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Published September 1, 1983

IDENTIFICATION
THAT

OF INTERFERON-3,

ACTIVATES

METABOLISM

HUMAN

AS THE LYMPHOKINE

MACROPHAGE

AND ANTIMICROBIAL

OXIDATIVE

ACTIVITY*

BY CARL F. NATHAN,* HENRY W. MURRAY,IMICHAEL E. WlEBE, and
BERISH Y. RUBIN

From The Rockefeller University, the Division of lnternational Medicine of the Cornell University
Medical College, and the Lindslev F. Kimball Research Institute of the New York Blood Center,
New York 10021; and the Developmental Hematopoiesis Laboratory, Sloan-Kettering Institute for
Cancer Research, Rye, New York 10580

* Supported by grants CA-22090 (to C.F.N.), AI 16963 (to. H.W.M.), and AI 17920 (to B.Y.R.)
from the National Institutes of Health, and grant RF 78021 (to. H.W.M.) from the Rockefeller
Foundation.
*To whom reprint requests should be addressed. Recipient of a research career award from the
Irma T. Hirschl Trust.
lRecipient of a Research Career Development Award in Geographic Medicine from the Rockefeller Foundation.
Abbreviations used in thispaper: IFN, interferon; IFNa, formerly leukocyte IFN; IFN/3, formerly
fibroblast IFN; IFN3,, formerly immune IFN; LK, lymphokine(s);LPS, bacterial lipopolysaccharide;
MAF, macrophage-activating factor; MLL-IFN3,, partially purified 1FN3, from human leukocytes
exposed to mezerein and lentil lectin; MNL, peripheral blood mononuclear leukocytes;PMA, phorbol
myristate acetate; R-HuS, Roswell Park Memorial Institute medium 1640 with penicillin, streptomycin, and human serum; SEA-IFN'r, partially purified IFN~, from human leukocytes exposed to
staphylococcalenterotoxin A; SEM, standard error of the mean.
670

J. Exe. MED.© The Rockefeller University Press • 0022-1007/83/09/0670/20 $1.00
Volume 158 September 1983 670-689

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In the later stages o f infection or after the host recovers, lymphocytes encountering antigens o f the infecting organism confer upon macrophages an enhanced
capacity to kill the same or unrelated pathogens (1). This process is t e r m e d
macrophage activation (2). O v e r a decade ago, antigen-stimulated lymphocytes
were f o u n d to release a glycoprotein(s) (3) that enhanced the activity o f the
hexose monosphosphate shunt in macrophages (4). Soon thereafter, supernatants
from antigen- or mitogen-stimulated lymphocytes were shown to a u g m e n t macrophage antimicrobial activity (5-9). More recently, it was demonstrated that
lymphoid supernatants increase the capacity o f both murine (10-13) and human
(14, 15) macrophages to secrete chemically reactive, incompletely r e d u c e d metabolites o f molecular oxygen, including hydrogen peroxide. T h e capacities o f
macrophages to secrete h y d r o g e n peroxide and to kill various microorganisms
are closely correlated (10, 1 1). In fact, reactive oxygen intermediates appear to
mediate much (though not all [ 15, 16]) o f the antimicrobial function o f activated
macrophages against such intracellular pathogens as Toxoplasma gondii, Trypanosoma cruzi, Leishmania, Candida sp., and mycobacteria (reviewed in r e f e r e n c e 17).
Thus, cell-mediated immunity to intracellular pathogens appears to d e p e n d in
large part on the secretion by lymphocytes o f a factor(s) that activates macrophage
oxidative metabolism and antimicrobial activity. In this p a p e r , the lymphokine
(LK) 1 meeting this description is called macrophage-activating factor (MAF).

Published September 1, 1983

NATHAN ET AL.

671

Materials and Methods
Culture of Monocvtes. Mononuclear leukocytes (MNL) were isolated from the venous
blood of normal adult donors as described (14, 27). 13-mm diam glass coverslips were
pretreated for 1 wk with 50% HNO3 before they were cleaned in ethanol as detailed (27).
For experiments with H202 release, 1 X 1 0 6 MNL suspended in 0.1 ml RPMI-1640
containing 100 U/ml penicillin, 100 #g/m] streptomycin and 25% fresh-frozen human
serum (R-HuS) were plated per coverslip. After 2 h, the coverslips were rinsed three times
in warm Eagle's minimum essential medium and transferred to 16-mm diam wells in 24well trays (Costar Data Packaging, Cambridge, MA) in 0.3 ml R-HuS. The medium was
replaced the following day (day 1), on day 3, and every 2-3 d thereafter. For experiments
with T. go~tdii, 12-mm diam coverslips received 1.5 x 106 MNL in R-HuS containing 20%
heat-inactivated human serum (Sabin-Feldman dye test-negative), and were cultured in
groups of three coverslips per 35-mm diam plastic petri dish. Cultures were rinsed after
2 h, and given fresh medium on day 1 and every third day thereafter.
Hydrogen Peroxide Secretiot~. Secretion of H~O~ in response to 100 ng/ml phorbol
myristate acetate (PMA) (Consolidated Midlands Co., Brewster, NY) was measured by the
fluorescent scopo]etin assay as described (27). The initial concentration of scopoletin was
selected so that <50% was oxidized. Six coverslips were used per data point: three for
peroxide secretion and three to measure adherent cell protein by the method of Lowry
et al. (28), with bovine serum albumin as the standard. Results were expressed as nmol
H,~O., per mg adherent cell protein.
Antitoxoplasma Activity. One million RH strain T. gondii trophozoites obtained from
infected mouse peritoneal exudates (29) were added to each 35-mm dish for 30 rain.
Uningested organisms were removed by washing, and coverslips were then cultured in
standard medium (without added lymphocyte products). At 4 h and 18-20 h after
infection, replicate coverslips were fixed, stained, and scored microscopically for number
of toxoplasmas per vacuole and per 100 macrophages (16, 29).
LK m~d IFN Preparatio~s. Buff), coats from 87 U of blood were pooled at the New
York Blood Center, and incubated for 48 h in serum-free RPMI-1640 containing 1 rag/
ml human albumin, 5 ng/ml mezerein, and 30 ~g/ml lentil lectin. At the concentrations
added to monocytes, mezerein and lentil lectin or supernatants containing them did not
themselves trigger H20~ release. Some of these supernatants were enriched for IFNy by

