.1 mol/l buffers at
pH 2, 4, 6, 8 and 10 for 60 min at 37C. Samples were
~,-, :. ` ''
-- 16 --
then dialysed against DPE3S and tested for activity. MF
was stable over this pH range. MF activity was lost
following reduction with 5% 2-mercaptoethanol.
The e~fect of proteinases on MF activity was
5 assessed by adding MF to 10 }lg/ml solutions of enzyme for
60 minutes at 37C. The proteinases were papain,
pronase, thermolysin and trypsin, which were used in
RPMI-1640, pH 7.4, and pepsin in RPMI-1640 adjusted to pH
4.0 with 1 mol/l HCl. The enzyme-MF mixtures or enzyme
10 controls were then added to DCF in RPMI-1640 containing
10% FCS as a source of proteinase inhibitors.
Proteinases alone had no effect on thymocyte survival,
whereas all five proteinases destroyed MF activity. Thus
the monokine is at least partly protein in nature. The
15 results of these studies are summarized in Table 2.
TABLE 2
Physical Characteristics of Monokine.
Parameter Monokine
Molecular weight Mr 36,000
Isoelectric point - 6.3 - 6.4
Heat stability Stable at 70C, activity lost - --
at 80C ~.
25 pH stability Stable between pH 2 and 10 - -
Proteinases Activity destroyed by papain,
pepsin, pronase, thermolysin ;~ -
and trypsin
5% 2-Mercaptoethanol Destroys activity `~
30 8 mol/l urea Destroys activity
- -
- 17 -
(b) Functional Properties.
MF was te~ted for its ability to stimulate 3T3
fibroblasts to proliferate both in the presence and the
absence of FCS. Proliferation of fibroblasts was
5 expressed as the mean of quadruplicates of counts per
minute of [3H]-thymidine uptake by the cells. As
controls, fibroblasts alone and fibroblasts with whole
macrophage supernatant were tested. The results given in
Table 3 indicate that while the whole supernatant has
10 stimulating activity both in serum-containing and in
serum-free conditions, MF has none under either
condition.
TABLE 3
r
Effect of Macrophage Culture Supernatants
15 on Fibroblast Proliferation
Sample Serum-free 10% FCS
(cpm) (cpm)
20 Control (fibroblasts alone) 1010 + 991880 + 315
Whole macrophage supernatant 5075 + 4344305 + 1300
Partially purified macrophage 903 + 125 1586 + 588
supernatant (MF)
.
25 Example 4 Thymocyte Viability Assay
Dense thymus cell fractions prepared as
described in Example 2 were divided into one ml aliquots
containing 1 x 107 cells. Test supernatants were added
as 50 pl samples to these cells and viability was
30 assessed at hourly intervals by the exclusion of trypan
blue (0.4% solution). Viability was expressed as a
percentage of the original cell number.
'~ :
.
- 18 -
The titre of the activity was defined as the
reciprocal of the maximum dilution of unfractionated
macrophage supernatant which gave complete protection at
four hours in the thymocyte viability assay. This
5 applied to 100 ,ul of supernatant added to 1 x 107 cells
in a total volume of 1 ml.
Unfractionated thymus cell suspensions show no
decrease in cell viability with time. However, depletion
of macrophages, e.g. by adherence to plastic, results in
10 apoptotic death of immature thymocytes in the suspension.
This was prevented by addition of the Mr 36,000 monokine.
Surprisingly, if the thymocytes were first
treated with corticosterone, cell death was increased.
The monokine evidently induces differentiation in a
15 population of Ia+ prothymocytes, which thereby become
corticosterone-sensitive. This is supported by results
described below with Con A.
Example 5 Further Characterisation of Synthesis and -
Release of the Monokine.
The time course of production for the factor, --
together with the effect of different stimuli at various -- -~
doses and at various times was examined. In addition, ~ -
the nature of the production of the macrophage factor was
studied using three different protein synthesis
25 inhibitors and trypsin treated and untreated lysed
macrophage preparations.
