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Glucocorticoid modulation of lymphokine-induced macrophage proliferation
CELLULAR
IMMUNOLOGY
67, 23-36 (1982)
Glucocorticoid
Modulation of Lymphokine-Induced Macrophage Proliferation’
MATTHEW R. DUNCAN,JOHN R. SADLIK,ANDJOHN Laboratory
of lmmunopharmacology, New Received
October
York,
Sloan-Kettering Institute New York 10021
7, 1981:
accepted
December
for
W. HADDEN Cancer
Research,
1. 1981
The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC,, of triamcinolone acetonide (TA): 2 X 10m9M), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (I&, of TA: 4 X lo-’ M), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC,, of TA: 3 X 10-s M), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms. in vitro
INTRODUCTION Glucocorticoids play a major role in the treatment of inflammatory and immunologically mediated diseases. The precise mechanism by which glucocorticoids attenuate immunologic inflammatory responses is still unresolved but the functions of the mononuclear phagocyte system are known to be sensitive to glucocorticoid action. In vivo the anti-inflammatory effects of glucocorticoids are associated with decreases in the number of macrophages present in the inflammatory site and with changes in their states of activation (l-3). The anti-inflammatory action of glucocorticoids could occur at any stage of macrophage involvement in inflammation, from the initial recruitment of cells in the bone marrow to the final effector stage. Recent studies in vitro indicate the regulation of macrophage function by soluble T-lymphocyte products, called lymphokines, to be a major locus of glucocorticoid action (4-7). However, controversy exists as to whether glucocorticoids act to suppress the induction phase of the immune response necessary for lymphokine synthesis or the effector phase induced by lymphokine action. Glucocorticoids have ’ This work was supported by grants from the National Institutes of Health (CA-08748, CA-20178). 23 0008-8749/82/030023-14$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.
24
DUNCAN,
SADLIK,
AND HADDEN
in various reports been shown either to solely prevent lymphokine synthesis by activated lymphocytes (6, 7), to only inhibit the interaction between lymphokine and effector macrophage (4, 5), or to inhibit both processes (6, 7). The previously described macrophage mitogenic factor (MMF)’ assay of lymphokine-induced macrophage proliferation in the guinea pig is an in vitro correlate of the macrophage proliferation observed during inflammation (8), as well as being an immune equivalent of the cytokine-induced macrophage proliferation produced by fibroblast macrophage growth factor (9). It provides a model system for the analysis of immunopharmacologic agents acting on the macrophage. We therefore attempted to clarify further the inhibitory mechanism of glucocorticoids on delayed hypersensitivity reactions using the MMF assay. Studies using the guinea pig system have particular relevance to human physiology as lymphocytes from both species are, unlike lymphocytes from rats and mice, resistant to direct cytolysis by glucocorticoids ( 10). Our studies indicate that physiological concentrations of glucocorticoids inhibit lymphokine synthesis by immune lymphocytes but that pharmacological concentrations are necessary to inhibit MMF-induced macrophage proliferation. In additional experiments, glucocorticoids were shown to further control macrophage proliferation by potentiating the action of high concentrations of MMF via a mechanism unrelated to prostaglandin synthesis. MATERIALS Production of MMF-Containing phocytes
AND METHODS
Supernatants by Guinea Pig Lymph Node Lym-
Hartley strain guinea pigs (200 to 400 g) were immunized with bovine y-globulin (BGG) in Freund’s complete adjuvant supplemented with heat-killed tubercle bacilli (H,,Rv) as previously described (8). Regional lymph nodes were harvested 14 to 17 days later and the cells were collected and cultured at a concentration of 1.5 X lO’/ml in Eagle’s minimal essential medium with 100 units/ml penicillin and 100 pg/ml streptomycin (referred hereafter as medium) without serum, with or without BGG (1 mg/ml). After incubation at 37°C in a 5% CO2 and air atmosphere for 36 hr the cell-free supernatants were collected and antigen was added to the control supernatant. In the subsequent experiments, supernatants preincubated with antigen (P) are compared to those reconstituted with antigen after culture (R). In other experiments molar concentrations of steroid hormones and/or indomethacin (Sigma Chemical Co., St. Louis, MO.) were added to the lymphocyte incubation medium. In these cases, harvested supernatants were extensively dialyzed against fresh medium to remove the steroids and/or indomethacin before testing for MMF activity. The antiglucocorticoid, 17a-methylprogesterone, was synthesized as described previously ( 11). * Abbreviations used: MMF, macrophage mitogenic factor; BGG, bovine y-globulin; P supernatant, supernatants preincubated with antigen; R control, supernatants reconstituted with antigen after incubation; HBSS, Hanks’ balanced salt solution; FCS, fetal calf serum; MIF, macrophage migration inhibitory factor; TA, triamcinolone acetonide; MCF, monocyte chemotactic factor; I(E)&,, concentration giving half-maximum inhibition (effect); IL.1, interleukin 1; IL.2, interleukin 2; MAgF, macrophage aggregation factor; MAF, macrophage-activating factor; CAMP, adenosine 3’:5’-cyclic monophosphate.