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T h e synthesis o f MAF by lymphocytes in trace amounts, and its secretion even
by cloned lymphoid populations in a d m i x t u r e with o t h e r LK, have frustrated
attempts to identify it physicochemically. O n e hypothesis is that MAF is interferon g a m m a (IFN3,). T h e r e is strong evidence that IFN3, is the LK that induces
the expression o f Ia or DR antigens on Macrophages (18, 19) and primes them
for a n t i t u m o r activity (20, 21). Various IFNs stimulate m o n o c y t e plasminogen
activator release (22, 23) and Fc r e c e p t o r expression (24, 25). H o w e v e r , none o f
these effects is known to be linked directly with e n h a n c e d antimicrobial activity.
Partially purified, leukocyte-derived IFNs may contain o t h e r LK. P o l y d o n a l
antibodies may neutralize not only IFN but the contaminants as well. Even p r o o f
that cloned, recombinant IFN can activate macrophages would not establish that
lymphocytes do so by means o f IFN. For all these reasons, the relationship
between MAF and IFN has remained undefined.
In this report, we made use o f unpurified antigen- and mitogen-induced LK,
partially purified lymphocyte-derived IFN3', p u r e IFN3, p r o d u c e d by bacteria
containing the cloned h u m a n gene (26), and a monoclonal antibody that neutralizes IFN3,. This combination o f reagents has p e r m i t t e d the identification o f
IFN3, as the LK that enhances both the p r o d u c t i o n o f h y d r o g e n p e r o x i d e by
h u m a n macrophages and their ability to kill an intracellular microbial pathogen.

manuscript in preparation). South San Francisco.Published September 1. manuscript in preparation). Test media were preincubated with antibody for 30-90 rain before use. 420 + 7 [day 3]. Dr. As expected (33-35). T o rule out the possibility that GIF-1 was toxic. Rubin et al. In addition. was obtained by stimulating buffy coat cells with staphylococcal enterotoxin A and purifying the supernatant by sequential column chromatography to a specific activity of 1 x 107 U / m g protein. which gave a single band on analytical polyacrylamide gel electrophoresis (personal communication. where P = nmol H 2 0 2 / m g cell protein after treatment of monocytes as indicated by the subscripts (LK = lymphokine or IFN3. these monocytes released 107%. and f u r t h e r suggested that the Downloaded from jem.) in comparison to a laboratory standard. and 630 -+ 70 [day 4] n m o l / m g cell protein). synthesized by E. 1983 672 INTERFERON-7 AS MACROPHAGE-ACTIVATING FACTOR ResuLts Elimination by Mo~oclonc~l Anti-IFN3. Complete inhibition was seen with six o f the seven LK preparations studied. as described (references 30. and 113% as much p e r o x i d e as those cultured in m e d i u m alone (the latter released 529 + 13 [day 2]. supernatants were collected from MNL cultures stimulated for 48 h with concanavalin A or toxoplasma lysate as reported previously (15). had a specific activity of 6 X 105 U/mg. W h e n tested on days 2. CA. GIF-1 antibody r e m o v e d all stimulatory activity f r o m the supernatants. The lot used. Another preparation. Control LK were obtained by adding concanavalin A to MNL cultures at the end of the 48-h incubation or by adding toxoplasma antigen at the outset of culture of MNL from SabinFeldman dye test-negative donors (15). and 4. coli(26) was provided by Genentech. respectively. we made use o f a monoclonal antibody (GIF-1) that neutralizes IFN-y but has no detectable effect on IFN0~ or IFNI3 (30). and B. Similar results were seen with supernatants o f buffy coat cells pooled from multiple blood donors and exposed to lentil lectin and mezerein. these supernatants all contained substantial titers o f IFN'y (Table I). Monoclonal IgGt antibody GIF-1 neutralizes human IFN3. Y. 15) or toxoplasma antigen (15) to enhance the peroxide-releasing capacity o f h u m a n macrophages. we added the antibody to monocytes for 3 d beginning on day 1 o f culture. SangHe Lee). Sheep globulins to human IFNc~ and to human IFN3 were National Institutes of Health (NIH) reference preparations GO 26-502-568 and GO 28501-568. Antibodies "toIFN. using a cytopathic effect inhibition assay with vesicular stomatitis virus in WISH (HeLa) cells (32). termed SEAIFN3.rupress.000 U/ml. In 8 o f 12 experiments.. R.. T o d e t e r m i n e whether the stimulatory activity in these unfractionated supernatants could be ascribed to their c o n t e n t o f IFN"y. 3.org on July 21. This d e m o n s t r a t e d the lack o f direct suppressive effects o f antibody GIF-1.. these are designated MLL-IFN% Details of the purification will be furnished elsewhere (M. Inc. 2015 sequential affinity chromatography to a specific activity of 1 x 106 antiviral U/ml. T h e 15 experiments summarized in T a b l e I are in accord with previous observations on the capacity o f lymphoid supernatants g e n e r a t e d with concanavalin A (14. Percent inhibition of peroxide-releasing capacity was calculated as 100(1-[PL~+A--PM] /[PLK--PM]). Chang et al. It should be noted that different antiviral activities were recorded for some of these preparations in other laboratories. M = medium control). IFN~ and IFN/3 could not be detected.. Antibody of the Ability of Unfractionated Lymphoid Supernatants to Enhance Peroxide Releasefrom Human Macrophages. Recombinant IFN3. For consistency. 31. A = antibody. M. but not IFNc~ or IFNI3 (31). In the remaining four experiments it was partially effective. 119%.. Wiebe. we have arbitrarily expressed all antiviral titers in this paper according to unitage of assays performed by one of us (B. . The GIF-1 hybridoma supernatant used here had a neutralizing activity of 5.

