Cytotoxicity and Anti-Inflammatory Activity of Methylsulfanyl-triazoloquinazolines
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Journal of Leukocyte Biology 40:801-813 (1986)
WORKSHOP REPORT
Comparison of Five Short-Term AssaysThat Measure Nonspecific CytotoxicityMediated to Tumor Cells by ActivatedMacrophagesStephen W. Russell, Judith L. Pace, Luigi Varesio and, in alphabetical order,Emmanuel Akporiaye, Elisabetta Blasi, Antonio Celado, Robert 0.Schreiber, Richard M. Schultz, Anita P. Stevenson, Carleton C. Stewart andSigrid J. Stewart
Department of Comparative and Experimental Pathology, University of Florida,Gainesville (S. WR., J.L.P.); National Cancer Institute, Frederick Cancer ResearchFacility, Frederick, Maryland (L. V, E.B.); Department of Immunology, ResearchInstitute of Scripps Clinic, La Jolla, California (A.C., R.D.S.); Department ofImmunology Research, Lilly Research Laboratories, Indianapolis, Indiana (R.M.S.);and Experimental Pathology Group, Los A/amos National Laboratory, Los A/amos,New Mexico (E.A., A.P.S., C. C.S., S.J.S.)
Five different short term assays (< 48 h) used to measure macrophage-me-diated, nonspecific cytotoxicity were compared under similar conditions in thesame laboratory using the same reagents. The purpose was to determine theextent to which results were comparable. Three of the assays were dependenton the release of a radioisotope to measure cytotoxicity, one was dependenton cell counting, and the last was dependent on flow cytometric quantificationof remaining viable tumor target cells after they had been exposed to macro-phages. The variables examined were the following: a) three different popula-tions of macrophages; b) four different kinds of target cells; c) two types ofradioisotopes; and d) two different agents that trigger the expression of cytoly-tic activity by primed macrophages. Recombinant gamma interferon was usedas the priming agent in all the experiments. There was unexpectedly goodagreement between the results of the various assays. No differences werefound among the different macrophage populations, the isotopes or the trig-gering agents. Perhaps the most important finding was that differences intarget cell susceptibility to killing by activated macrophages, which were ap-
Received and accepted May 14, 1986.
A. Celada is now at the La Jolla Cancer Research Foundation, 10901 North Torrey Pines Road, La Jolla,CA 92037. R. Schreiber is now at the Department of Pathology, Washington University School of
Medicine, 666 5. Euclid Avenue, St. Louis, MO 63110.
Reprint requests: Stephen W. Russell, Department of Comparative and Experimental Pathology, Box J-
145, JHMHC, University of Florida, Gainesville, FL 32610.
© 1986 Alan R. Liss, Inc.
802 Russell et al
parent in assays of less than 24 h duration, disappeared when the same kindsof targets were compared in assays of greater than 40 h duration. The resultsof this study are an important first step toward standardizing the way in whichmacrophage-mediated, nonspecific cytotoxicity is measured in short-term as-says, laboratory to laboratory.
Key words: macrophage, cytotoxicity, short-term assays, assay standardization,gamma interferon
INTRODUCTION
Several different short-term assays, ie, those in which target and effector cells
are coincubated for less than 48 h, can be used to measure nonspecific cytotoxicity
mediated by activated macrophages. With each of these different assay types, inves-tigators have used dissimilar sources of target and effector cells, as well as different
reagents. It has been impossible to know, therefore, whether data from one laboratory
to another are comparable. This problem was addressed at a laboratory workshopheld in March, 1985 in Los Alamos, New Mexico under the auspices of the Reticu-
loendothelial Society and the University of Florida. The objective was to compare the
principal types of short-term assays of macrophage-mediated, nonspecific cytotoxicity
under the same conditions, by using a range of effector populations and target cells.
The results and conclusions from the workshop are presented here.