(a) Macrophages were prepared as in Example 1, and
were stimulated with either LPS (1-20 ug/ml) or MDP
(0.5-2.5 ~ug/ml). Supernatants were collected at various ---~
30 time intervals between five minutes and forty-eight hours -
according to the experiment. In the case of the
unstimulated macrophage supernatant, an identical
procedure was followed without the addition of the
stimulant, and the supernatant was collected two hours
35 after the replacement of serum-free medium.
~ ' - :
~.;.: ,.. - , ., .. ,, .- - :, ., - . : - - - -
-- 19 --
Varying concentrations of LPS and MDP were
added to freshly established peritoneal macrophage
cultures, and the supernatant was collected after two,
eight or twenty-four hours incubation and tested for
5 activity in the thymocyte viability assay. The results
are shown in Figure 3. For LPS, there was a
proportionate increase in titre from 1 to 10 ~g LPS and
then an abrupt increase from ten units at 10 ,ug LPS to
500 units at 20 ,ug LPS. There was no demonstrable
10 activity at eight or at twenty-four hours after the
addition of LPS. The response to MDP was quite
different in both the titre achieved and in the
persistence of the response, so that for 2.5 yg MDP,
although the titre was only one unit, it persisted for at
15 least twenty-four hours. Controls were set up to test
the effect of the stimulants alone or in combination with
the partially purified macrophage factor. Neither
stimulant protected the thymocytes or affected the
activity of the macrophage factor.
20 (b) The time course for the release of the
macrophage factor was examined using 20 ,ug/ml LPS as the
stimulant. As shown in Figure 4, low level activity was --~
released from the macrophages within five minutes after
the addition of the stimulant. After one hour the
25 activity had increased ten-fold, with the peak response --
of 500 units being reached at two hours. The activity
decreased to 100 units at three hours, while from four
hours to forty-eight hours there was no demonstrable
activity. The yield as assessed by the number of units
30 of protective activity in the thymocyte viability assay
is greatest in response to 20 ~g/ml LPS when the
supernatant is collected two hours after the cells are
challenged. Increasing the culture time beyond two hours
led to a decrease in the number of units of activity at
35 three hours and a loss of protective activity beyond that
time and up to forty-eight hours.
,.~
- 20 -
(c) Effect of Inhibitors of Protein Synthesis.
Macrophage supernatants produced in the
presence of inhibitors of protei~ synthesis were prepared
as follows: the macrophages were allowed to adhere and
S were then washed to remove any traces of serum. The
protein synthesis inhibitors were used in the following
concentrations: actinomycin D 1 ~g/ml, puromycin 100
~ug/ml, cycloheximide 10 ~g/ml.
Inhibitors were added to macrophage cultures at
10 the same time as 20 ~ug LPS and the supernatants were
collected at time intervals up to two hours. They were
dialysed to remove low molecular weight inhibitors and
then tested for protective activity in the thymocyte
viability assay. The results shown in Figure 5 show that
15 in the presence of each of the three protein synthesis
inhibitors, protective aetivity was demonstrable in the
culture supernatants at five and ten minutes after LPS
challenge. By fifteen minutes, however, activity had
been lost in the puromycin and cycloheximide treated
20 cultures, whereas it was still present in the cultures
which had been incubated with actinomycin D. From thirty -
minutes to two hours, activity was lost in all of the
treated cultures. -~
The early release of low level activity from -~- -
25 five minutes onwards suggested that the factor was
already present within the macrophages and was released.
In addition it was shown that lysates of unstimulated
cells also contained protective activity, although this
activity was not titratable. Addition of the protein - -~
30 synthesis inhibitors, actinomycin, puromycin and
cycloheximide to cultures at the same time as the LPS
demonstrated that de novo protein synthesis was necessary
for production of the factor. In contrast to cultures -
with LPS alone, protective activity could not be detected
35 beyond ten minutes with puromycin and cycloheximide or
after fifteen minutes with actinomycin. Hydrolysis of
IP
~-"v .. ~ " . ,, . : ,,
- 21 -
surface proteins by trypsinization prior to washing or by
lysis of thè cells failed to remove the protective
activity, indicating that the factor was not a surface
protein on the macrophage, but wa9 probably cytosolic.