104 105 ND~ 6. I n t h r e e e x p e r i m e n t s .r e l e a s i n g capacity typically o b s e r v e d o v e r t h e first 3 . I Antibody GIF-1 used at 60-600 U/ml of neutralizing capacity for antiviral activity. 2. 146 95 ND 666 _ 67 (15) 3. 1 Prep.9. which was added to macrophages at a 10-fold dilution. Human macrophages were exposed to the indicated concentration of LK for 3 d beginning on the 3rd to 12th d of culture.1 2.2 78. titer . f r o m c o n t a m i n a t i n g T cells. and stimulated with PMA to measure H202 release. 189 2. 1 H20~ release by LK-treated cells* U/ml U/ml nmol/mgprotein~60 min 100 1-10 H~O2 release.5.9 1.8 -+ 0. 2. except that LK was not added.. washed. 1. 1983 NATHAN ET AL.8. Different batches of toxoplasma lysate were used for the two antigen-stimulatedsupernatants.2. 0. 6964 1094 1.500 0 67-100 100-300 100 0 339. Supernatants from MNL of Sabin-Feldman dye test-negative donors incubated with toxoplasma antigen were used at a 10% concentration on macrophages.org on July 21. experimental*/ controla cells Inhibition by monoclonal antibody! % 1.912 511 172.8. Control cells were tested in parallel with those in note *.Published September 1. SEM for H20~ release by triplicates averaged 8.7 _ 0.000 100 321. 4. a n t i b o d y r e s u l t e d in H 2 0 2 . 673 TABLE I Stimulation of Human Macrophage Peroxide-releasing Capacity by Unfractionated Lymphoid Supernatants: Prevention by Monoclonal Antibody to IFN'y Stimuli for LK production* Lentil lectin + mezerein Prep. ** Not tested.6% of the mean for individual experiments. 1022 529 157.6 (15) 124_ 19 (12) 170 * MNL from individual donors (for concanavalin A or toxoplasma antigen preparations) or buffy coat cells pooled from 87 donors (lentil lectin + mezerein) were incubated with the indicated stimuli and the supernatants collected as described in Materials and Methods. 2015 Prep.7 0. in w h i c h 3 0 % by v o l u m e o f u n f r a c t i o n a t e d L K was m u c h less s t i m u l a t o r y t h a n 1%.394 2. 2 ControlH Overall means -4-_ SEM (N): Concentration IFN~.1 4. Prep. ** MNL received concanavalin A just before harvesting the supernatant. Inhibition calculated as described in Materials and Methods.7 141. 3.9.4 d o f c u l t u r e o f a d h e r e n t M N L m a y n o t b e s u s t a i n e d b y IFN3.9. 12. 4. T a b l e I). T h i s possibility was also r a i s e d by t h e d o s e .0~ 1. used 1FN3. 1. each in triplicate for both H~O2 release and adherent cell protein. a d h e r e n t cell p r o t e i n was 1.5.7. O n t h e a v e r a g e .000 2.rupress.2 85. 1 Prep. 529.000 0 400-600 134 0 514.3. T h i s s u g g e s t e d t h a t l y m p h o i d s u p e r n a t a n t s m a y c o n t a i n factors s u p p r e s s i n g m a c r o p h a g e o x i d a t i v e m e t a b o l i s m . Values for individual experiments performed on different days or with different donors' macrophages. i n c u b a t i o n o f m a c r o p h a g e s in s u p e r n a t a n t plus a n t i IFN3. 2 Prep.520.305. preparations made with different cells on different days. 1. 5.r e s p o n s e p r o f i l e s h o w n in Fig.8 4. 89 49. 1052 368.3 t i m e s g r e a t e r a f t e r 3 d Downloaded from jem.000 3. 3 Control** Toxoplasma antigen Prep. 1.919. 657.r e l e a s i n g capacity s u b s t a n t i a l l y l o w e r t h a n t h a t o f m a c r o p h a g e s i n c u b a t e d in m e d i u m a l o n e ( i n h i b i t i o n > 1 4 0 % .3. 2 Concanavalin A Prep. e l e v a t e d H 2 0 2 . 113.311 ~ 1.000 1.

i n d u c e d l y m p h o i d s u p e r n a tants tested that c o u l d e n h a n c e h u m a n m a c r o p h a g e p e r o x i d e . The Rockefeller University) had no effect (not shown). T h e results o f 13 e x p e r i m e n t s with two i n d e p e n d e n t l y d e r i v e d p r e p a r a t i o n s e n r i c h e d in IFN~. The open symbols are results with the corresponding preparations exposed to 120-350 neutralizing U/ml of monoclonal anti-IFN~ antibody.I i 11"/ I O 600 fi t~ OO4 4011 T . I / * 200 . 2015 ~ 0 .Published September 1. 1983 674 INTERFERON-y AS MACROPHAGE-ACTIVATING FACTOR ~oo 1~o0o I'll / // E o (D soo . Steinman. T h e f o r e g o i n g e x p e r i m e n t s implied that IFN3. An unpurified supernatant from buffy coat cells stimulated with mezerein and lentil lectin (solid triangles) had 102 U/ml antiviral activity. washed. Effects of Partially Purified IFNy.rupress. I f so. An equivalent amount of monoclonal antibody to a mouse H2 antigen (the kind gift of Dr. Means _ SEM for triplicates. i n c u b a t i o n o f m a c r o p h a g e s in the l y m p h o i d s u p e r n a t a n t s t h a n in c o n t r o l m e d i a (n = 15).3 1 3 10 _ O 30 100 300 I F'NY.o r a n t i g e n . Enhancement of macrophage peroxide-releasing capacity by three IFN"r-containing preparations and its prevention by monoclonal anti-IFN~' antibody.r e l e a s i n g capacity. An independently purified preparation from buffy coat cells stimulated with staphylococcal enterotoxin A (solid squares) had an activity of 6 x 103 U/ml or 1 X 107 U/mg protein. was the sole f a c t o r in the m i t o g e n . and stimulated with PMA to measure H20~ release.I F N ' y a n t i b o d y (data n o t shown).. Human macrophages were exposed to the indicated concentrations of IFN~ on days 4-7 of culture. Ulml FIGURE 1. R. a r e s u m m a r i z e d Downloaded from jem.org on July 21. t h e n native I F N y s h o u l d have M A F activity. A fraction obtained from this supernatant (solid circles) had an antiviral activity of 104 U/ml or t x 10~ U/rag protein.# 0. N e i t h e r the elevations n o r the r e d u c t i o n s in a d h e r e n t cell p r o t e i n seen in individual e x p e r i m e n t s w e r e consistently a f f e c t e d by m o n o c l o n a l a n t i .

