A
30LL31~
?AJl0~
IJJUJEJ 11Th
Honors rhesis
\;'Uarter, 198J.
Undergr~duate
3prin~
IVilli9..:: .-\.. Harter
Ball 3t2.te Uni versi ty
~,=tmcie, Indi2.na
Thesis Director
-
SUMMAR.Y:
Tests were perrurmed in search or a soluble, tumor cytoto:<ic factor produced by ;nacrophE-ges.
lOOi bi ti,)rl ')1'
t'I.A.I!lOr gro'",th was also investig'3.ted.
Peri ton'2?.l macrophe.g'::'s
fro.a C57Bl/6 mice were cultured with lipo901ysaccrJ.<?ri,:':?s (LP.3)
0r ir:dcmethacin, a.nd the resulting s'I.A.per:lE:. tan t s were pi tted
agg,inst 1e''Vis lung carcinoma (1LC) radioactively labe 11ed 'Ni th
either JH-thymidine OI' ~'Cr.
TritiQ~ tests for~ell ,sr'),';t':l
ended inconc 1 usi vel v but hi:l ted tna t ir.done thacin aids inh i cit)r(s) in:(1erent in ~acroph;-J.ge3.
5'1 Cr tests
for cytutoxiClty
srlowed that indometh<3.cin promotes t-'lJ!lOr de:.l th but acts very
briefly; 1PS also promoted, but later and to a lesser degree.
A brief, ele~.er:tar.f review is included.
Sver since the discoverYJf ;nacrophages, science h2.S mONn
t~at
~acrophages
tis3ue.
are capable of ingesting or killing
cancero~s
Indeed, it is preCisely this talent that cause.i their
discovery (12).
Curren.tly, efforts to find or create 30:11e
means of freedom
fro~
phage
func·~ions.
'rhe function ·Jf tumorcytotoxici ty follows
several pathways, the
and
~ttack;
mechanism.
mor cell s
I
cancer have accelerated stUdies on macro-
s~~ll
siill~lest
of which perhaps is recognition
explain later in more detail
~bout
the
contact between a macrophage and qnj number of tu1:1:"lS
l:J.r mode of
been impl ic:J. ted to be required for thi s particu-
~illing
(3).
Both tlleory and data allow that
3.
toxic substance (s) is secreted by the :nacropnage to effect
killing.
:::Vidence demonstr:3. tes that tnis sUQstance might be
available for extraction.
Sxperiments in neighboring fields
suggest prostaglandins could have a relationship with this
SUbstance.
I have worked on three sets of experiments, looking for the
soluble factor mentioned.
Two of these tests are specific:
tumor growth inhibition and tumor death.
-
Radioactively label-
led Lewis lung carcinoma (LLC) cells were incubated with supernatants from peritoneal macrophages cultured in media with
added activators and inhibitors.
Macrophages are diverse cells.
immQ~e
systems in several forms.
They function in
mam.mali~_n
They are a part of antibody-
depende:-i t <::ell-media ted cytotoxici ty (ADGC), immediate anaphylactic rea<::tions, delayed-type hypersensitivity reactions, and
natural cytotoxicity.
modes of a(:tivity:
This means that macrophages have a few
phagocytosis, antibody-dependent recogni-
tion of target cells, and antibody-free rece
cells.
-~ition
of target
Antibody-free abilities are poorly u..."1derstood (3d).
"The most prominent functional property of the macrophage is
its ability to recognize foreign or damaged material," (38).
In fact, it is this independent defense that began my set of
experiments.
A detailed recollection of macrophage structure or capabilities would be superfluous to include here, so I shall attempt
to include a relatively few, helpful descriptions.
Mature
macrophage:3 usually do not replicate, and can live for several
months in humans.
Their plasma membranes contain receptors
for the Fc portion of antibody molecules and for some types of
complement (38).
The surface of the cell changes depending on
the most recent origin.
Some surface structures allow the,:. to
adhere to containers (plastics, glass) as they do to non-self
materiel (.30).
In some marmer, macrophages bound to twnor
cells destroy the tissue, most likely by cellular secretions,
though the mode is unclear (13).
chemicals:
Macrophages secrete many
hydrogen peroxide, argenine, complement (3), neu-
tral proteases (16), and many others not
here.
n~cessary
for mention
(5).