MATERIALS AND METHODS
For the purpose of this presentation, under the conditions employed, macro-phages that were exposed to gamma interferon will be referred to as “primed. “ This
connotes that they were prepared to become cytotoxic, but that they did not reachtheir full cytolytic potential unless a second, “triggering” agent was added. It is
emphasized that it is possible to get full activation of macrophages without the use of
a triggering agent [7, 11]; however, the induction of cytotoxicity is always more
efficient when both signals are present.
Assays
The characteristics of the various assays are summarized in Table 1 and de-
scribed elsewhere in detail (see individual references in Table 1) . The basic differ-
ences among those that are based on the release of a radioisotope were: the shape of
the well bottom; the time of preincubation of macrophages with stimuli; whether ornot stimuli were removed prior to adding targets; and the duration of exposure of the
targets to macrophage monolayers.The flow cytometric assay, which is detailed elsewhere [10], is based on the fact
that i) Hoechst 33342 (Calbiochem, La Jolla, CA), a blue fluorescent vital dye, can
be used to quantify DNA in viable cells, while propidium iodide stains DNA in deadcells; ii) fluorescently labeled antibodies, such as MAC-l [9], can be used to distin-
guish macrophages from tumor cells by cell surface labeling; and iii) remaining viable
tumor cells can be quantified by comparing their number to a known concentration of
fluorescent beads included in the sample. Briefly, after incubating targets with the
macrophages, remaining cells were labeled at 37#{176}Cwith Hoechst 33342, treated with
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804 Russell et al
trypsin/EDTA if adherent, and collected. Cells were then incubated at 4#{176}CwithMAC- 1 antibody (produced from hybridoma cells M 1/70. 15. 1 1 .5, American Type
Culture Collection No. T1B128; generously supplied by Dr. G.C. Saunders, LosAlamos National Laborotory) and labeled with fluorescein isothiocyanate (Miles-
Yeda, Ltd. , Israel). After next staining with propidium iodide (Calbiochem), they
were mixed with a known concentration of 10 jsm-diameter fluorescent beads (Super
Bright, Coulter Electronics, Hialeah, FL) and analyzed in a flow cytometer equipped
with dual argon lasers (CR-b, Innova 90-5; Coherent, Palo Alto, CA). Blue (Hoechst,
UV excitation), green (FITC MAC-i labeled macrophages, 488 nm excitation) and
red (propidium iodide-stained cells and beads, also 488 nm excitation) emissions were
collected. Beads were discriminated from cells on the basis of small-angle light scatter
and high intensity emission of red fluorescence. Because beads were present at a
known concentration, cell concentration could be calculated by comparison. Results
obtained with this assay after 44 h of incubation were a composite of target cell
growth inhibition and cytolysis.
Many of the details of the macrophage-mediated cytotoxicity assay that is based
on visual assessment [8] are similar to those of the flow cytometeric assay described
above (Table 1). Cells remaining at the end of the incubation were harvested by
repeatedly jetting liquid up and down in the wells, after which the contents wereremoved, centrifuged, and resuspended in trypan blue dye to mark dead cells. Viable
tumor target cells were enumerated in a hemacytometer using a phase contrast
microscope. Macrophages were distinguished from tumor target cells based on differ-
ences in size and morphological appearance.
The control for nonspecific killing in each assay was target cells incubated in
medium alone, ie, without the inclusion of either priming or triggering agents.