5 These results are shown in Figure 6.
Example 6 Effect of Restimulation of Macrophage
Cultures
Restimulation of cultures with LPS was examined
to see whether yield of the monokine could be increased.
In addition, the abrupt decrease in titre from
a peak at two hours after stimulation to no detectable
protective effect at four hours was studied by examining
the effects of restimulation with LPS at two, four and
six hours of culture.
The abrupt decrease in titre of protective
activity after 2 hours could have been due to
prostaglandin production by the macrophages. The
spontaneous production of prostaglandin E2 by thymic
phagocytes in culture is greatly enhanced by adding LPS
20 to the cultures; the prostaglandin response to LPS is
reduced 10-fold by indomethacin (Papiernik and
Homo-Delarche, 1983).
We found a further peak of protective activity
when 10 5M indomethacin was added to cultures at 4 hours
25 together with LPS and new medium, but not when cultures
; were restimulated in the absence of indomethacin.
-~; Supernatants were taken after two hours from
cultures stimulated with 20,ug/ml LPS. At this time, the
cultures were restimulated with 20 ~g/ml LPS alone or in
30 combination with 10 5M indomethacin. The supernatants
were collected at four hours and the cultures were
restimulated. This procedure was repeated at six hours
and the final supernatants were collected at eight hours.
~'
,
.,", . ~
~'s.:; . .. :
... ~ .
The results are shown in Figure 7, and indicate
that macrophages were anergic to further stimulation. In
this context, it was noted that 10 nM of prostaglandin
E2, added together with LPS at 20 ~g/ml to fresh
5 macrophage cultures, totally removed the activity at two
hours. Indomethacin (10 5M), added together with LPS to
fresh cultures, did not alter the peak titre at two
hours; however, in the presence of this inhibitor of
prostaglandin synthesis macrophages which were
10 restimulated at four hours did release low titre activity
at six hours of incubation. There was no effect of
indomethacin, however, on restimulation at two hours or
at six hours.
Example 7 Partial Purification of the Monokine
Macrophage supernatant was prepared as in
Example 1, and filtered through an XM 300 membrane, to -~
remove the bulk of the lipopolysaccharide, and then
through a PM 10 membrane. The fraction above the ~ ~-
membrane was retained and sterile filtered as described. -~
20 The molecular weight was determined by gel filtration
fractionation on Sephacryl S200, and the activity was -
localized to an Mr 36,000 fraction (Yield = 80% of loaded ~
sample). The macrophage factor was further purified by - -
ion exchange chromatography on DEAE Sephacel (Pharmacia). -~
25 The active fraction (Sml) from Sephacryl S200 was loaded ~
onto a DEAE Sephacel column (1.6 x 60 cm), and the ~ -
activity eluted from this column in 2 x 2 ml fractions at - -
120-124 ml after a gradient of 200 mls of 0.05 M Tris (pH
8.0) + 200 mls of the same buffer with 0.5M NaCl was
30 applied (Yield = 50% of loaded sample). The active --
fractions were pooled and sterile filtered, and half of
the active fraction was dialysed against lM (NH4)2S04 at
pH 7.0 in preparation for chromatography on
Phenyl-Sepharose CL-4B (Pharmacia). A 7 ml sample was
35 applied to 10 mls of Phenyl-Sepharose in a 0.9 cm
diameter column with adaptor, the matrix having been
equilibrated with lM (NH4)2S04, tpH 7.0). The flow rate
was 20 ml/hour and a 100 ml gradient of lM ~NH4)2S04, (pH
7.0) + 100 mls H20 was applied immediately after the
5 addition of the sample and 2 ml fractions were collected.
The activity was recovered in 2 fractions, which
represented 2~ of the total gradient (Yield = 27% of
loaded sample).
Example 8 Receptor Site for the Monokine.
During the course of optimizing procedures for
the depletion of Ia-positive cells it was observed that
MRC OX6, in the absence of complement, promoted thymocyte
survival (Figure 8a), thus mimicking the effect of MF.