Secretion varies inversely with macrophage growth
For our purposes concerning tumor cytotoxicity, there are
three categories of secretions:
toxic chemicals, tumor growth
inhibitors, ~nd DNA precursor inhibitors (13).*
are inhibited, however, by PBS in vitro (3).
,-
Secretions
Activated macro-
phages secrete a yet-elusive sUbstance of about 45,000 daltons
molecular weight (5,15) which might be my killing mechanism.
*~. prevention of incorporation of uri dine or labelled
ioaodeoxyuridine (21,28).
2
las
1-
One common explanation for the mechanism of tumor cytotoxicity by maerophages without antibody mediators requires contact between both cells.
It has been proposed that a great
deal of aceommodation is offered by the target surface.
are two levels of
attacr~€nt,
There
based on the strength of their
membrane-membrane bond (3).
There is no proof that these two
levels are related to stages of ma:·ropb.a.ge activation.
'rhe
lesser level might be involved only in cellular recognition.
In both
instanc~s,
contact is not flush; there is a narrow
pocket formed, not unlike placing two craters lip to lip_
This pocket is approximately 100
is tighter.
A wide,
narrower if contact
It is assumed, then, that cytotoxic chemicals are
released here as a conservative practice (3).
Research is
presently underway to find more about this particular process.
Macrophages are only moderately Pllocrnacious; their bell igerence must be bought with many signals both inside and outside
a host's immunologic systems.
Once activated, they can kill
or restrain foreign tissue (25).
(1,)2).
Activation has many stages
Resident macrophages have been given no stimulus of
any kind, much like "native" cells.
Macrophages can be elici-
ted to an area by chemoattractants and irritants (38), but
activation has not yet been accomplished.
There are at least
two levels of activation, primed and complete, depending on
their ability to destroy tumor cells (32).
This process 4ight
be due to differentiation rather than ttmodula tions" (2).
Acti-
vated 'macrophages have ruffled plasma membranes, phagocytize
better, adhere better, have more endocytic vessels, and as a
rule usurp inflammed regions from resident macrophages (27).
They are, then, super macrophages.
Activation of macrophages can be caused in vivo by infection (4,19,38), in vitro by LPS (2,15), lymphokines to an extent (2,13), double-stranded RNA (13), phytohemagglutinin from
other macrophages stimulated with ConA (1), calcium ionophore
A23187 (15), and endotoxins (1,3,4,6).
Activity is lost in
vitro after 24 hrs. (31) but can be maintained for several
-
iays
~i~h
re~2~~ed
co~sta~t
or
~ntigen
addition of
( 13)
-
pilY-
"
tohemagglutinin (31), lymp~okines (2), Iore andotoxins (3),
~ore
LFS (32) •••• thos~ chemicals
th~t
somehow
~he
~or~
ac~i-
to
Go:-r.ITIon expl"',TI2. tion
for the ::;-r:o,Qu:-l loss of activity is th::1.t 3,ctivc'.ted ='lcrO:::;!::':.'s-es
Dro(iu~e ,.?
gro'-,.p of chemi -;', 'ls n,.,:r:ed pro2tc:g12,ndins
(:2(~)
,::t.i Cf'c
}::'e sec'::'eted to res:.ll'3.te ths':'1.:',elves 'oy i::--i"doitlr:.g th~=ir own
',.
(-,"')
actlVlTr12S
~J,~-.
,1ithin 24 hrs., :::acropf':.c'g,;,s cultured in
,.
vitro
r!la~·:e
::'.1:
'.:.ccw!:1J.l~,tion
of
?rostagl?n,~.ins
levels; t~erefore, activity is shut off
u9 to e:fective
(35,37,39).
Frost::=.;L-:.ndi:'lsJ..re 'lliphatic aciis, and .:me of t:::eir
f'...trlC-
t i:)ns--:l£c c (' o~~i.:. ',.::e re;ula t;ion--fl.(:'~s alre'::.d,iGeen ,Herl tiJne d (.3 3) •
i:3 tI~c::_t PG~2 is prev3.lent in cancerous tissues (3).