compared with 12 + 2 in the controls.-rich fractions contained 1.0. After 2 more days in IFN% peroxide secretory capacity fell to 652 + 53. Peroxide-releasing capacity usually (but not invariably) peaked on the third day of exposure to partially purified IFN% and thereafter declined somewhat.706.0. 2102 919 _ 155 (13) Inhibition by monoclonalantibody! % 2. For example. 5.4 and 1. 675 TABLE II Stimulation of Human Macrophage Peroxide-releasing Capacity by Partially Purified Native IFN'y: Prevention by Monoclonal Antibody to IFN'y Preparation H~O~release by. compared to 393 _+ 20 for controls. SEM for H202 release by triplicate cultures averaged 9.8-fold.3.-treated macrophages on days 7-9 sometimes exceeded that secreted by samples of the same monocytes on day 0.3 antiviral U / m l . 41. 1). 1236.Published September 1. 652. Downloaded from jem. However.0ll See Table I. **LK induced with staphylococcalenterotoxin A and fractionated as described in Materials and Methods. Effects ofRecombina nt IFN'y. 11. compared to 101 _+ 25 for the controls. despite replenishment with fresh IFN% However. when IFN7 was removed on day 8. cultures incubated in IFN~.9. These kinetics closely parallel those reported earlier for unfractionated lymphoid supernatants (14).-mediated enhancement of macrophage peroxide-releasing capacity by an average of 97% in seven experiments (Table II).IFN-treated cells tivity(U/ tration (nmol/mg p r o mg protein) IFN~. 15. 3.7~ 102. and peak effects were seen at 30 and 100 U/ml. 1549.4. In two titrations of preparation SEA-IFN-r (Fig. 1479. On the average. T o establish conclusively that IFN3.3 times as much adherent cell protein as those in medium alone (n = 13).1. 2015 in Table I I. 435 Overall means + 8. macrophages treated with IFN~. This was unaffected by the antibody (not shown). 50% of maximal stimulation of macrophage peroxide-releasing capacity followed incubation in 0.org on July 21. 12261 211. 1983 NATHAN ET AL. 8.5.5 + 0. I F N-r-rich fractions enhanced macrophage peroxidereleasing capacity 8.5. Excludes the highest value (435). Thus. 12. ** LK induced with mezerein + lentil lectin and fractionated as described in Materialsand Methods. On the average. the longest period tested.367.8. Specific ac.1. and to evaluate the possible contribution of other lymphoid cell products that might copurify with . 80.8 _ 1. 103. on macrophage peroxide release was reversible. peroxide releasing-capacity of cells continuously exposed to IFN7 remained markedly elevated for at least 5 d. secretion on day 11 fell to 23 _+ 4. Monoclonal anti-IFN'r antibody inhibited IFN~. 6.479 + 124 n m o l / m g p r o t e i n / 6 0 min. the enhancing effect of partially purified IFN~.5% of the mean in individual experiments.5. 1241 H202 release experimental* controli cells 2./** 1 X 106 10-242 SEA-IFN3. could activate niacrophages for enhanced hydrogen peroxide release.used tein/60 min) U/ml MLL-IFN'. 122 24.Concen. 108.rupress. 6.482. from days 5-8 released 1.381. 1168. Peroxide released by IFN~.~ 1 X 10 7 30-300 392. 7.8 (12)M 97 _+ 10 (7) SEM (n): .

The potent MAF activity of recombinant IFN'y is illustrated for three independent titrations in Fig. 3).org on July 21. and 1000 U/ml.3. recombinant 1FN3. Enhancement of macrophage peroxide-releasing capacity by pure. T h e dashed line (solid triangles) indicates results in the presence of monoclonal antibody to native IFNy added at (from left to right) 3.009. with peak responses at 0. 10. Two experiments are shown in which macrophages were exposed to the indicated concentrations of IFNy on days 6 . and 1.9 (open circles and triangles) and one experiment in which exposure was from days 5-8 of culture (open squares). The positions of the three dose-response curves varied considerably. AS M A C R O P H A G E . we next tested the effects of pure IFNy produced by bacteria transformed with the cloned buman gene for this LK.. Half-maximal stimulation of macrophage peroxide-releasing capacity followed 3 d of exposure to 0.A C T I V A T I N G FACTOR \ 0 £ 1" 200 oL< J" -3 -i -.rupress. some of the macrophages were exposed to 100 U / m l of IFN7 for 10 min on day 6 of culture.3 U / m l of IFNT. or I00 times the neutralizing activity for the indicated concentration of native ]FN7. 2015 IFNy. Their peroxide-releasing capacity was at least as great as that of . With continuous exposure to IFN% peroxide-releasing capacity remained elevated for at least 6 d. and then cultured in standard medium for 3 more days. Means of triplicates are indicated. The variability in the dose-response curves might be due in part to the use of different donors for the macrophages and the serum in each experiment. Another possible contributory factor is the fact that the time to peak response varied from 2 d of exposure (not shown) to as long as 4 d (Fig. 3. but fell toward baseline within 3 d of removing the recombinant IFN'r (Fig.14. o i i 3 . 10. 0.Published September 1. extensively washed to remove IFNy. as already noted for native IFN% In the experiment illustrated in Fig. SEMs averaged 13% of the mean. 2. Downloaded from jem. IFN~° I ~ l o ( U / ml ) FIGURE 2. 1983 676 INTERFERON-'). 3).