As expected, this antibody had no effect on the survival
15 of the Ia-negative DCF. When incubated with isolated
thymic epithelial cells at 1.2 x 106 per ml, it promoted
the release of thymocyte survival activity. In contrast,
MRC OX3, which recognizes a rat strain-specific epitope
on Ia, failed to protect.
The possibility that MF also bound Ia was
probed initially by attempting to deplete MF activity by
absorption with different types of cells, as outlined
earlier. MF was absorbed at 0C, using 3 cycles of --
incubation for 1 hour with 1 x 107 cells. As shown in
25 Figure 8b, the DTEC effectively removed the MF activity.
Spleen cells also removed the activity, whereas spleen
cells depleted of Ia-positive cells did not. RBC were
also ineffective. Similarly, MF was absorbed on to
spleen cells which had previously been incubated at 0C
30 with MRC OX6 or MRC OX3, and the supernatant was tested
for protective activity. In order to negate the
possibility of carry over of MRC OX6 into the viability
assay, the antibody was labelled with 125I. The specific
activity of the dialysed product was 2.09 x 105
35 disintegrations/min per ~g protein. No carry-over of
~5"` ". ". ,' ., ' ~ " , ,
,'', . ' " ' ~ ' ' ' ' , " ' . '
':' . " . . '', ' ' :
''.',"'.' :" ',', ' ,
- 24 -
radioactivity was detected. The results shown in Figure
8c indicate that prior binding of MRC OX3 did not prevent
absorption of MF, whereas prior binding of MRC OX6 did
so. This provided evidence that MF bound to surface Ia
5 at a site which was the same as or nearby the site
recognized by MRC OX6. The finding that MRC OX3 did not
block absorption of MF mitigated against an
intermolecular steric blocking of MF by MRC OX6. The
binding site of the MF was further examined by studying
10 the competitive effect of univalent Fab MRC OX6
antibodies prepared as described. Fab OX6 at 2.5 ~g/ml
failed to protect the DCF in the thymocyte viability
assay. Pre-incubation of the DCF with this Fab antibody ~
preparation for 15 min. at 37C did, however, effectively -;
15 block the protective action of MF, indicating a close -~
relationship between the binding site of the antibody and
the macrophage product (Figure 9).
':,: " ,~
Example 9 Mediation of the Protective Effect Via
Thymic Epithelial Cells
The DCF was depleted of Ia-positive cells by
treatment with MRC OX6 and complement as described -~
earlier. Similarly, DTEC were prepared by lysis of -~
thymocytes using MRC OX52 and complement. Cell ~-
populations were characterized as described in Table 1. ~-~
There was good agreement between the number of
cells positive for keratin and those remaining after -
lysis of thymocytes in the DCF. The presence of
macrophages and non-epithelial dendritic cells in this
fraction was excluded on the basis of the buoyant nature ~ -
~30 of these cells and that all of the dense cells were
accounted for by either anti-keratin staining of
epithelial cells (12.8%) or MRC OX52 staining of -
thymocytes (87.2%). In addition, none of the cells in
this fraction gave a positive reaction for lysozyme or
35 for acid phosphatase. Furthermore, the purified
:- '. -
~i~ ' '
:
- 25 -
epithelial cells appeared to be a uniform population by
electron microscopy. The DTEC maintained viability over
24 hours in culture while the dense Ia-negative
thymocytes showed 100% mortality over this time period.
5 Aliquots of MF (50 ~1) failed to protect 1 x 107
thymocytes depleted of Ia-positive cells (Figure 2).,
The incubation of 50,ul MF with 1.2 x 106 DTEC (in
proportion to the percentage in the DCF) did, however,
result in the release of protective activity by these
10 cells (Figure 10).
Thus removal of the Ia-positive cells in the
DCF left a population which displayed the described loss
of viability over 4 hours. The addition of macrophage
supernatant failed to prevent cell death, as did the
15 addition of supernatant from unstimulated thymic
epithelial cells. The addition of supernatant from
thymic epithelial cells which had previously been
incubated with MF was protective, suggesting that a
cascade was occurring in which macrophage products were
20 acting on thymic epithelial cells, resulting in the
release of a factor responsible for the survival of
thymocytes.