If tfEn
:1.
tl.<.m,ar can :2ar:i)ulateJ.. !:lost' siefenses into aeing £'Jole:1 ':",:::d
into being disordered, the jrowth has
~
good ch2nce at surviv'llhis voltu:J.e of
~
FIT2:,..., must 'oe
inGre::-.s~l
11)C(-£Jld
3..:::2.l.::"'cS~
_
lV:::::Jhokine :lctive..tion
u
_
(2) • .\.lthough it _~1ig:C~t not aLv'?ys be sis:.'1ificant in vivo (-to),
lik~
in vi tr£ s i;udies
logic processes
~ni
-:;hi ~ ?id our und'9!"standing of i:n.'TIuno-
~ould ~id
in findin;
~or~
options for
cancer ther8.T)y.
',rherelre :.':.?-.ny
subs~
?nce s :mo',';n to ':Je toxic to tumor cells,
:lnd I shall list a Ia-;; whicD.
UOina (LLG).*
.~,l~8
~l~,ru~"'_ging
to Le'.vis L,mg carci-
.-i.d.riamyacin iYl_hioi ts two.or €rowti::
~.ifluorome thylorni thine
(23), DL- 0 ( -
(Jjrr;:O) is de tri:ne~~ t2,l to tU,'.nor proli-
feration (7), lev9.n i:3?,lso 'ietricental (10), melIlhalen is
toxic ~'1der hee..t (20), ~1~Jl-lyso9hospholi9ids prevent LLC
met~stases or spre2ding
(9),
polyinosinic:polycytidylic acid
aids indomethacin ani lYIDDhokines in toxicity (14), and 5fluorouracil stops IN..\. syntn9sis in tWilors (2,11).
*?or an excellent .:iescrip'tion of L-::wis lunA: carcinoma, I refer
to an~rticle by T. 3ato, et. aLe (33).
-
In spite of the wealth of sUbstances avail9ble for study,
or perha9s
bec~use
:J.et[>~8.cin.
.since indometnacin
of said extensiveneas, I
bloc~.-cs
'Nay of :::rostp_glar_dL: syntl18sis,
ch~se
~ne:
indo-
the cyclooxygen.2.se pc. th-
it C2..!.'l reinsta.te macroph::',ge
toxici ty to tUr.'1lor cells (35, J;l) •
IndJt2let:'lacin is a .!.'lonsteroid,
a::;.tii::L:"l8.mma tory drug (26) ..::apaole of sto~.ping hormone-lik'"
'?.c ti vi ty
0
f
lym:.~ao(;;tes
an?,logu,es (29), a f hel:;ing prali fe:'']. tiJn of
2
(36), and 0:' ::aaintair:.ir~g toxicity of 2,:,.cra)r: '"es
PGE
L: vit~·o beyond 24 hours (39).
2arlier tests h2.,ve given ·:::,s
~'luch as -+O7~ reductio;~ Jf tW!lor g:'owth in :nice
.3tr9. tion in vivo still
vi tro '.vork is
~ill
'et
:'0 t
:l,3.S
(17).
) . ci"1ini-
it "'ficul ties (24,29), tnougf'_ in
cOLi,~le
':;e.
:-i:ope:\llly, these eXDeri:ner. -:.3
advance this.
~,!:acrop1::age s '.vere col~_ ect ed fron C5731/6 mi ce ·.vhic.h 'fiere injecte~
with O.2ml CFA interperitaneally two days
tone;'1.l Na sf'~i{'. g.
'rhey were plated
Or!. to
to peri-
polystyrene 'Nell s
(Falcon, 3ecton Jickinson J: Co., Oxr..ard,
'Nell density. *"
~rior
c;d
in 3. 7xl0 5 cells/
leside:1.t m.8.cropc..ages Nere extracted wi thout
prior treatment of mice.
'rhe first set of tests '1'12..S to J.eter-
:nine the time fr2..J.e in '."lhicn o2.xi:nwn secretion existed; this
set; ?l2.S prelimiru?xY.lnd dat3. are not shmVIl.
~,:acroph'3.ges
were
incuba ted ei tiler wi thDut or 'Ni til ?p.g/ml LPS ·.vhile varying the
'?.ccur:lule.tive tl::ne spans.