I ~. T h e dashed line indicates results for cells which were removed from IFN7 on day 9. cells exposed continuously to 100 U / m l IFN~. The remarkable potency of recombinant IFN7 compared with. E °E '°°/I'll \\ £ 20oe 6 7 8 9 10 11 12 Days FIGURE 3. washed four times in standard medium. 2015 01 . respectively. 2 with antibody concentrations ranging from 3 to 100 times the amount required to neutralize an equivalent antiviral activity of native IFNT.native IFN7 in some experiments raised the possibility that the former might contain a co- Downloaded from jem. Antibody GIF-1 also failed to inhibit the antiviral activity of recombinant IFN3. monoclonal antibody GIF-1 had no inhibitory effect on the MAF activity of recombinant IFNT. Results were similar in a second experiment using a 2-h pulse. Means + SEM of triplicates.org on July 21. then incubated in the latter for 3 d before assay. This is illustrated in Fig. washed four times.rupress. 1983 677 N A T H A N ET AL. T h e open triangle indicates cells which were pulsed with 100 U / m l IFN7 for 10 min on day 6. and incubated in standard medium until assay on day 9.0oo o C- L 600 Q. Fresh medium of the same type was added on day 8 or 9 for cells assayed on days 11 or 12. for the entire 3-d period. Kinetics of response to recombinant 1FNT. Macrophages on day 6 of culture were incubated in standard medium (solid circles) or 100 U / m l of IFN7 (open circles) for the indicated times before assay of H20~ release.Published September 1. (data not shown). In contrast to the results with native IFNT.

unstimulated h u m a n m a c r o p h a g e s cultured for 1 3 .3 3 313_+ 11 0 1. An effective but suboptimal dose o f r e c o m b i n a n t IFN3.SEM for triplicates. but to some extent by o x y g e n . Secretion o f h y d r o g e n p e r o x i d e by m a c r o p h a g e s was undetectable or barely detectable after incubation in either native or r e c o m b i n a n t IFN3.3 0 313 _ 11 0 0. We next tested w h e t h e r I F N ~ also a u g m e n t s the antimicrobial activity o f m a c r o p h a g e s against an intracellular p a t h o g e n . when the cells were p r e i n c u b a t e d for 3 d with unfractionated mitogen. unless a s e c r e t a g o g u e such as P M A was a d d e d (data not shown).0 100 478 _+8 * Macrophages were treated with IFN3. T h e a b o v e e x p e r i m e n t s established that IFN3. § Recombinant IFN3. or a Costimulator in Recombinal~t IFN3'* SEA-IFN3.org on July 21. T h e killing o f this parasite by m u r i n e m a c r o p h a g e s is closely related to their capacity to secrete h y d r o g e n p e r o x i d e (1 1) a n d is m e d i a t e d largely by oxidative mechanisms (1 1. did not give g r e a t e r than additive effects when c o m b i n e d with a suboptimal but effective dose o f the native product. hTduction of Antitoxoplasma Activity. we used the p r o t o z o a n ..3 100 448 _+ 12 390 30 1.. mixing e x p e r i m e n t s were carried out as shown in T a b l e III. T. and s u p p o r t e d intracellular replication (5.* Recom.. As a test organism.1 0 139 _+28 0 0. when neutralized by monoclonal antibody a n d a d d e d to r e c o m b i n a n t IFN3. .. Downloaded from jem. ([SEA-IFN3" + Aby]'. T h u s . As shown in Fig.or . AS MACROPHAGE-ACTIVATING FACTOR TABLE Ill Evide~. * Partially purified. 4. 36).5 t o x o p l a s m a s / v a c u o l e at 20 h).ce Against a Suppressor Factor in Partially Purified IFN3.2 3 d b e f o r e infection killed > 5 % o f ingested toxoplasmas in the first 4 h.0 100 320 _+9 302 6 0. In contrast.). I Monoclonal antibody G|F-I (does not neutralize recombinant IFN3. 29).. gondii.15) + 15. from days 6-9 of culture.Published September 1.i n d e p e n d e n t processes as well (16. 1983 678 INTERFERON-3.§ Aby| H~O2. nmol/mg protein/60 min Predicted value for additive effects~ U/ml U/ml U/ml 0 0 0 15_+ 11"* 6 0 0 178 + 16 30 0 0 367 _+ 16 30 0 100 85 _+8 0 0. did not give less than additive effects. ** Means --. An optimal dose o f native IFN3. T o test these possibilities. where 15 is the value in medium alone.0 0 332 + 8 0 1.rupress. native IFN'y elicited with staphylococcal enterotoxin A (see Table II). neither costimulator n o r suppressor factors could be detected by mixing.1 0 199 _+ 13 383 30 0..15) + (Recom. enhances the oxidative metabolism o f h u m a n macrophages.t h e cardinal criterion for m a c r o p h a g e activation. 2015 stimulator or the latter m i g h t contain a suppressor of MAF activity.