Reagents and Cells
Mice were female C3HeB/FeJ, 6-8 weeks old, from Jackson Laboratories (Bar
Harbor, ME). Macrophages were either derived from bone marrow 14 days earlier,
as previously described [3], or were elicited from the peritoneal cavities of mice by
each of the following means: i) 10 ng bacterial lipopolysaccharide (LPS; lipid A-rich
fraction II of phenol-extracted E. coli 0111 :B4 [5]) injected intraperitoneally in 1 ml
pyrogen-free saline 24 h before exudate cells were harvested by peritoneal lavage, or
ii) 1.5 ml proteose peptone (10% solution; Difco, Detroit, MI) injected intraperito-
neally 3 days before harvest. When peritoneal exudate cells were used they wereallowed to adhere to the bottoms of wells for up to 4 h, depending on the assay,
before being washed vigorously and repeatedly to remove nonadherent cells. Target
cells were of the following kinds: P8155 (provided by SWR/JLP), a subline of
mastocytoma that is carried in vitro and is highly susceptible to macrophage-mediated
cytotoxicity; P815R (provided R.M.S.), a subline of mastocytoma that is relativelyresistant to killing but not to growth inhibition; L5178Y (provided by L.V. and E.B.),
a T cell lymphoma; and EMT6 (provided by C.C.S. and A.P.S.), an adherent
mammary carcinoma cell line. Each had been growing in the workshop laboratory inthe same lot of assay medium for at least 1 week before the experiments were
performed. Each target cell line was changed into fresh medium the day before it was
used. The medium used (H-MEM) was modified Eagle’s minimal essential medium.
It was prepared from a powdered mix (Auto-POW MEM) and supplemented with
sodium bicarbonate (2 mg/ml), glutamine (2 mM) (all from Flow Laboratories,
Comparison of Short-Term Cytotoxicity Assays 805
MOUSE MACROPHAGE TARGET RADIOISOTOPE TRIGGERSTRAIN CELL AGENT/P$1$$ �zZ,: _____ LP$
.6f � ______________51 �, LP$Bone Marrow #{163}..-� � ,,
DerIved �c:zzz ________ 5’ c� -� �
/ :�e�::::�::�___
I ._-‘#{149}�#{149}�� ps1� _____________ #{149}�c�
C3H3B/FeJ � PEC/LPS 4EE�:�‘�‘ ::�\ ___
\ ,4? 111bi LP$
\ �#�#��__#{149}, p�j� � � �
\ �‘-� -� Ill �, �_‘�z::z:z:�...� .�i::::::: �:: : �
�NEMT, � ____Fig. 1 . Schematic diagram of the experimental design of the radioisotope-based assays. Assays not
dependent on radioisotopes were designed similarly, except that the column titled Radioisotope wasdeleted. Abbreviations include: PEC, peritoneal exudate cells; LPS, bacterial lipopolysaccharide; HKLM,heat-killed Listeria monocytogenes; PP. proteose peptone; P8155, sensitive subline of mastocytoma cellline; and P815R, resistant subline of mastocytoma cell line.
McLean, VA), 15 mM HEPES (Sigma Chemical Co. , St. Louis, MO), injectable
penicillin G potassium ( 100 U/mI) and injectable streptomycin sulfate ( 100 j�g/ml),
both from Pfizer (New York, NY). Fetal bovine serum (FBS) was obtained from
Sterile Systems (Logan, UT) and used at 10% v/v with H-MEM to make the medium
in which target cells were cultured and in which assays were performed. All tissue
culture media and sera used in this study were negative for detectable endotoxin, as
determined by assay [4] with Limulus amebocyte lysate (Associates of Cape Cod,
Woods Hole, MA) at a sensitivity level of 0.07 ng/ml.
Radioisotopes used to label tumor target cells were sodium 5tchromate (ICNBiomedicals, Inc. , Irvine, CA; specific activity 145 mCi/mg Cr) and ‘ ‘ ‘indium-oxine(Medi-Physics, Inc. , Richmond, CA; specific activity 420 Ci/mg In). Purified mouse
gamma interferon (1.9 x iO� international reference U/mg) was made by recombinant
DNA technology [3] and provided by Genentech, Inc. , South San Francisco, CA.
Triggering agents included the LPS described above, provided by Dr. D.C. Morrison,
University of Kansas, Kansas City, KS, and heat-killed Listeria monocytogenes
(HKLM; iO� organisms/mi; provided by R.D.S. and A.C.).
Experimental Design
Each radioisotope-based assay contained the samples diagrammed in Figure 1.