GLUCOCORTICOIDS
Preparation of Partially
AND MACROPHAGE
PROLIFERATION
25
Puri$ed MMF
Partial purification of MMF was accomplished by concentration of supernatants by vacuum dialysis and gel filtration using Sephadex G-100 chromatography as previously described (8). These procedures yielded an active fraction in the range of 35,000 to 70,000 daltons. Active fraction volume was adjusted to one-fourth the volume of original crude supernatant applied to the column. Dilution values given for MMF are relative to the volume of the original supernatant. Preparation of Macrophages
Peritoneal exudate cells used for assay of MMF activity were collected from normal, nonsensitized guinea pigs 72 hr after ip injection of 20 ml of sterile light paraffin oil (Fisher Scientific Co., Fairlawn, N.J.). Animals were sacrificed by cervical dislocation. Exudate cells were collected in 150 ml of cold Hank’s balanced salt solution containing 100 units/ml of penicillin and 100 pg/ml of streptomycin (HBSS) and were washed twice in HBSS by centrifugation at 220g for 10 min at 4°C. Assay of MMF
Activity
The proliferative effect of MMF-rich supernatants or partially purified MMF was assayed by tritiated thymidine uptake into microcultures (Microtest II-Falcon 3042, Falcon Division, Beckton, Dickinson, Oxnard, Calif.) of guinea pig peritoneal macrophage monolayers as has been previously described (8). One-tenth milliliter of suspension of macrophages (2 X 106/ml) in HBSS was added to wells of a microtest plate. Attachment proceeded for only 15 min at 37°C in COZ incubator. Attachment for such a short time circumvented the contamination with fibroblasts that can be seen if longer times are used for attachment. Wells were washed three times with HBSS, containing 10% fetal calf serum (FCS) to yield a monolayer of >98% pure macrophage (8) and 0.2 ml medium containing 20% FCS (Microbiological Associates, Bethesda, Md.), and various dilutions of MMF preparations were added to each well. The culture plates were incubated at 37°C in a humidified atmosphere of 3% CO2 in air for 3 days. Thymidine incorporation was determined by a terminal 24-hr pulse of [3H]thymidine (2.50 &i/ml, sp act 20 Ci/mmol, New England Nuclear, Boston, Mass.). At termination of culture the plate was frozen at -70°C and thawed and refrozen twice before processing with a multiple automatic sample harvester (Otto Hiller Co., Madison, Wise.) and liquid scintillation spectrophotometry. Assay of MMF activity in the presence of steroids or indomethacin was done by adding dilutions in medium of 10e2 M ethanolic stock solutions directly to the microcultures. These low concentrations of ethanol had no effect on the assay. In those experiments where a comparative quantitative assay of MMF was performed, it was accomplished by comparing the counts per minute (cpm) caused by a l/32 dilution of a MMF supernatant prepared in the presence of steroids and/or indomethacin to a linearized dilution curve (l/16 to l/256) of a concomitantly prepared standard MMF supernatant. Assay of Macrophage Migration
Inhibitory
Factor (MZF) Activity
Inhibition of migration of guinea pig peritoneal macrophages by MMF containing supernatants was assayed and quantified as previously described ( 12).
26
DUNCAN,
SADLIK,
AND HADDEN
160-
140
$ E
-
120-
8 x
loo-
I 0
60-
2 tii ,”
60-
: 40
-
20-
I
lo-"
IdO
10-g
I 10-a
TRIAMCINOLONE
I 10-7
ACETONIDE
# 10-g
I lb-s
10-d
(molar)
FIG. 1. Effect of varying concentrations of triamcinolone acetonide on the production of MMF by guinea pig lymph node cells in the absence (-) or presence (- - -) of indomethacin (1 Om5M). Points and vertical bars represent the mean f SD of two separate experiments of five replicate determinations of [3H]thymidine incorporation by macrophage monolayers stimulated by a l/32 dilution of MMF-rich supernatants generated in the presence of molar concentrations of triamcinolone acetonide. MMF production was determined as described under Materials and Methods and is expressed as a percentage of MMF produced by cultures containing neither steroid nor indomethacin.