Lewis lung c:rcinome. (L1C) cells "Nere cultured in liquid
-
:nedia, RPr,U (GIBC,) Le.borg.tories, Grand Island, lITY) with
?BS (KC 3iological, Lenexa, KA), 2.nd transferred
These ...rere -::.dherent cells grown in polystyrene
Glass ,'/orks, Corning, l\j"y).
passed in vivo (TA
3
3.S
IO;~
necessary.
flas~s
(Corning
The third set of tests used cells
).
*Macro~)h2,gas are sUEIIDoned to th.e lJeri tone:?l cavity by the CFA.
Of all ::natarial and cells, only :nacrophages ',Vill orihere to the
surface of t!'~e wells.
5
In the first set, tumor cells were labelled with l"/Cr*
(ICN Chemical
& Radioisotope Division, Irvine, CA) by incuba-
tion for one hour to test for cytotoxicity.
three times.
These were washed
They were incubated for 24 hrs. with macrophage
supernatants:
20,000 cells and 0.2ml supernatant per 0.6ml
total solution per sample.
Supernatants from these were count-
ed in 3. f3-cOl.mter t'or cytotoxici ty.
;!lining maximum release of
The control sample deter-
slCr was obtained by lysing tumor
cells with Na-deoxycholic acid.
The second set attempted to measure tumor cell growth when
exposed to macrophage
3uperr~;ants.
These tests
ar~
primarily
not my own, 'out :.ire included as a sister analysis to my other
two tests.
Induced and r9sident macrophages were collectei:.ls
the first set.
Cells were divided into four groups:
medium
.llone, ::nedi '..llIl ;vi th 5J.lg/ml LPS, medium with lp.g/ml ind.omethacin,
'3.nd
~ejium
collec~ed
o. 2:nl
with both LPS and indomethacin.
over various spans.
Supern:..-'itants were
rumor cells were incubatei with
supernatant and 2Jlcuries of JH-thYiT:idine per sample. **
'rumor cells were then dried and read.
The thlrd set tested tumor cytotoxicity from macrophage
sup e rna tants.
Thi s ·.vas perfor:ned simi l'l.rly to the first set,
·,vi"'eh a few adjus tments.
Extracted macrophages were incubated
lon their wells with altered media; there were four divisions
:.lS in the second set.
All four
~inds
of medium (plain, LPS,
indomethacin, and LPS + indomethacin) cont3.ined no FBS.
rhese
supernatants were then incubated with tumor cells for 12 hrs.
Released 5"1 Cr was read in a'T-counter.
* S'/Cr is absorbed by live, heal thy twnor cells in a short
period; it is forfeited upon the cell's death.
**JH-thymidine is a labelled nutrient useful for DNA synthesis
that is taken up by a cell as it multiplies. Counting tritiwn
from cells measures the 3.mount of growth during the "pulse. 1t
6
-.
a.2:SULTS
]a tao
from. tce se cond set are :=,olmd L1 the table.
lcti vi ty
'/la.? ;ne:J.su:red as :;;ercent reduc""Gion of growth with 3H-thymidine.
In lookin,s at ti'd s, we
3ee the. t ITl2_xim.e.l inhi bi tion appe2rs
C::'.!'l
"to fall be tWeen the firs t :lnG 24 hr. of :nacro pi'..age culture.
Gradu8.1ly, this ';.cti vi:;y see.::.s 1;::> decline over time.
Superl12.-
t:?nts did hE.ve a not;iceable effect frotJ. st.'3.ndard, medi1..un solutions, out th-a addition of LPS or indometr..acin oecarne negligible in 3.idir.g rete.rrJa tion rsg2.rding c:J 1_mterY2.rts wi -;r_ .:l?cro';;!'...2.gs s.
~te siden t
:1.3,croph2€eS beg2.n ir.hi bi tion more
but othen'lise behaved as irlduced ::12crophages.
be tin. t aeti vi ty
COi:le~1
~uici<ly,
'rhus it miEht
from tile T..<..-':.cropilage :lni is not affected
by these extra substance3.