Example 10 Production of the Thymic Epithelial Cell
- Factor
The thymic epithelial cell factor was induced
with either the macrophage factor or an antibody to a
common determinant on Ia, as follows: Whole thymus cell
suspensions were treated with MRC OX52 and complement for
thirty minutes at 37C. The remaining cells were then
30 fractionated on Percoll and the dense cell fraction which
contained keratin-positive thymic epithelial cells was
collected. These cells were divided into three aliquots
and were cultured in tissue culture flasks at a density
of 1 x 108 cells/ml in RPMI 1640 medium. The first
35 aliquot served as a control, the second aliquot was
~
.
~,, . ~ .
.- .. ~
"
; - 26 -
stimulated with 2.5 pg/ml of MRC OX6 and the remaining
suspension was stimulated with the macrophage factor ~at
dilution equivalent to 1/250 of the original macrophage
supernatant). The cultures were incubated for three
5 hours, after which the supernatants were collected. The
resultant supernatants were tested in the thymocyte
viability assay using a dense cell fraction depleted of
Ia positive cells. The macrophage-induced thymic
epithelial cell factor (MF-TECF) was active at a
10 concentration of 1/5000 and the antibody-induced -
epithelial cell factor ~MRC OX6-TECF) protected the
thymocytes at a dilution of 1/750 in the thymocyte
viability assay. The control supernatant from
unstimulated thymic epithelial cells was inactive in the
15 thymocyte viability assay.
.,. ~,. . .
Example 11 Properties of the Thymic Epithelial Cell - ;
Factor
The protective activity of the macrophage
factor-thymic epithelial cell factor was destroyed by
20 incubating the factor (at a ljlO00 dilution) with 1 mg/ml -
of trypsin. Activity was also destroyed by boiling the
factor prior to testing it in the thymocyte viability -~
assay, suggesting that it was probably protein. ~ -
The molecular weights of both MF-TECF and MRC
25 OX6-TECF were determined by gel filtration fractionation
on Sepharose CL-6B (Pharmacia). The results are shown in
Figure 11. A column (100 cm x 2.6 cm) was calibrated -
with ferritin (Mr 440,000), aldolase (Mr 158,000),
albumin (Mr 68,000~ and ovalbumin (MR 43,000) in PBS with
30 0.1 M NaCl and was run with a 1 ml sample under total
identical conditions. The MF-TECF eluted at a molecular
weight of 320,000 and the MRC OX6-TECF eluted at 760,000.
A possible binding site for the MF-TECF was
suggested by experiments using a monoclonal antibody to
35 rat T helper cells which bound at CD4 (Sera Lab, Clone
W3/25). Whole thymus suspensions were incubated with
this antibody, Ia positive cells were removed and the
remaining cells were fractionated on Percoll for use in
the thymocyte viability assay. The addition of MF-TECF
5 to this dense cell fraction failed to protect the
thymocytes, suggesting that the availability of CD4
molecules was important in this process.
Example 12 Characteristics of the Release of the Thymic
Epithelial Cell Factor.
The MF-TECF was prepared as previously
described but supernatants were collected from cultures
at various intervals between five minutes and three hours
and tested in the thymocyte viability assay. As shown in
Figure 12, low titres of activity are produced in the
15 first fifteen minutes following which the titre increases
steadily up to three hours. The addition of one of three
protein synthesis inhibitors, actinomycin D, -
cycloheximide or puromycin had no effect on the
production of MF-TECF, indicating that protein synthesis
20 was not required.
These experiments suggest that the thymic
epithelial cell factor, when induced by the macrophage
factor, has a molecular weight of around 320,000.
Induction of the thymic epithelial cell factor with MRC
25 OX6 (the antibody to a common determinant on Ia), results
in the release of a protein of molecular weight 760,000.
The large molecular weight of the protein together with
the fact that the molecular weight of the product varied
according to the method by which it was induced,
30 suggested that the thymic epithelial cell factor might in
fact be an Ia-ligand complex that is shed from the thymic
epithelial cell. If this does occur, then the selection
of thymocytes for which apoptosis is arrested could be
based on their ability to bind the Ia-ligand complqx that
35 is shed. This appears to be related to the expression of
,. . . .