RESULTS Effect of Glucocorticoids on Production of MMF
by Sensitized Lymphocytes
To determine if glucocorticoid inhibition of cell-mediated immunity might in part be due to a suppression of MMF production during the induction phase, guinea pig lymph node lymphocytes were stimulated with BGG and cultured in the presence of triamcinolone acetonide (TA) (10-i’ to 10m4 M). After 36 hr of culture, supernatant samples were harvested, dialyzed against fresh medium, and assayed for MMF activity. BGG-stimulated lymph node cells were found to be extremely sensitive to the presence of triamcinolone acetonide (Fig. 1). A concentration-dependent triamcinolone acetonide-induced inhibition of MMF production was observed. Cultures containing 1 X lop4 M triamcinolone acetonide produced less than 10% of the MMF produced by control cultures, while the concentration inducing half-maximum inhibition (I&,) of MMF production was estimated as 2x 10-9M. Since prostaglandins are also potent inhibitors of several lymphocyte functions including the production of some lymphokines and prostaglandins are produced
GLUCOCORTICOIDS
AND MACROPHAGE TABLE
27
PROLIFERATION
1
Effect of Various Steroids on the Production of MMF and MIF by Guinea Pig Lymph Node Cells Steroid (1 Om6M) None Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone Cortisone Progesterone Testosterone 17@‘-estradiol Cortexolone ( 1O-5 M) TA ( 10m6M) + cortexolone ( 10e5 M)
MMF production” (% control) 100 + 29f 23+ 30 f 58f 49f 89 f 85k 92f 93f 91f 65 f
8* 8 5 12 7 9 13 11 8 9 9 11’
MIF production” (% control) 100 f 11 35 f 14 92+ 10 102 f 12’
’ MMF-rich supernatants were produced in the presence of steroids or combination of steroids listed and assayed at a l/32 dilution for macrophage monolayer proliferation and at a l/2 dilution for inhibition of macrophage migration as described under Materials and Methods. MMF and MIF production are expressed as a percentage of MMF or MIF produced by cultures without steroids. b Values are the means of six determinations for MMF and three determinations for MIF + SD. ‘Significantly different (P < 0.05) from triamcinolone acetonide (TA) 10e6 M by two-tailed Student’s t test.
during lymphokine generation (13), we examined the effect of triamcinolone acetonide on MMF production in the presence of indomethacin ( 10e5 M), an inhibitor of cyclooxygenase-mediated prostaglandin synthesis, to eliminate interference by endogenously generated prostaglandins. While indomethacin constantly augmented the BGG-induced production of MMF by 50 to 60%, a concentration-dependent triamcinolone acetonide-induced inhibiton of MMF production was observed even in the presence of indomethacin (Fig. 1). Moreover, the concentration-response curve paralleled that generated in the absence of indomethacin indicating that triamcinolone acetonide inhibition of MMF production and prostaglandin biosynthesis are likely unrelated processes. Evidence indicating that the inhibition of MMF production was truly a glucocorticoid-specific phenomenon is displayed in Table 1. In specificity experiments, sensitized lymph node lymphocytes were cultured with BGG in the presence of nine steroid hormones at a concentration of lop6 M. Only cultures treated with active glucocorticoids showed depressed MMF production, whereas an inactive glucocorticoid analog (cortisone) and the sex hormones (progesterone, testosterone, and estradiol) had no inhibitory effects. Moreover, the active glucocorticoids inhibited MMF production according to their relative potencies as inhibitors of lymphocyte activation ( 14) and inflammation (15). Studies with lymphocytes from several species have established that most effects of glucocorticoids are mediated by specific macromolecular binding proteins, referred to as receptors (16). Since we observed inhibition of MMF production at concentrations of triamcinolone acetonide reported to saturate receptor binding it is likely that glucocorticoid inhibition of MMF production occurs via a receptor-
28
DUNCAN,
SADLIK,
AND HADDEN
o----0%--...J \\ I
‘\ ‘\ \\I
0
\
\ :
\ \ !