Assays that did not require radioisotopes were performed similarly, except that
samples in the column titled Radioisotope were deleted.
RESULTS
Comparison of Dose Response Curves Developed With the Different Assays
The panels on the left in Figure 2 show dose response curves for the three
different radioisotope-based assays. They were similar despite differences in the
806 Russell et al
OO� D:7 , �I.
� , �
O?,__0 0.03 �.O9 0.27 �.8I 2.4 7.5 � 0.03 0.09 0.27 � 2.4 �5
Gamma Intsrf#{149}ron(UftnI)
Fig. 2. Dose response curves generated with the various assays by using proteose peptone-elicitedperitoneal macrophages, P81SS target cells (labeled with 51Cr in assays based on release of radiolable),
increasing concentrations of recombinant gamma interferon, and a constant dose (10: 1 ratio of organismsto macrophages) of the triggering agent, heat-killed Listeria monocytogenes (HKLM). The panels in theleft column are based on 51Cr release (A=SWR/JLP assay, B=LV/EB assay, and C=RDS/AC assay;
see Table 1); panel D is derived from the flow cytometric assay; and E depicts results from the visual,
cell counting assay.
lengths of time that macrophages were preincubated with activating agents, whether
stimuli were removed or left in the wells when target cells were added, and whether
wells were flat or round-bottomed. Each response developed in the same range of
interferon concentrations and each was basically sigmoidal in shape. The resultsobtained with the flow cytometric and visual cytotoxicity assays can be compared in
the pair of panels on the right side of Figure 2. Although the data were generated by
very different means, the dose response curves were similar in appearance and in therange of concentrations over which they developed. It can also be appreciated thatthere is little difference between these two dose response curves and those based on
radioisotope release (panels A-C). All five assays gave similar effective dose 50
(ED50) values for gamma interferon, ie, that dose which primed macrophages to
express approximately 50% maximal cytotoxicity when a triggering agent was added.
Comparison of EfTector Macrophage Populations
Figure 3 summarizes the cytotoxic performance of the various macrophagepopulations tested, ie, bone marrow-derived macrophages, LPS-elicited peritoneal
macrophages, and proteose peptone-elicited peritoneal macrophages. The perfor-
mance of each cell population was assessed in each of the five different assays using
Gamma Intirferon (U /m�)
Comparison of Short-Term Cytotoxicity Assays 807
Fig. 3. Dose response curves developed with different macrophage populations, using each of the five
assays. Bone marrow-derived macrophages (0-0); proteose peptone-elicited peritoneal macrophages(0-0); and LPS-elicited peritoneal macrophages (LI1-EIIJ). Target cells were of the P8155 mastocy-
toma subline. All monolayers were exposed to triggering concentrations of LPS (1 ng/ml). The panelsin the left column are based on 51Cr release (A = SWR/JLP assay, B = LV/EB assay, and C = RDS/AC
assay ; see Table 1); panel D is derived from the flow cytometric assay ; and E depicts results from thevisual, cell counting assay.
P8155 mastocytoma cells and LPS as the triggering agent. The dose response curves
obtained developed over approximately the same range of gamma interferon concen-
trations, regardless of the kind of assay. The assay using round bottomed, 6-mm
diameter wells may have been more capable of detecting low concentrations of gamma
interferon than the others. The one instance in which there was virtually no dose
response (panel C) was one in which the LPS trigger alone caused high levels of
release with all three macrophage populations. In those assays that were not dependent
on the release of radioisotope (panels D and E), macrophages cultured from bone
marrow may have been significantly more responsive.
Comparison of 51Cr and 111InOx as Radiolabels for Target Cells
The panels in Figure 4 depict the results obtained with each of the three
radioisotope-based assays when the only difference was the radiolable used, ie, 51Cr
or “InOx. Results in each panel were similar, with the only possible difference being
the slightly lower specific release values obtained with ‘ ‘ ‘InOx in two of the three
assays.