'rhe th:"rd set has jean ,.:lec:.suT-3d by a
S'I
Cr-!'elease ass2.Y
using tne following ec;.uEtsion:
(
~ Activity
= 100
s~mple
e~is3ion
cOlUlts from sample's)
standg.rd
x
( total
~ossible
SICr cytotoxicity pro;iied better results.
grow-::;h ir:hibi tion, there '.vas
su-:::.e~a tants
rele2se )
As in tumor cell
s:--::a1l mec:sure of::tctivi ty in
3.
incuOa ted alone--::1'3 di tun ?_r.:i
.--"1:"'1
cro lJi~:"'6e s only.
jiedium stored ',Ii tl:. LPS and. -:;i tilOU.t ll2'.c:copne,ges appec:-"rs "GO be
sliglltly detrime:'ltal; r?tlier, it
ing tu,'TIor cells
"3.S
siHl:,Jle
though, f2.red better
t~1::1n
'<'12,3
~edium.
sL:~le
,::.ot
'3.S
efficie~:t
i:1 kill-
LFS-sti:nul:::.ted I!l2.cr::>ph?ses,
.i1edium.
Indomethacin:tcteci uncomfortably lUlstable, yet ?ided toxicity
co~siderably
(see gr'3.ph).
rapidly from 3 to 6 hrs.
Its success is short, f'3.11ing
7,Iy samples of macropnages incubated
wi th both 5pg/ml LPS 3.nd Ip.g/ml indomethacin v3.ried unsatisfactorily bet'Neen tests, though it usually perfor:-:le,i midling '.vi th
LPS ['.nd indomet!l.Bcin cultures individually •
unreliable yet for inclusion here.
7
-------------_._------
Results -.vere too
DISCUSSION
Absence of positive results
is always discouraging.
My
goal to find a soluble secretion from activated macrophages
has not concluded successfully,
al though n.)t because of the absence of the factor.
Macro-
~
• ••
phages can. be manipulated by
endotoxins and indometh::;.cin,
the extert of which is uncertain.
I
:ave shown in coarse
data that lndometh2cin assists
.~------~-----------.-----( ...... )
product':'on ot" the factor immedia tely following admini ::::tr-c"j.-
r ...... ;~1
~.s .. i . s .....
#3. c-'c. ....~lc. .. c<, ... ~-
4-0 -ftc -c..l:_- .... ;r~-~ ~J.i""~+--~·
l'\c.":_ ... 1" ( 0 ) , L1'S ( A ) • ~"ci..-c.t'I._: ..
( . ) • ~o~ ~ ..
(*' ). "2.Je.. h '-.,,~.
I .......
tion to macrophages, and that
...,s
its efrect holds over ror a
so
short time afterward.
I regret the shortness of
time which prevented me from
continuing research.
My own
skills and experience, however,
are the true culprits.
Since
so much is left to be finished,
..
a list or future suggestions
follows here.
Testing for tu-
mor growth inhibition needs to
•
.~-----,--------------,--------
be perfected, for the techniques are not yet qualified.
Certainly, narrower time limits
To";"i~
ID>~".'"
;~ .. ~\ .... -
( . ),
""'--t. oc.4- -3. "I~ r.1c..o.sc. ~ ...
... ~ .. cj.,,;" ... 1 s .. _plc. s h ...... r.ls.
.... ~ (,0), \..PS ( .. ),
b.+~
~-
of incubation are imperative.
It has been discovered that ma-
J:"ol._..H...u,;o..
d ..... ~j C% ). 12 ~l..,.. k +c. ... ~ .
crophages are able to depress
3
-
remarkably tritium counts (28);
should this be true, our results from experiment set 2
could become drastically altered.
~.
Care ought to be taken
:r
'"~
·Ni:en collecting macrophage
IL
.
1:
SUperl12.tan ts, for there is some
col
evidence that the factor is
fragile and labile (13).
confir~ ~y
.
Ie
1
.
41
For
at
the cytotoxic tests, extra
data would
)
"
•
;"'
results.
.11
:Jther act i"'l:3. tors ':CJul::l be used,
but plenty is needed 3till with
indomethacin.
fhe~e
.~----,----------+~-----{lor.}
c.
1
is little
question that the factor exists,
but the "wnere" and "how" renain puzzles.
",..0""1.
~dded
"U"''1.
.,,1.,
~L" -2 . • ~ .. p .... ~ ....
._,.t.
s~_4 ..... J,.