0 t
,;.11,‘o-To ,;a ,;a ,;a TRIAMCINOLONE
,f-.
ACETONIDE
FIG. 2. Effect of varying concentrations of triamcinolone acetonide on guinea pig peritoneal macrophage proliferation stimulated by a suboptimal (l/ 16) dilution of partially purified MMF in the absence (p) or presence (- - -) of indomethacin ( 10e5 M). Points and bars represent the mean f SD of two separate experiments of six replicate determinations of [ ‘Hlthymidine incorporation. Monolayer proliferation in the absence of steroid was 74,918 cpm; in the presence of indomethacin, 110,735 cpm; R control was 3942 cpm.
mediated process. This theory is supported by the observation that cortexolone, an antagonist of glucocorticoid-receptor interaction (16), blocked the triamcinolone acetonide-induced inhibition of MMF production, although inactive alone (Table 1). Furthermore, assay of supernatants for concomitant production of MIF revealed that cortexolone alone had no effect, but in combination with triamcinolone acetonide blocked the glucocorticoid-induced inhibition of MIF production (Table 1). Effect of Triamcinolone liferation
Acetonide
(1O-’ M) on MMF-Induced
Macrophage
Pro-
Because glucocorticoid-induced inhibition of MMF production might not be solely responsible for previous reports demonstrating suppression of macrophage proliferation in inflammatory exudates (l-3), we also investigated if glucocorticoids
GLUCOCORTICOIDS
AND MACROPHAGE TABLE
PROLIFERATION
2
Relative Glucocorticoid Inhibition of Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Suboptimal (l/16) Dilution of MMF Steroid
Estimated I&O of) 4.0 4.1 4.2 7.5 9.2
Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone ’ ICso were estimated for each listed steroid from concentration-response in Fig. 2.
x lo-’ x lo-’ X 1O-6 x lo+ X lo-’
curves generated as detailed
inhibited the effector phase of delayed hypersensitivity via a suppression of MMFinduced macrophage proliferation. In these experiments guinea pig peritoneal macrophage monolayers were stimulated with various dilutions of partially purified MMF and cultured in the presence or absence of a tentative receptor saturating concentration of triamcinolone acetonide (lo-’ M) (17). After 3 days of culture, monolayers were harvested and assayed for [ 3H]thymidine incorporation. Macrophage proliferation in response to MMF was concentration dependent and generated a bell-shaped concentration-response profile similar to that previously reported (8). Triamcinolone acetonide was found to be both a negative and a positive regulator of MMF-induced macrophage proliferation. Triamcinolone acetonide (IO-’ M) inhibited the macrophage proliferation induced by suboptimal dilutions (l/64 to l/16) of MMF, while potentiating supraoptimal dilutions (l/4 to l), resulting in a net shift of the concentration-response profile toward higher concentrations of MMF (data not shown). Effect of Glucocorticoids on Macrophage Proliferation timal (l/16) Dilution of MMF
Stimulated by a Subop-
To further investigate the observed triamcinolone acetonide (lo-’ M)-induced inhibition of suboptimal MMF-stimulated macrophage proliferation, macrophage monolayers were stimulated with 1/ 16 dilution of MMF in the presence of varying concentrations of triamcinolone acetonide. A concentration-dependent triamcinolone acetonide-induced inhibition of macrophage proliferation was observed (Fig. 2). Proliferation in cultures containing 1 X 10m4 M triamcinolone acetonide was only 25% of that occurring in control cultures, and the I& was estimated at 2 X lo-’ M. A similar concentration-dependent inhibition of MMF-induced macrophage proliferation was observed with triamcinolone acetonide at MMF dilutions of l/32 and l/64 (data not shown). Since exogenous prostaglandins are inhibitors of MMF action (8) and MMF preparations increase prostaglandin synthesis (9) the triamcinolone acetonide-induced inhibition of MMF action was investigated in the presence of indomethacin, to prevent interference by endogenously generated prostaglandins. As previously reported (8), indomethacin ( 10m5 M) consistently augmented the MMF-induced macrophage proliferation by increases of 50 to 60% (Fig. 2) while inhibiting the
30
DUNCAN,
SADLIK,
AND HADDEN
TABLE 3 Antagonism by Cortexolone of the Glucocorticoid Inhibition of Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Suboptimal (l/16) Dilution of MMF Steroid None Cortexolone ( 1O-’ M) Triamcinolone acetonide ( 10m6M) TA ( 1Oe6 M) + cortexolone (lo-’ M)
Macrophage proliferation’ (% control) 100 94 34 64
+ + f +
7b 5 8 8’
a Macrophage proliferation is expressed as a percentage of proliferation stimulated by MMF without steroid (75,825 cpm). b Values are the means of six determinations + SD. ’ Significantly different (P < 0.05) from triamcinolone acetonide (TA) 10m6M by two-tailed Student’s t test.