Comparison of Different Tumor Cell Lines as Targets
Analysis of the four different tumor target cells in the five different assays
yielded the data that are presented in Figure 5. Two important points are immediately
J
0
G���ma k�ta1�oi� ( U/mi)
808 Russell et al
Fig. 4. Dose response curves obtained using proteose peptone-elicited peritoneal macrophages, P8155target cells radiolabeled with either 51Cr (0- 0) or ‘ ‘ ‘InOx (#{149}-#{149})and the three assays that depend
on radioisotope release (A =SWR/JLP assay, B=LV/EB assay and C =RDS/AC assay). All monolayers
were exposed to triggering concentrations of LPS (1 ng/ml).
apparent. First, among the assays that are dependent on radioisotope release, in this
case 51Cr, P8155 was much more susceptible to killing than were the other tumor cell
types. The only other target to show low, but interpretable, levels of release in these
assays was L5178Y. The second observation of significance is that all target cells
were killed equally well in the assays that were independent of radioisotope release
as a measure of killing (rows D and E). In these assays, coincubation of macrophages
with targets was longer, ie, > 40 h, than in any of the radioisotope release assays ( <
24h). The dose response curves obtained, even though the means of assessing killing
differed in these two assays, were of similar shape and developed over approximately
the same range of gamma interferon concentrations for each target cell type.
Comparison of Target Cell Killing in 24 and 48 h Radioisotope Release Assays
To ascertain whether the key difference between the radioisotope and non-
radioisotope assays was the duration of coincubation between macrophages and
targets, the two target cell types that were not killed at 24 h were re-examined in a
longer term radioisotope-based assay, after both 24 and 48 h of coincubation with
macrophages.
Leakage of 5tCr from target cells occurs at a sufficiently high rate that assays
greater in duration than 24 h are not feasible because of unacceptably high background
levels. The rate at which � � ‘InOx is released spontaneously from target cells is slower,
allowing assays of up to 72 h to be performed (A.C. , personal communication).
Figure 6 compares specific release values when each of the two target tumor cell
types, labeled with ‘ � ‘InOx, was coincubated with activated macrophages for either
75
50
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70
TARGET TUMOR CELL TYPE
P#{216}I5R LSI7SY
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I& 5o�
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I.0i .03 .09 0.27 0.81 2.4
Comparison of Short-Term Cytotoxicity Assays 809
u,,�J �- -
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25 0.0.0-0�0
0/100
�w�
O�O.O.O-O ‘�.‘�-(,
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.01 09 .a 7.5.0 .09 31 7.50i .09 .a m.� og .a i�Gomma frd.rferon(U/mO
Fig. S. Effect of proteose peptone-elicited peritoneal macrophages stimulated with increasing doses ofrecombinant gamma interferon and a constant, triggering dose of LPS ( 1 ng/mI) on each of four different
target tumor cell types, as measured by five different cytotoxicity assays. Each row is a different assaytype (A= SWR/JLP assay, B= LV/EB assay, C=RDS/AC assay, D=CCS/APS assay, and E=RMSassay). Target cells were radiolabled with 51Cr in assays that are dependent on radioisotope release(rows A-C).
100 P815rss
25
0 � .d .03 .09 0.27 0.81 24 0o-�o--o--o
Gommo Interferon (U/mI)
Fig. 6. Comparison of results obtained using two different target tumor cell types in an �InOx-release
assay after 24 hr (0-0) and 48 h (#{149}-#{149})of exposure to proteose peptone-elicited macrophagesstimulated with increasing concentrations of recombinant gamma interferon and a constant, triggeringdose of LPS (1 ng/ml). The target cells were: P815R (left panel) and EMT6 (right panel).