:G' ., ~ ~
,,. , : .: ': !
- 28 -
CD4 by the thymocytes since pre-incubation of immature
thymocytes with anti-CD4 antibodies inhibits the ob~erved
protective efect of the monokine-induced thymic
epithelial cell factor.
It was not possible to absorb the monokine with
the monoclonal antibody W3/25 to CD4, indicating that it
is unlikely that this macrophage product is soluble CD4.
Example 13 Stimulation of Thymocyte Maturation by the
Monokine.
The immature thymocytes in the dense cell
fraction do not proliferate in response to Con A. The
effect of the monokine on their ability to respond to Con
A was therefore tested.
The dense thymic fraction was incubated at 1 x
15 106 cells per well with 2.5 ,ug of MRC OX6 or MRC OX3.
The proliferative response to Con A was measured. While
MRC OX3 had only a slight effect, there was a
substantial increase in proliferation with both MRC OX6
and 2 hours whole macrophage supernatant, and an additive
20 effect when the two treatments were combined, as shown in
Figure 13. This trend was reproduced over eight
experiments.
Incubation of the dense thymic cell fraction
with partially purified macrophage supernatant (Mr 36,000
25 fraction from chromatography on Sephacryl S200) also
resulted in an increase in the proliferative response to
Con A. This fraction was used at a concentration which
demonstrated protective activity in the thymocyte
viability assay, and three different preparations were
30 tested. All preparations showed a similar increase in
proliferation, as shown in Figure 14, and the effect was
reproduced over five experiments.
The depletion of adherent cells from whole
thymus suspensions resulted in a decrease in thymocyte
35 proliferation in response to varying levels o~ Con A and
'
- 29 -
0.5 ~g o~ LPS compared with a control population. The
mean o~ the ratios of the control population to the
depleted population was 2.27 + 0.6. The addition to each
well of 25 ~1 of whole macrophage supernatant (prepared
5 from 1 x 106 cells stimulated with 20 ,ug/ml LPS and
harvested at 2 hours) restored the response to that of
the control. The purity of the macrophage cultures was
confirmed by staining for lysozyme and acid phosphatase.
The means of the ratio of the control response to the
10 restored response was 1.0 + 0.07.
The restoration of the response with macrophage
supernatant did not appear to be due to IL-l.
Supernatant treated with 10 mmol/l phenylglyoxal to
remove IL-l activity was still capable of restoring the
15 response to control levels. Similarly, the response was
restored by the addition of Mr 36,000 factor from
chromatography on Sephacryl S200, at a concentration
which was active in the thymocyte viability assay. These
results are shown in Figure 15.
In other experiments it was found that 0.5 ~g
LPS increased thymidine uptake in response to a range of
concentrations of Con A, but that the shape of the
dose-response profile was unchanged. Similar results
were obtained with MDP (1 ,ug/106 cells) or streptococcal
25 cell wall fragments (0.5 ,ug/106 celIs). Conversely, in
the presence of a constant amount of Con A (0.25
~ug/well), 1 pg of LPS sharply stimulated thymidine
uptake, with gradual further increase up to 1 mg LPS.
Again, similar results were obtained using MDP or
30 streptococcal cell wall fragments.
Fractionation of the thymus cell suspension on
a density gradient of Percoll confirmed that the buoyant
fraction which contained mature thymocytes, macrophages,
epithelial cells and non-epithelial dendritic cells
35 proliferated in response to Con A and 0.5 ~g of LPS. The
dense fraction containing immature thymocytes and dense
~i~
r",',' ..
~ .
~~. ' ' " , .
'
., ' '~ .
- 30 -
epithelial cells responded only weakly to Con A and LPS.
Recombination of the two fractions restored the response
to a level equivalent to that of whole thymus, showing
that cells had not been lost in the separation procedure.
5 Figure 16 shows the response of the buoyant, dense,
recombined and whole fractions to Con A and LPS.