M",I: .. _
Co), LPS ( • ), l: .. J.. ... ' .. " .... ~M
(.), •• Hocl..-.. ')t,..." fk ...... ,'4c..~l.. k. .... i-.
:nacrophages, stri?ped bare Nith
trJ9sin, could be
:AI.:\o:Iol ...
,.&I .. ~ ...... ~A4: ...•..... 1
For instance
to
OF TUMOR GROWTH INHIBITION
-Relative CPM-
-Incubation Timesmedium
LPS
178+- 4.9
182
4.3
3.0
53
2.0
53
60 12.6
67+- 0.0
170-to169
96
69
202 +-19.0
117
2.8
1.1
114
105 11.4
7.5
49
103+-21.0
indo • .
both
166-to-23.1
3.1
4.2
47+- 2.1
1631--13.6
171
1.9
2.7
47
56
1.3
2.2
64
68+- 9.4
167 10.6
).6
39
38
5.7
40
3.9
38+- 6.4
187 +-12.9
55
4.5
7.5
93
81 13.7
7.2
29
75+- 6.8
214+-12.2
115
4 • .3
112 12.3
5.a
52
56
3.1
82 -to-17.9
213+-18.7
8.1
53
103 22.2
7.2
91
48
3.5
123+- 1.7
301icited macs
standards
1.5 hrs.
24 hrs.
48 hrs.
72 hrs.
114 hrs.
5.6
6.4
J.O
44
J.esident macs
standards
hr.
hr.
hrs.
hrs.
hrs.
0-1
1-2
2-4
4-24
24-43
9
tumor cells
~long
with supernatants; or macrophages, killed
intact by earr3.geenan (22), could instead be added to tumor
cells with supernatants; or indomethacin cou.ld be fed to the
~ice before macrophage acquisition,
cells.
:\iany, many, and more
~nd so be added to tumor
experime~ts
the invitation.
10
arc:: waiting--I offer
ABBREVIATIONS
CFA
Complete Freund's adjuvant.
ConA
A lectin which
CPM
Counts per minute.
FBS
Fetal calf serum, or fetal bovine serum.
3H
Tritium.
LLC
Lewis lung carcinoma.
LPS
Lipopolysaccharides, ~lso known as endotoxin; stuff
from gram-negative bacteria, used as an activator
(38) •
macs
Macrophages.
sups
Supernatants, the upper or liquid portion of a
solution or culture.
stimul~tes
some lymphocytes; mitogen
(38).
ACKNOWLEDGMENTS
I would like to thank Dr. M. Rita Young for her inexhaustive patience and encouragement. She and Mrs. Meunier have
helped me immensely in these endeavors and in "life."
1.
Adam!3, Dolph 0., "Effector Mechanisms of Cytolytica11y
Activated Macropr~ges, I. Secretion of Neutral Proteasel3 and Effect of Protease Inhibitors," J. of ImmunoloQ. 124(1980) :286.
---.
2.
Adam13, D.O., "Macrophage Activation and Secretion:
mary," Federa tion Proc.eedings 41 (1982): 2193.
3.
Adams, D.O., ,/.J. Johnson, and P.A. J1arino, "Mechanisms
of~arget ae~og'.li tion and Destruction in Macrophagemediated I'um,)r ~ytot')xici ty," Federation Proceedings 41
Sum-
~1982):2212.
4.
Adams, Dolph 0., Kuo-jang i(aa, Ro::iericlc if'arb, and Salva":)re V. Pizzo, ".2:ff~ctor ~t1ech ~ni3ms of Cytolytica11y
_~cti fa t~d Macrophages, I I. Secretion of a Cytolytic Factor oy Activated ~acrophages and Its ~elationship to 3ecreted Neutral Proteases," J. of Immunolog;£ 124(1980):
293.
5.
Aksami t, 3.obert
~Ji ne s Produce
l785.
6.
Arenzana-Seisdedos, F., and J. Vireli~ier, "Interferons
as Macropli.a.ge-activating ?!J.ctors: 2.. ~nced Secretion of Interleukin 1 by Lipop,:,lysacc:C-""'''cride-s1;imulated
Human MO:locytes," Eur. J. of I::'illl·.molJ~Y: 13(1983) :437.
7.