PROTEOSE PEPTONE ELICITED BONE NARROW DERIVED
Gamma Intsrfsron (U Mi)
810 Russell et al
Fig. 7. Comparison of LPS, I ng/ml, (0-0) and HKLM, 10: 1 ratio of organisms to macrophages,(#{149}-#{149})as triggering agents on either proteose peptone-elicited peritoneal macrophages (left column)
or bone marrow-derived macrophages (right column). Each row, A-E, corresponds to a different assay,the letter designations corresponding to those given in the legend for Figure 5. The target tumor cell wasP8155, labeled with 51Cr in radioisotope release assays.
24 or 48 h. There was a marked difference between the extent to which each of the
cell types was killed at the two time points: at 24 h, there was again little to no
killing, compared to controls, whereas there was evidence of extensive cytolysis at
48 h.
Comparison of Two Different Kinds of Triggering Agents
From data presented to this point, it is clear that each of the two kinds of
triggering agents, LPS or HKLM, had the capacity to elicit killing from macrophages
primed with gamma interferon. The two are directly compared in Figure 7 for two
different populations of macrophages. On proteose peptone-elicited peritoneal mac-
rophages, there was good concurrence between the results obtained using the two
triggering agents. In the one instance where there was a discrepancy (panel C), there
was a high background release level with LPS in the absence of gamma interferon,
which prevented the lower segment of the dose response curve from developing.
Dose responses did not develop as clearly using macrophages derived from
bone marrow, compared to proteose peptone-elicited macrophages. Two of the
radioisotope-based assays (panels A and B) gave good agreement between the LPS
and HKLM trigger. The third (panel C) could not be interpreted due to the high
background release level mentioned earlier. In the flow cytometric (panel D) and
visual (panel E) assays, bone marrow-derived macrophages gave a distinct difference
Comparison of Short-Term Cytotoxicity Assays 811
between the dose response curves that were developed using LPS as a triggering
agent, compared to those which were obtained using HKLM. A similar, though far
less striking, separation between the curves was obtained using proteose peptone-
elicited macrophages.
DISCUSSION
There were few technical difficulties experienced in performing the assays. As
a consequence, most of the data sought were obtained. The participants want to
emphasize the need for caution in interpreting the results, however, because the data
are the product of a single set of experiments. Even with this caveat, it is clear that
an extensive amount of new information has been obtained that should make short-
term assay results more comparable laboratory to laboratory.
Perhaps the most striking finding was that apparent differences in the suscepti-
bility of tumor cells to macrophage-mediated, nonspecific cytotoxicity were decreased
-.--- - or eliminated when targets were coincubated with macrophages for periods longer
than 40 h; only the sensitive P8155 was killed efficiently during periods of exposure
that were 24 h or less. The dose response curves generated with P8155 after 24 h or
less were similar to those obtained with any of the four target cell types after 40 h.
Therefore, it should be a point of future research to ascertain whether or not P8 l5S
can be used as a faithful, early indicator of results that would be obtained with other
target cells in longer-term assays.
The data also clearly allowed the conclusion that not all sublines of the P815
mastocytoma are the same. For example, P815S was killed readily in < 24 h, while
P815R was not. One pair of participants (R.D.S. and A.C.) noted that their own P815
subline (not tested in these experiments) is sensitive but remains so only if it is passed
periodically in mice. By comparison, the P8155 subline retains its sensitivity to killing
without animal passage. Thus, the issue of differences between target cell sublines
must be considered as an important one when striving for standardization.
The discovery that the various cytotoxicity assays all gave approximately the
same ED50 for gamma interferon was another important result. This finding suggests
that it may not be necessary to use a single assay to achieve standardization. This is
providing, of course, that as yet undetected, minor differences do not exist between
assays that would complicate comparisons.
Little difference was found during the workshop that would distinguish 51Cr
from � � ‘InOx-with one important exception. Because of its slow rate of spontaneous
release, � � � InOx could be used in assays that had to be continued for longer than 24 h
to detect target cell injury. Table 2 compares the advantages and disadvantages of the
two radiolabels, as they were listed by the workshop participants.