These results indicate that macrophage products
are responsible for the increased proliferative responses
of thymocytes to co-stimulation with Con A and microbial -~
10 products. The data support the efficient uptake of
picogram quantities of a variety of bacterial components
by macrophages, which comprised less than 0.1~ of the
thymic cell suspension. The evidence for the r~le for
macrophages in the augmentation of the proliferative
15 response was provided by a diminished response following
depletion of adherent cells, and by the restoration of
the activity by products from highly purified peritoneal
macrophages. The active macrophage component was shown
to be the Mr 36,000 protein, which did not have IL-l
20 activity as determined by a fibroblast proliferation
assay.
. .
Example 14 Effects of the Monokine in vivo.
It is known that the cytokines IL-l, TNF, and -~-
interleukin-6 can induce liver epithelial cells to
25 secrete acute-phase plasma proteins. Macrophages are a
~; major source of these cytokines. ~ -
A single intravenous injection of the -~ -
partially-purified Mr 36,000 protein into rats induced a
significant elevation of plàsma fibrinogen and
; 30 ~2-macroglobulin levels.An injection of heated protein -`
caused no response.
The amount of protein injected was equivalent
;; to that released by 1 x 105 macrophages. This small
amount of monokine produces a substantial effect in the
- -.:: '
`i
- 31 -
animal by a cascade phenomenon whereby responding
macrophages secrete one or more cytokines, which in turn
activate the acute phase response.
Example 15 An Automated Bioassay for the Monokine.
In order to monitor large numbers of samples
conveniently, an assay which can be carried out in
microtitre plates and in which many samples can read
simultaneously is desirable.
We have developed an assay based on the
10 conversion of a soluble, yellow compound ~MTT) to an
insoluble blue formazan by enzymes in living cells. The
insoluble product is dissolved in isopropanol, and
estimated spectrophotometrically. The assay can be
carried out in 96 well microtitre plates of the type used
15 for ELISA assays.
Two complementary variations in technique were
used. The assay is based on the cleavage of the
tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyl tetrazolium bromide) into a blue
20 coloured product (formazan) by the mitochondrial enzyme
succinate dehydrogenase (Denizot and Lang, 1986). The
conversion takes place only in living cells, and the
amount of formazan produced is proportional to the number
of cells present.
Thymic suspensions were either fractionated on
Percoll as described earlier, or depleted of adherent
cells by incubating 1 x 108 thymic cells in Sml RPMI 1640
medium in a 75 cm2 tissue culture flask for one hour at
37C. This depletes the suspension of thymic macrophages
30 and destabilizes the population so that the immature,
dense thymocytes undergo apoptotic death.
Using the dense ce-ll fraction from Percoll
gradient separation, cells were washed into RPMI 1640
medium, without serum or phenol red, at a density of 2 x
35 107 per 0.9 ml. Dilutions of macrophage supernatant or
..,~
.,, ~ ,
:
- 32 -
of the fractions containing monokine activity were added
at 100 ~ul/2 x 107 cells. The cells were then aliquoted
in replicates of eight into flat bottom 96 well culture
plates at 2 x 106 cells/100 ,ul. The effective range is
5 0.5 x 106 to 5 x 106 cells/well but 2 x 106 cells are
preferred. The final row of cells received cycloheximide
at 10 ,ug/ml instead of macrophage products. This arrests
the protein synthesis necessary for apoptotic death, and
provides a positive control at 100% viability. The
10 plates were incubated at 37 for three hours and then MTT
was added as 50 ,ul of a 1 mg/ml solution in RPMI 1640
medium. After one hour of incubation at 37C, the plates
were centrifuged at 800 x g for 10 minutes and the
untransformed MTT was removed by inverting, flicking and
15 blotting the tray. Two hundred microlitres of
isopropanol was then added to each well, the plates
briefly shaken and the formazan read at 540 or 560 nm
(preferred) test wavelength against a 690 nm reference
wavelength in an ELISA reader.