In the experiment illustrated in Fig.. and inhibited the replication of surviving organisms (1... toxoplasmacidal activity was evident after treatment with as little as 1 U / m l IFN3. H~II~L IL.. .. antibody was used at 300-600 neutralizing U/ml. 5.Published September 1. .. .. Means + SEM for the indicated number of experiments. . Under these conditions. 5). exp. . macrophages that had been in culture for 13 d were exposed to IFN3. 1983 NATHAN ET AL. but not with polyclonal sheep antibodies against human IFN~ or IFN/3 (Fig. Monoclonalanti-IFN3.. T h e induction of antiparasitic activity was abrogated by treatment of the unfractionated supernatants or the partially purified IFN3' with monoclonal anti-IFN3.8-1. Toxoplasmacidal activity of macrophages incubated in lymphokine (LK) preparations with or without antibody to the indicated types of IFN. ... anti -"f 2 I 4 ~ none II killed at 4 h 40 50 60 70 Con A II II II toxo II I I I Illlllllll Illl Illlllllllllll I I [ !. 679 Percent toxoplasmas Aby LK No...2 . " FIC. Pure.rupress.org on July 21..URE4. Unpurified LK were induced with concanavalin A (Con A) or toxoplasma antigen (toxo).. there was variability in tile optimal period of preincubation in recombinant I F N y to activate macrophage Downloaded from jem.. . for 3 more days before challenge with the parasite. . . Partially purified native IFN3~preparation SEA-IFN3"had an activityof l0 T U/mg protein.._ SEA II Recom. Recom. . antibody. . recombinant IFN3.. antigen-induced LK or partially purified native IFN% they killed 38-60% of the initially ingested parasites within 4 h. 0 20 10 30 7 1- anti -T 2 I- anti-T "4 IlI- anti -~ anti -/3 1 anti-T 1 1 t II anti -~x 2 _. As with enhancement of peroxide-releasing capacity.. 2015 . 4). 4) in a dose-dependent m a n n e r (Fig. .LL 12LL222. . Li II anti-/3 2 . Addition of 300 U / m l of recombinant IFN3' had no effect when it was added after infection of the macrophages (not shown). . recombinant IFN3'.... Anti-IFNa and anti-IFN# antibodies were used at 600 neutralizing U/ml. Direct exposure to 10% concanavalin A-induced LK or to 300 U / m l of partially purified native I F N y for t h at 37 ° C did not affect the ability of toxoplasmas to survive and replicate when subsequently ingested by unstimulated macrophages (data not shown).. Incubations were in 100-300 U/ml IFNy for 2-3 d beginning on days 13-23 of culture...9 parasites/vacuole at 20 h). also markedly enhanced macrophage antitoxoplasma activity (Fig. . .

Thus. the cells tended to spread in a more disklike fashion and often had highly rounded. Decreased spreading of human monocytes incubated in IFNa or IFNfl has been attributed to inhibition of their maturation into macrophages (37. 1983 680 I N T E R F E R O N .org on July 21. Monocytes treated from days 0 to 2 with 100 U / m l IFNy killed 42% of the organisms at 4 h and displayed 1. 7 a). The effects of recombinant IFN3. 6 summarizes three such experiments. killed 23% of ingested organisms by 4 h and limited the replication of the remainder to 2. 7 c). 7 b).0 per vacuole by 20 h. Addition of monoclonal anti-IFNy forestalled these shape changes (Fig. Peak activity was seen from 2-3 d after adding IFNy. the morpho- Downloaded from jem. with flat nuclear regions that were dark by phase-contrast microscopy (Fig. 2015 o eu L. Macrophages incubated in control medium were usually bipolar. toxoplasmacidal activity. Effect o f the concentration o f recombinant IFNy on the toxoplasmacidal (solid circles) and toxoplasmastatic (open triangles) activity o f human macrophages after 3 d exposure to IFN'r beginning on day 13 o f culture. but were not always evident by the time peroxide-releasing capacity was elevated.Published September 1.4 40 < O~ E 3o 0 ~ 2o . U / rnl FIGURE 5. or fan-shaped. The same shape changes were seen after prolonged exposure to recombinant IFNy (Fig.y AS M A C R O P H A G E . were less dramatic when IFNy was added from the outset of culture (day 0) to monocytes that were challenged with toxoplasmas on day 2. We examined the effects of applying IFNy-containing media to cells that had already differentiated into macrophages.4 toxoplasmas/vacuole at 20 h. 7 d). In this case. Effects of IFNy on Cell Shape. 10 . multipolar.2 .1 0 0. 38).1 1 10 ~ 100 IFN~. refractile nuclear regions (Fig.rupress. the untreated controls.A C T I V A T I N G F A C T O R 6 --I l- o 50 Ul 3 "0 . In unfractionated lymphoid supernatants or in partially purified native IFNy. which could release copious H20~. Fig.

2015 20 .org on July 21. Time course of induction of toxoplasmacidal (solid circles) and toxoplasmastatic (open triangles) activity of macrophages exposed to 300 U / m [ recombinant IFN'y beginning on days 10.. suggests that macrophage activation may be one of the primary physiologic functions of this LK.rupress.. Under the conditions tested. logic alterations did not appear to be necessary for enhanced peroxide-releasing capacity. induction of antiviral resistance. With recombinant IFN% activation of macrophage peroxide-releasing capacity was stimulated to 50% of the maximal value with a geometric mean concentration of 0. I O' 0 0 1 2 Days incubation in IFN~ 3 FIGURE 6.4 (~ 3o 3 E ~) 'o_ 2 10- 1 o ~3r L I> 13. The remarkable sensitivity of the oxidative metabolism of human macrophages to enhancement by IFN3. then the 50% maximally effective concentration of the preparation used here can be estimated at 0.SEM for three experiments at each time point except two experiments at 3 d. 16.or mitogen-stimulated human leukocytes was IFN3'. Discussion We conclude that IFN7 is a potent activator of human macrophage oxidative metabolism and antimicrobial activity.Published September 1. 39.s . Means +. 6 pM). is the LK for which the most sensitive bioassay is available.a O I/~ "0 4o . or 23 of culture. It is now unarguable that more than one LK activity (as defined by effects on Downloaded from jem. Assuming that recombinant IFN7 is a nonglycosylated dimer of 34. 34. 42).4-63 picomolar (geometric mean. Thus it is of special interest that it capacitates macrophages to release a microbicidal product (H202) whose chemical composition is also known. 1983 N A T H A N ET AL. 6O A 681 [ • --4 1-. 40) of known structure (41. namely. the only such activator consistently detected in the medium of antigen.1 antiviral U/ml. IFN-).5o .292 daltons. IFN7 is the first secretory product o f T iymphocytes (33.