The triggering agents LPS and HKLM behaved similarly. The apparently
greater efficiency of LPS as a triggering agent, compared to HKLM, cannot be
accepted on the basis of the workshop results, primarily because dose response
titrations were not performed with each of the triggering agents on each of the
macrophage populations prior to the workshop. Titration is necessary because the
maximal amount of each triggering agent that can be used without activating macro-
phages directly must be known. It is likely that the maximal concentration of each
that is useful as a triggering agent will vary with the source of the macrophage
population and the duration of the assay. The need to perform such titrations cannot
812 Russell et al
TABLE 2. Advantages and Disadvantages of “Indium
Compared to 5tChromium in Short-Term Assays of Macrophage-Mediated Non-Specific Cytotoxicity
Advantages of � � � InOx Disadvantages of � � � InOx
Shorter labeling time Much shorter half-life
Less inherent cytotoxicity Much more expensivea
Higher specific labelingAllows smaller well size,
conserving macrophages andreagents
Lower background releaseMore adaptable to time needed
to killdifferent targets
aThis disadvantage is less pronounced if several investigators establish
a contract in common.
be overemphasized. First, as the concentration of triggering agent increases toward
the maximal nonactivating concentration, the gamma interferon dose response curve
shifts to the left [6]. Thus, if LPS and HKLM were not used in these experiments at
their maximal nonactivating doses, any apparent dose response differences between
them might disappear when they are compared at the appropriate concentration. Also,
in terms of standardization, units cannot be calculated accurately unless dose response
curves are shifted fully to the position they will occupy in the presence of the maximal
nonactivating dose of triggering agent. Second, and just as important, is the consid-
eration that titrations must be performed to eliminate activation caused by an excess
of triggering agent. When there is appreciable activation caused by the triggering
agent alone, the lower portion of the sigmoidal curve is raised and flattened (eg, see
Fig. 4, panel C, or Figs. 6A, 7C, or 7D), with the result that units of priming activity
calculated using half maximal release values are spuriously high. Thus, from several
standpoints, there is the need to determine the maximal nonactivating concentration
of a given triggering agent as a part of assay standardization.
The demonstration that bone marrow derived macrophages can mediate cytotox-
icity in all five assays suggests another means by which assays could be better
standardized laboratory to laboratory. The bone marrow-derived population is rela-
tively homogeneous compared to elicited populations of peritoneal macrophages, both
from the standpoint of stage of macrophage differentiation and lack of other, contam-
mating cell types [3]. In particular, this approach could eliminate the potential
problem of cytotoxicity mediated by NK cells. Furthermore, large numbers of
macrophages can be obtained from a relatively small number of mice, making the
approach a cost effective one. The participants emphasize, however, that specific
requirements and defined reagents will likely be necessary if functionally similar bone
marrow-derived macrophages are to be produced laboratory to laboratory.
A recommendation was also made by the participants that, in radioisotope-
release assays, the value for the total uptake of radiolabel be used in calculating
specific cytotoxicity, rather than values that are based on repeated freezing and
thawing or detergent lysis of target cells. The rationale for the recommendation stems
from the need to eliminate differences laboratory to laboratory in the way that
maximal release values are produced.
Comparison of Short-Term Cytotoxicity Assays 813
Finally, there was unanimous agreement among the participants that some
agency must make a gamma interferon standard available to investigators if they are
to compare their results most effectively.
In conclusion, although much more remains to be done before comprehensive
recommendations can be made, the results obtained at this first workshop on short-
term assays have suggested that the goal of standardization is likely attainable.
ACKNOWLEDGMENTS
The participants would like to thank the technical and secretarial staffs of the
Los Alamos National Laboratory for many things, without which the workshop would
have been much less successful. Support for the workshop came primarily from the
Reticuloendothelial Society and the University of Florida, as well as the National
Flow Cytometry Resource (Grant number DRR 01315) and research grants CA3 1199,
CA 33593, CA 37187 from the National Institutes of Health.
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