Formazan generation in test samples was read
with reference to both cycloheximide-treated positive
controls and unprotected controls. The conditions for
using adherent cell-depleted whole thymic populations
were identical, except that the incubation step was for
25 seven hours or sixteen hours (preferably seven) before
adding the MTT.
The assay using fractionated dense thymic cells
involves rapid apoptotic death and therefore a short
incubation period while the assay using depleted adherent
30 cells involves a slower rate of death and longer
incubation time. It does, however, have the advantage
that it is easy to establish, and is applicable to -~;-
screening large number of samples. ^`
- 33 -
Example 16 Distinction Between the Monokine and
Previously known Macrophage-derived
Factors.
The characteristlcs of IL-l and TNF in the rat
5 have been described ("Lovett et al, 1983; Schmitt et al,
1986; Vilcek et al. 1986; Rupp et al. 1986).
The failure of the monokine of this invention
to stimulate fibroblast proliferation in either the
presence or absence of serum (Example 3(b)) suggests that
10 it is neither IL-l nor TNF. This is supported by the
homogeneity of the factor with respect to both Mr and
isoelectric point; both IL-l and TNF have a number of
molecular forms (Smith et al., 1986; Wood et al. 1985).
Phenylglyoxal is an inhibitor of IL-l activity
15 (Klamfeldt, 1985; Krakauer, 1985). Whole macrophage
supernatant from cultures incubated for 2 hours with 20
,ug LPS/ml were treated with 10 mmol phenylglyoxal/ml.
This treated supernatant was able to restore the response
of adherent cell-depleted thymus cell cultures to Con A,
20 as shown in Figure 15.
The time course for the production of this
factor differs from that of IL-l or lymphocyte activating
factor in mice, humans and rats. Mizel (1981) showed
that mouse peritoneal macrophages stimulated with phorbol
25 myristate acetate reach a peak level of production of
IL-l around twenty-four hours, and that this then
plateaus. Similarly, Wood e~ al, (1983) collected
supernatants from a Balb/c macrophage line, 2-3 days
after the cells were stimulated with LPS from Escherichia
30 coli in order to harvest lymphocyte activating factor for
testing in the thymocyte proliferation assay. Human
monocytes and macrophages stimulated with LPS show a
maximum yield of lymphocyte activating factor at
twenty-four hours with very little production prior to
35 two hours as assessed by the ability of the supernatants
to augment proliferation of mouse thymocytes in response
.,, ~, . -
~" .
- 34 -
to phytohaemagglutinin (Treves et al. 1983). Bird et al.
(198S) prepared IL-1 from Sprague-Dawley rat peritoneal
macrophages by culturing the cells for forty-eight hours
in the presence of 10 Jug/ml LPS. Examination of the time
5 requirement for TNF production by LPS induction of human
monocytes showed that cytotoxicity reached plateau values
within two hours (Kornbluth and Edgington, 1986). The
monocytes, however, need to be primed two to three days
prior to challenging with LPS, with an agent such as
10 tubercle bacilli (Carswell, 1975; Mestan, 1986). This
was not necessary for the early production of the factor
of the present invention.
While it is recognized that the macrophages
will produce a large number of soluble factors in
15 response to challenge with LPS, the early release
together with the abrupt decrease in production at two
hours of this factor would appear to be different from
other well recognised monokines.
::
In summary, the monokine of this invention acts
20 on thymic epithelial cells to cause the release of a
second factor, thought to be a complex between the
monokine and Ia antigen, which in turn promotes the
survival and differentiation of immature thymocytes.
The monokine would be useful in:
~ -,- ,. . .
(a) Stimulating the maturation of the immune -~
system e.g. in children with congenital or acquired ---
immune deficiency; AIDS patients; patients being treated -~
with radiotherapy or cytotoxic drugs;
(b) Selective stimulation of immunity;
(c) Selective suppression of inflammation or ;
autoimmunity.
It will be clearly understood that the
invention in its general aspects is not limited to the ~-
specific details referred to hereinabove.
~ .
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7. Dinarello, C.A. (1984). Interleukin-l. Rev.
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~.'
: . ~ ,, :
,,~ - .
, . ~ . .
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~ -chain genes in the thymus; implications for the
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