the recombinant IFN3. Four qualifications require emphasis. the term "MAF" has been applied to factors that induce a variety of physiologic changes in macrophages. it is now clear that a single LK can cause pleiotropic effects in the same cell population. D.org on July 21. or if present. were masked by inhibitors or dependent on IFN3. are only a few of an unknown number of alterations induced in the same cells by the same Downloaded from jem. as already noted. Nathan. and M.. C. used in our studies has also been reported to enhance the expression of DR antigens (19) and Fc receptors (25) on monocytes. Morphology of IFNT-treated macrophages by phase contrast microscopy. in order to activate macrophages. control cells in standard medium on day 8.rupress. and to activate monocytes to kill tumor cells (43). Our experiments do suggest that other MAFs were either not consistently present in active amounts in the lymphoid supernatants we studied. that can induce the same changes in macrophages as described here. treated from days 6-12 with 100 U/ml recombinant IFN3. .. IFNa. macrophages exposed from days 5-8 to partially purified native IFNT.Published September 1. Third. unpublished observations). In addition. First. We have used the term in a restricted sense. A. Second. Murray. B. 2015 FIGURE 7. our studies are fully compatible with the possibility that there may be other factors besides IFN3. H. and our results do not bear on the issue whether MAF as defined in other ways or in other species is IFN7 (reviewed in reference 21). B. same as B but a4so exposed to monoclonal antibody against IFN3. Wiebe. the biochemical and functional changes we measured in macrophages in response to IFN3. induces antiviral activity in a variety of cell types and enhances the capacity of macrophages to secrete H 2 0 2 and to kill toxoplasmas. a product of non-T leukocytes. is of special interest in this regard (C. IFN3. 1983 682 INTERFERON--y AS MACROPHAGE-ACTIVAT1NG FACTOR different target cells) can be ascribed to the same molecule. Thus. For example. Rubin.

Recombinant IFN~. it was necessary to determine directly whether native. and inhibitors of IFN antiviral activity are reportedly produced by stimulated lymphocytes (47). Monoclonal antibody GIF-1 neutralized both the antiviral and macrophageactivating effects of native IFN~. was enriched to at least the same degree as antiviral activity. These carbohydrates are not necessary for the expression of at least some of the antiviral (45) or MAF activity of IFN% However. the partially purified native IFN~' could have been contaminated with a factor suppressing macrophage oxidative metabolism. no cells other than macrophages could be identified by morphologic criteria. yet neutralized neither of these effects of the recombinant bacterial product. Exposures to IFN~' as brief as 10-120 min led to substantial activation when macrophages were tested 3 d later. It is possible that the carbohydrate groups contribute to the epitope seen by the antibody. In most of these cultures. remained. varied from 2-4 d. This preparation displayed especially potent MAF activity. Neutralization of MAF activity in crude lymphoid supernatants by the monoclonal anti-IFN~. but was elevated for at least 6 d when IFN~. Accordingly. 2015 molecule. First. The antibody might interfere with the function of this domain by steric hindrance. was usually considerably more potent than the equivalent concentration of native IFN'y. was the only human protein and probably the only protein. phages to display an optimal response to recombinant IFN~. IFN~. activity.rupress. without neutralizing recombinant IFN'y made it possible to perform mixing experiments. There are at least three possible explanations for this apparent discrepancy. After 3 d of exposure to IFN% peroxide secretory capacity fell to barely detectable levels when IFN~. was added starting on the 3rd through the 23rd day of culture. the carbohydrate moieties may border a domain necessary for IFN~. purified to apparent homogeneity from bacteria transformed with the cloned gene for human IFN3. The time required for macro.Published September 1. There are believed to be two carbohydrate chains on native IFN7 (26. in which native IFN3. namely. 683 Downloaded from jem. such as a receptor-binding site. For a definitive answer. However. antibody was strong evidence that MAF was IFN% However. preparations plus antibody GIF-1 were added to recombinant IFN% No . antibody.. the epitopes recognized by monoclonal antibodies may sometimes be shared by seemingly unrelated proteins (44). 16) may also be IFN% Finally. these preparations of IFN"r were not pure. Such a suppressive factor has been observed in the culture medium of murine lymphoid cells (46). we turned to a preparation in which IFN~. we have not formally excluded the possibility that the effects of IFN3. and the possibility remained that a contaminant contributed to MAF activity. was removed. The ability of monoclonal antibody GIF-1 to neutralize native IFN~.org on July 21. results were similar whether IFN~. 1983 N A T H A N ET AL. Using two independent protocols for induction and partial purification of IFN% MAF activity was readily demonstrated. based on antiviral activity. 42) and none on the recombinant product. Experiments are in progress to determine whether the lymphokine that enhances oxygen-independent antimicrobial activity in human macrophages (15. 41.. Nonetheless. leukocyte-derived IFN~ had MAF activity. and was again neutralized by monoclonal anti-IFN~. on macrophages could have been mediated by another type of cell contaminating the cultures.