formation of cyclooxygenase products from labeled arachidonic acid (data not shown). However, a concentration-dependent triamcinolone acetonide-induced inhibition of MMF-stimulated proliferation was observed even in the presence of indomethacin, and the concentration-response curve paralleled that generated in the absence of indomethacin (Fig. 2). Thus triamcinolone acetonide inhibited MMF action through a mechanism unrelated to prostaglandin biosynthesis. As indicated by the results in Table 2, the inhibition of suboptimal MMF action was a glucocorticoid-specific phenomenon. Only active glucocorticoids inhibited MMF action, whereas cortisone and cultures containing lop5 M progesterone, testosterone, or estradiol had no inhibitory effect (data not shown). The active glucocorticoids inhibited MMF action according to their relative anti-inflammatory potencies. Although IC&‘s were about 100 times higher than the concentrations reported to give half-maximum saturation of glucocorticoid receptors in mouse and rabbit macrophages (17), it is likely that the inhibition was receptor mediated as the glucocorticoid antagonist, cortexolone, reversed the triamcinolone acetonideinduced suppression of proliferation (Table 3). Effect of Glucocorticoids on Macrophage Proliferation Stimulated by a Supraoptimal (I/2) Dilution of MMF A more detailed study of the observed triamcinolone acetonide ( 1Oe7M)-induced potentiation of macrophage proliferation stimulated by supraoptimal dilutions of MMF was also carried out. Macrophage monolayers were stimulated with a l/2 dilution of MMF in the presence of varying concentrations of triamcinolone acetonide. A concentration-dependent potentiation of macrophage proliferation was observed, although high concentrations of triamcinolone acetonide ( 10e5 to lop4 M) caused less potentiation than intermediate concentrations ( 10d7 to lop6 M) (Fig. 3). Proliferation in cultures containing 10e6 M triamcinolone acetonide was twofold that occurring in control cultures. An ECSo of 2 X 10e8 A4 was estimated from the ascending portion (lo-” to 10m6M) of the concentration-response profile. Glucocorticoids inhibit the production of prostaglandins in various cell types, including macrophages, by blocking the release of arachidonic acid from phospholipids (18). Since prostaglandins also inhibit MMF action (8) and MMF preparations increase prostaglandin synthesis (9) we investigated whether the triamci-
GLUCOCORTICOIDS
AND MACROPHAGE
31
PROLIFERATION
70.
60
I: . ii :: : ii :: :. .1ii::::;:1 ;i ::ii1::.-ii:: J
ii :: :: ii ii :: :: :: :: :: ii
. i: :: :: :: :: ;; :::: :: .* :: / ::
i 10-l’ IO”0 TRIAMCINOLONE
1o-g
10-8 ACETONIDE
i; ii :: :: :: :: :: :: :: :: ::. . :: ii :: :: :: ii ::. . 1: :: ; ::
::. . ii :: ii i; ii I; :::: ::. . ii ii .::. .::. :::: :: f ..
I-’
lo-’
J
:: .. :: .. :: .. ii .. ii :: ii ::. . ii ii ii ; :: lo-)
i
:: :: :: :: :: :: ii :: :: :: .. ii :: ;I
lo-.
IMOLAR)
FIG. 3. Effect of varying concentrations of triamcinolone acetonide on guinea pig periotoneal macrophage proliferation stimulated by a supraoptimal (l/2) dilution of partially purified MMF in the absence (Ll) or presence (0) of indomethacin (lo-’ M). Columns and bars represent the mean f SD of three separate experiments of six replicate determination of [3H]thymidine incorporation. R control was 2985 cpm.