Bartholeyns, Jacques, "Treatment of ~Ietastatic Lewi3 Lung
Garcinoma with DL-ex-Difluoromethylornithine," ~r. J.
of Cancer and Clinical Oncology' 19(1983):567.
8.
Bennett, A., et.al., "Prostaglandins and Human Lung
nomas," Bri TI 8hJ. of Cancer 46 (1982) : 388.
9.
Berdel, Wolfgang E., and Paul G. Munder, "Metastatic
Growth of Le','/is 3LL Lung Carcinoma in Mice 'rrea ted ',vi th
Alky1-1ysophospholipids and Lysophospholipid-induced
Peritoneal Macrophages," Anticancer Research 1(1982):
397.
10.
Bubis, J.J., et.al., "Effects of Levan on the fate of ~x­
perimenta1 Hematogenous Metastases of Lewis Lung Carcinoma," Experimental Molecular Pathology 36(1982):287.
11.
Connolly, Kevin M., et.al., "Host Interactions in the
Effects of 5-fluorouracil on 2hrlich Ascites Tumor
Cells," Cancer Research 42(1982):4921.
~., and K. Jin Kim,
:3. Cytoto xin," J. of
"Macrophage '::ell
ImmQ'1.o logy 122 (1979) :
~arci-
12.
Currie, Graham, "Immunological Aspects of Host Resistance
to the Development and Growth of Cancer," Biochem~ et
BioEhys. Acta 458(1976):135.
13.
Currie, G.A., and Connie Basham, "Activated Macrophages
Release a Factor which Lyses Malignant Cells But Not
Normal Cells," J. of Experimental .edicine 142(1975):
1600.
14.
Droller, Michael J., and Diana Gomolka, "Inhibition of
Tumor Growth in Association with Modification of In
Vivo Immune Response by Indomethacin and Po1yinoslnic:
polycytidylic Acid," Cancer Research 42(1982):5038.
15.
Drysdale, Beth-ellen, Charles M. Zacharchuk, and Hyun S.
Shin, "Mechanism of Macrophage-mediated Cytotoxicity:
~roduction of a Soluble Cytotoxic Pactor," J. of Immuno~l 131(1983):2362.
16.
Gira1di, !., G. Sava, M. Kopitar, J. Brzin, A. Suhar, and
V. Turk, "Spleen Intracellular Inhibitors of Neutral
Proteases," Eur. J. of Cancer and Clinical Oncology 17
(19.31) :1301.
17.
Hial, Valdemar, Zdenka Horakova, Richard E. Shaff, and
Micha.el A. Beavc:n, "Alteration of tumor Growth by Aspirin and Indomethacin: Studies with Two Transplantable
Tum,:>rs in Mouse (sic)," Eur. J. of Pharmacology 37 (1976):
367.
18.
Hibbs, John B. Jr., "Activated Macrophages as Cytolytic
Eff,:!ctor Cell s, II. Requirement for Local Persistence
of Inducing Antigen," TransElantation 19(1975):81.
19.
Hopper, K.E., and J ••• Cahill, "Immunoregulation by Macrophages: 2. Separation ot" Mouse Peritoneal Macrophages
Having Tumoricidal and Bactericidal Activities and
Tho:3e Secreting .i:'rostaglandin K and Interleukin 1,"
J. Reticuloendothelial Soc. 33(1983):443.
20.
Joiner, M.C., G.G. Steel, and T.C. Stephens, "Response of
Two Mouse Tumours to Hyperthermia with CCNU or Melphalan," British J. of Cancer 45(1982):17.
21.
Kaplan, Alan M., et.al., "Mechanism of Macrophage-mediated
Tumor Cell CytotoxICity," J. of Immunology 121{1978}:
1781.
22.
Kopper, L., T. VanHanh, and K. Lapis, "Experimental Model
for Liver Metastasis Formation using Lewis Lung Tumor,"
J. of Cancer Research and Clinical Oncology lU3(19~2):
Jr.
-
-
23.
Madonna, M.-, T. Colombo, M.G. Donelli. and G. Dagnino,
"In:E'luence of Adriamycin on Growth Kinetics o:t" Lewis
Lung Carcinoma and Its Lung Metastases,n Oncology (BaselL 40(1983) :124.
24.