C. using the same culture conditions as in the present work (C. The reasons for these variations are not well understood. 62). pure IFN/3 was not used. However. salmonellae (58). These include some of the most prevalent chronic infections. 61.Published September 1. unpublished observations). malaria (55). lepromatous leprosy.org on July 21. no costimulator activity could be demonstrated. There is precedent for variations in ratios of different IFN effects when comparing natural and recombinant IFNs (49). The same lymphoid supernatants contained IFN~ but not IFNa or IFNB. such as tuberculosis. The capacity of host lymphocytes to secrete IFNy mounts in parallel with other manifestations of delayed-type hypersensitivity and cell-mediated immunity (34. In contrast.rupress. Effectiveness after such dilution virtually rules out a contribution by traces of LPS in the IFN% or by any other minor contaminant. a suppressive factor seemed to be present in the unpurified LK preparations when they were used at high concentrations. Furthermore. Thus it seems appropriate to ask whether provision of IFNy might favorably affect the course of diseases in which persistent parasitization of macrophages is a prominent feature. concanavalin A. staphylococci (59). 2015 suppression of the macrophage-activating effect of the latter was observed. gondii (51-54). Sevastopoulos) (limit of detection. we have not been able to enhance human macrophage H 2 0 2 release with LPS. This is consistent with the evidence presented here in support of the hypothesis that IFNy mediates macrophage activation during the latter responses. There have been almost no previous reports on the effects of IFN on macrophage oxidative metabolism.05 ng/ml). Rickettsia (56). or staphylococcal enterotoxin A secreted a factor (macrophage-activating factor. that one molecule of recombinant IFN~. A second possible explanation for the relative potency of recombinant IFN3. (50) reported that IFN/3 inhibited the superoxide-releasing capacity of mouse peritoneal macrophages. shigellae (57). and leishmaniasis. or MAF) that enhanced the capacity of human macrophages to release H~O2 and to kill toxoplasmas. The MAr Downloaded from jem. This preparation was effective after a dilution of more than 10%fold. on a protein basis. perhaps because of differences in posttranslational modifications. may either have more potent MAF a n d / o r less potent antiviral activity than one molecule of native IFNy. SEA-IFNy and recombinant 1FN3' had similar MAF activity. Nathan. In fact. Finally. mezerein plus lentil lectin. including T. 0. Dr. Boraschi et al. It will be of interest to determine the relation between the inhibitory activity in that study and the factor suppressing macrophage oxidative metabolism that was detected in medium conditioned by a variety of cell types. such as bacterial LPS (48). in mixing experiments. including fibroblasts (46). . and mycobacteria (60). is that there could be a costimulator contaminating it. However. Thus. However. no LPS was detectable in the purified recombinant IFNy by the limulus amoebocyte lysate test (personal communication. 1983 684 INTERFERON-'r AS MACROPHAGE-ACTIVATING FACTOR Summary Human blood mononuclear leukocytes stimulated with toxoplasma antigen. a third possibility must be considered. numerous reports have suggested that IFN may enhance antimicrobial activity of a variety of cells against pathogens other than viruses. 39. The nature of such a suppressive factor and its possible role in anergic states warrant further study.

The capacity of macrophages to secrete HzOu after incubation in partially purified native IFN3. andJ. 6175. R. Inc. but the effect of IFN3. Med. Schreiber and J. Nathan. 1. David. 137:275. G. Native IFN3' partially purified by two independent protocols to specific activities of 1 x 106 and 107 U / r a g protein was enriched in MAF activity at least as much as in antiviral activity. Sylvia Anderson. Exp. was reversed within about 3 d of its removal. J.. IFN3. W. L. The MAF activity of the partially purified native IFN~. 8. S. R. Remington.6 + 1. B. In vitro induction of nonspecific Downloaded from jem. and J..org on July 21. had potent MAF activity.) from bacteria transformed with the cloned human gene for this lymphokine. Vil~ek for communicating results prior to publication. Philadelphia. B. References Mackaness. F.. G. Mackaness. 5. IFN3.1 antiviral U / m ] of recombinant IFN3. David. and Richard Bonomo for their skillful assistance. preparations was abolished by monoclonal anti-IFN3. editor.8-fold at peak response and enhancing their ability to kill toxoplasmas from 2. F.Published September 1. 1969. Exp.4 d after exposure to IFN3. Thus. M. L. Krahenbuhl. Alterations of macrophage functions by mediators from lymphocytes. The mechanism of macrophage activation. J... 129:973. Pawlowski for advice. C. G. Karnovsky. 3.. Peroxide secretory capacity and toxoplasmacidal activity of macrophages peaked 2 . and appeared to be the only factor consistently capable of doing so in the diverse LK preparations tested. Exp. C.8-fold) and sometimes equaled or exceeded the capacity of freshly harvested monocytes. Remold. ! 33:1356. 2015 activity of six of seven unfractionated supernatants was completely eliminated by a monoclonal antibody that neutralizes IFN% and MAF in the remaining supernatant was almost completely neutralized. for their generous provision of recombinant IFN~. . in particular Sang-He Lee and Costa Sevastopoulos. M. Finally. Mudd. Activation was usually but not invariably accompanied by characteristic changes in cell morphology. We thank Genentecb. The influence of immunologically committed lymphoid cells on macrophage activity in vivo. and R. In Infectious Agents and Host Reactions. Receivedfor publication 4 May 1983. 1971. Saunders Co.4% for treated cells. Attainment of 50% of the maximal elevation in peroxide-releasing capacity required a geometric mean concentration of 0. Med. Spitalny and N. Peroxide-secretory capacity remained elevated during at least 6 d of continuous exposure. H. 685 Zanvil Cohn is warmly acknowledged for encouraging us to undertake these studies and for his critical advice and review. which is estimated to be -~6 picomolar for this preparation. J. Recombinant IFN3.J. Carol Deboer. S. Med.. 4.. Nathan.. andJ. and Judy Adams and Anna Szuro-Sudol for aid in figure preparation. G. (mean peak stimulation. Inc. activates human macrophage oxidative metabolism and antimicrobial activity. B. and for helpful suggestions.rupress. We are grateful to Nancy DeSantis.3% for untreated cells to 54 + 0. 1971. Tobie Overdank. 1973. Denise Cartelli.8-fold) was greater than with unpurified ]ymphokines (3. Chang for purification of MLL-IFN3. Characterization of a lymphocyte factor which alters macrophage functions. 2. 1970. stimulating the peroxide-releasing capacity of macrophages an average of 19. of >99% estimated purity was isolated (at Genentech. 1983 NATHAN ET AL.

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