nolone acetonide-induced potentiation of macrophage proliferation was due to inhibition of macrophage prostaglandin synthesis. We found this not to be the case, as triamcinolone acetonide caused a concentration-dependent potentiation of MMFdriven proliferation even in the presence of a total blockade of prostaglandin synthesis by indomethacin (Fig. 3). Results indicated that the potentiation of the action of supraoptimal dilutions of MMF was a glucocorticoid-specific event (Table 4). Only cultures treated with active glucocorticoids showed increased proliferation, whereas, inactive analogs (cortisone) and sex steroids (progesterone, testosterone, estradiol) had no potentiating effects. The active glucocorticoids increased MMF-driven proliferation according to their relative potencies in other glucocorticoid-sensitive assays. As noted for glucocorticoid inhibitory actions on MMF production and action at suboptimal MMF concentrations, glucocorticoid antagonists also reversed this glucocorticoidinduced phenomenon. The antiglucocorticoids, cortexolone, progesterone, and 17 a-methylprogesterone, while inactive alone, all inhibited the triamcinolone acetonide-induced potentiation of MMF-driven proliferation (Table 4), suggesting that this glucocorticoid response is also receptor mediated. DISCUSSION Previous investigations have shown that great numbers of mononuclear phagocytes are present in sites of immunologic inflammation in viva ( 19-21). The pop-
32
DUNCAN,
SADLIK, TABLE
AND HADDEN 4
Effect of Various Steroids on Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Supraoptimal (l/2) dilution of MMF Steroid (lo-’
Macrophage proliferation” (W control)
M)
None Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone Cortisone Progesterone Testosterone 1‘IO-estradiol Cortexolone (lo-’ M) Progesterone (lo-’ M) 17a-methylprogesterone ( 1Oe5 M) TA (lo-’ M) + cortexolone ( 1Om5M) TA (lo-’ M) + progesterone ( low5 M) TA (lo-’ M) + 17a-methylprogesterone
( 10m5M)
100 f 126 251+ 8 230 3~ 15 174 + 10 147+ 9 113+ 4 95 + 14 97+ 13 103 * 10 102 f 12 94+ 4 72+ 16 79* 12 102 f 13’ 95 f 14’ 99+ 9
’ Macrophage proliferation is expressed as a percentage of proliferation induced by MMF without steroid (18,652 cpm). b Values are the means of three separate experiments of six determinations each + SD. ’ Significantly different (P < 0.05) from triamcinolone acetonide (TA) lo-’ M by two-tailed Student’s t test.
ulation of mononuclear phagocytes present in these inflammatory sites is controlled by both the immigration of circulating blood monocytes into the lesion and by the cell division of the recently immigrated monocytes and resident macrophages within the lesion (19-21). The number of macrophages undergoing replication varies with the site of inflammation, ranging from a limited 5% proliferation in cutaneous granuloma to much higher values in sites with numerous resident macrophages such as liver and peritoneum. Both the immigration and the proliferation of these mononuclear phagocytes are thought to be controlled by lymphokines. Combined in vivo-in vitro evidence has suggested that monocytes/macrophages are first attracted to sites of antigen T lymphocyte interaction by the lymphokine monocyte chemotactic factor (MCF) (22), retained in such sites by MIF (22), and then induced to proliferate by MMF (8, 9) or MMF-like factors (24). The anti-inflammatory action of glucocorticoids may be mediated through the macrophage as glucocorticoid administration has been shown to profoundly reduce the number of macrophages in inflammatory sites in vivo (l-3). Studies in vitro suggested that glucocorticoids reduce the population of inflammatory macrophages by suppressing lymphokine-mediated processes (4-7). However, it is not clear whether glucocorticoids exerted their major suppressive effect on lymphokine synthesis by lymphocytes or on the macrophage response to lymphokine. The results of experimentation detailed herein, using MMF-induced macrophage proliferation as in in vitro model, suggest two sites of glucocorticoid modulation in delayed
GLUCOCORTICOIDS
AND MACROPHAGE
PROLIFERATION
33
hypersensitivity reactions. However, glucocorticoids had their major suppressive effect on lymphokine production by lymphocytes, inhibiting both MMF and MIF generation at physiological concentrations, while larger pharmacological concentrations were required to regulate MMF-driven macrophage proliferation. The postreceptor mechanism by which glucocorticoids inhibited MMF production by sensitized lymphocytes was investigated only insofar as to show cyclooxygenase prostaglandin products were not involved. However, since the conditions used to generate MMF also resulted in the production of other lymphokines, including MIF (Table 1) and MCF (data not shown), observations from other studies in which glucocorticoids inhibited lymphokine production permit some speculation. The production of both MCF and MIF has been reported suppressed by near physiological concentrations of hydrocortisone (6, 7). The observations that inhibition of MCF production occurred only if hydrocortisone was added early in culture and that hydrocortisone also caused a parallel concentration-dependent inhibition of lymphocyte proliferation suggest that glucocorticoids inhibit lymphokine generation by suppressing some early event involved in lymphocyte activation. One early event in lymphocyte activation known to be inhibited by physiological concentrations of glucocorticoids is the release of the monokine interleukin 1 (IL. 1) from antigen/mitogen-stimulated macrophages (25). IL. 1 is obligatory for stimulated T-lymphocyte production of the lymphokine, interleukin 2 (IL.2), whose action culminates in T-lymphocyte proliferation (26). Since the presence of viable macrophages is obligatory for the production of other lymphokines such as macrophage-activating factor (MAF) and MCF (27), it is possible that IL.1 may be necessary for the production of most lymphokines, including MMF, and that glucocorticoids universally inhibit T-cell lymphokine production by suppressing IL. 1 release. However, other studies indicate that nonviable macrophages can support the generation of some lymphokines, including interferon (28) and MIF (29) and thus the exact locus of glucocorticoid inhibition of lymphocyte activation remains uncertain. Whatever the mechanism, most glucocorticoid actions are believed to be initiated by glucocorticoid binding to specific cytosol receptors and mediated by the interaction of this activated steroid-receptor complex with selected chormosomal sites, resulting ultimately in control of the synthesis of specific protein products (16). Observations from three separate experiments suggest that the glucocorticoid-induced inhibition of MMF production was a receptor-mediated phenomenon. First, the ICsO for triamcinolone acetonide inhibition of MMF production was equivalent to the concentration of triamcinolone acetonide required for half-maximum saturation of lymphocyte receptor binding sites (30). Second, the inhibition of production caused by other glucocorticoids paralleled their relative receptor affinities ( 14, 15). Third, cortexolone, an antagonist which displaces glucocorticoid-receptor binding (16) also blocked the inhibition of production of both MMF and MIF by triamcinolone acetonide. While we must conclude that glucocorticoids exert their major suppressive effect on the initiation phase of immune responses, additional experiments described herein also indicate that glucocorticoids have a regulatory function at the level of the effector mechanisms. Glucocorticoids were found to exert both a negative and a positive effect on MMF-driven macrophage proliferation, depending on the concentration of MMF present.
34
DUNCAN,
SADLIK,
AND
HADDEN
Macrophage proliferation stimulated by suboptimal dilutions of MMF was inhibited by glucocorticoids in a concentration-dependent and potency-related manner (Fig. 2 and Table 3). However, it is unlikely that the observed inhibition represents a physiological mechanism, as the estimated ICSO’s are 100 times the reported glucocorticoid concentrations at half-maximum receptor saturation in mouse and rabbit macrophages ( 17). Similarly, other investigators have shown that pharmacological concentrations of glucocorticoids were necessary to inhibit the action of several lymphokines including guinea pig macrophage aggregation factor (MAgF) and MIF (4-7) and rat IL.2 (31). Nevertheless, suppression of the effector phase of cell-mediated immunity by glucocorticoid inhibition of MMF-induced macrophage proliferation may still be partially responsible for the observed antiinflammatory effects of glucocorticoids as pharmacological doses of glucocorticoids can easily produce an in vivo concentration of 10e5 A4 (32). Even though pharmacological glucocorticoid concentrations were necessary to inhibit MMF-induced macrophage proliferation, the fact that the inhibition was concentration dependent, glucocorticoid specific, and blocked by a glucocorticoid antagonist suggests that it was receptor mediated. The events distal to receptorgenome interaction responsible for the glucocorticoid inhibition of MMF action are not known, but could involve an alteration in MMF receptors, resulting in a decreased MMF signal transmission. In contrast to the suppression observed with suboptimal dilutions of MMF, glucocorticoids potentiated the macrophage proliferation induced by supraoptimal dilutions of MMF. The observed potentiation was not only significant but also occurred at close to physiological concentrations (ECSO triamcinolone acetonide 3 X lo-* M). The potentiation exhibited all the characteristics of a receptor-mediated glucocorticoid action, being concentration dependent, potency related, and suppressed by glucocorticoid antagonists (Fig. 3, Table 4). The postreceptor mechanism responsible for the glucocorticoid potentiation of supraoptimal MMF action is unknown and may simply be due to a decrease in MMF signal transmission, However, the observation that high concentration of MMF stimulated only limited macrophage proliferation, resulting in a bell-shaped concentration response curve (8), suggests that macrophage proliferation induced by supraoptimal dilutions of MMF is subject to inhibitory controls not operative at lower MMF concentrations. This is true for mitogen-induced lymphocyte proliferation where high concentrations of mitogens induce elevated intracellular levels of adenosine 3’:5’-cyclic monophosphate (CAMP) which acts to limit lymphocyte proliferation (33). Similarly, it is possible that high concentrations of MMF may induce an antiproliferative factor that acts to limit MMF-driven macrophage proliferation. Alternatively, an antiproliferative factor could be a minor contaminant in partially purified MMF present in effective amounts only at higher MMF concentrations. In either case, glucocorticoids could potentiate the action of supraoptimal MMF by suppressing the action or production of the antiproliferative factor. Evidence for two separate factors controlling supraoptimal MMF action (i.e., MMF and an antiproliferative factor) is suggested by the fact that ECso of triamcinolone acetonide for the inhibition of suboptimal MMF action is a log greater than that for potentiation of supraoptimal MMF and by the observation that the highest concentrations of triamcinolone acetonide did not give maximum potentiation of supraoptimal MMF action.