McCarthy, James P., et.al., "Acute Intrinsic Renal Failure Induced by Indomethacin: Role of Prostaglandin Synthetase Inhibition," Mayo Clinic Proc. 57(1982):289.
25.
Meltzer, Monte S., et.al., "Interaction of BeG-activated
Macrophages with Neoptastic and Nonneoplastic Cell
Lines In Vitro: Cinemicrographic Analysis," Cellular
ImmunoIOgy 17 (1915): 30.
26.
Narisawa, Tomio, M. Sa. to, M. Sano, and T. Takahashi, "Inhibition of Development of Methylnitrosourea-induced
Rat Colonic Tumors by Peroral Administration of Indomethacin," GAl'fN 73(1982):337.
27.
North, Robert J., "The Concept of the Activated Macrophage," J. of Immunology 121l1978):806.
28.
Pasternack, Gary R., Robert J. Johnson, and Hyun S. ::>hin,
"TUmor Cell Cytostasis by Macrophages and Antibody In
Vitro," J. of Immunology 120l1918):15bO.
29.
Rinaldi-Garaci, Cristina, et.a1., "Induction of Serum
Thymic-like Activity in Adurt Thymectomized Mice by a
Synthetic Analog of FGE l , " Cellular Immunology 12(1982):
97.
30.
Rivnay, B., E. Gorelik, S. Segal, and M. Shinitzky,
"Plasma Membrane Microviscosity of Lewis Lung Carcinoma
Cells Derived from Local Growth and Pulmonary :'I[etastases," Invas. Metas. 1(1981):99.
31.
Ruco, Luigi P., and Monte S. Meltzer, "Macrophage Activation for Tumor Cytotoxicity: Development of ~acrophage
Cytotoxic Activity Requires Cocpletion of a Sequence of
Short-lived Intermediary ~eactions," J. of Immunology
121 (1918): 2035.
32.
Russell, Stephen w., et.al., "Functional Characteri--z:ation
of a Stable, NoncytOIytic Stage of Macropha~e Activation
in 'IUmors," J. of Experimental Medicine 146(1978):1511.
33.
Sato, T., et.al., "Ultrastructure of the Lewis Lung Carcinoma," Eur.7. of Cancer and Clinical Oncology 18(1982):
369.
34.
Schultz, R.M., N.A. Paulidis, iV.A. Stylos, and M.A. ChirigOSt "Regulation of Macrophage 'l'umoricidal Function: A
RolEl f"or Prostaglandins ot" the E Series," Science 202
(1978):320.
35.
Shaw, James 0., et.al., "Macrophage-mediated Tumor Cell
Killing: Lack of Dependence on the Gyclooxygenase ~'ath­
way of Prostaglandin Synthesis," J. of Immunology 123
(1979):50.
36.
Skoldstam, Lars, David Zoschke, :3.nd Ronald Messner, "Contra~;ting Effects of Prostaglandin E 1 and Indomethacin
in ••• ," Clin. Immunol. Immunopathology 25(1982):32.
37.
Snider, Mary Ellen, Richard H. Fertel, and Bruce S. Zwilling, "Prostagl~ndin Regula tio~ of Macroph.a.ge Function:
~ff€!ct of
2ndogenous and Exogenous Prostaglandins,"
~ellular ImmuYl::Jlogy 74 (19~2 ) : 234.
38.
stites, Daniel P., John D. stobo, H. Hugh Fudenberg, :ind
J. Vivian Wells, Basic and Clinical Immunology, Los
\ltos, CA, Lange Medical Publications,I982.
39.
Taffet, steven M., :ina stephen 'N. Russell, "Macrophagemediated Tumor Killing: aegulation of 3xpression of
Cytolytic Activity by Prostaglandin 3," J. of Immuno10ffJ.. 126 (1981) : 424.
40.
Tilden, Arabella, ':lnd Charles M. Balch, "Immune Modulatory Effects of Indomethacin in Melanoma Patients Are Not
Rela.ted to Prostaglandin Ez.-mediated Suppression,"
Surgery (st. ~ouis) 74(1983):528.
41.
Young, M.R., 3.nd :li. Dizer, "Enhancement of Immune ?unction
and 'rumor Growth Inhibi tion by Antibodies Against Prostaglandin E1.'" Immunological Communications 12(1983):
11.