GLUCOCORTICOIDS
AND MACROPHAGE
PROLIFERATION
35
While the nature of the antiproliferative factor remains speculative, one class of substances known to inhibit macrophage proliferation are the prostaglandins (8). Furthermore, MMF preparations are known to increase prostaglandin production (9) and glucocorticoids have inhibited prostaglandin synthesis in starchelicited macrophages (34). However, the glucocorticoid potentiation of supraoptimal MMF-induced macrophage proliferation was not due to a suppression of prostaglandin synthesis, as triamcinolone acetone caused a concentration-dependent potentiation even in the presence of indomethacin. Thus, the exact nature of the antiproliferative factor remains unknown but additional preliminary experiments indicate that the factor is a heat-labile component present in both MMF-rich supernatants and partially purified MMF preparations. Heating MMF preparations at 70°C for 2 min ablated the effect of high concentrations of MMF to induce less than optimum proliferation and a plateau-shaped response curve resulted. Triamcinolone acetonide (lo-’ M) failed to potentiate the action of high concentrations of this heat-treated MMF but rather was inhibitory at all MMF concentrations. The relationship of the observed potentiation of supraoptimal MMF-induced macrophage proliferation to the regulation of delayed hypersensitivity reactions in vivo is not clear. However, if the heat-labile antiproliferative factor is a physiological product involved in the inflammatory response, then the observed potentiation may be operative in vivo. If such is the case, then the anti-inflammatory action of glucocorticoids might be entirely due to the suppression of lymphokine synthesis during the induction phase as the net effect of glucocorticoids on the lymphokinemediated effector phase would depend on the in vivo concentration of MMF, with glucocorticoid suppression occurring at low MMF levels but glucocorticoid augmentation occurring at higher MMF concentrations. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
14. 15. 16. 17. 18. 19.
Thompson, J., and van Furth, R., J. Exp. Med. 131, 429, 1970. North, R. J., J. Exp. Med. 134, 1485, 1971. McCue, R. E., Dannenberg, Jr., A. M., Higuchi, S., and Sugimato, M., InfEammation 3, 159, 1978. Weston, W. L., Claman, H. N., and Krueger, G. C., J. Immunol. 110, 880, 1973. Balow, J. E., and Rosenthal, A. S., J. Exp. Med. 137, 1031, 1973. Wahl, S. M., Altman, L. C., and Rosenstreich, D. L., J. Immunol. 115, 476, 1975. Wahl, S. M., and Rosenstreich, D. L., In “Mitogens in Immunobiology” (J. J. Oppenheim and D. L. Rosenstreich, Eds.), pp. 537-553. Academic Press, New York, 1976. Hadden, J. W., Sadlik, J. R., and Hadden, E. M., J. Immunol. 121, 231, 1978. Hadden, J. W., Sadlik, J. R., Englard, A., Warfel, A. W., and Hadden, E. M., In “Biochemical Characterization of Lymphokines” (A. L. de Week, Ed.), pp. 235-242. Academic Press, New York, 1980. Claman, H. N., Moorhead, J. W., and Benner, W. H., J. Lab. C/in. Med. 78, 499, 1971. Duncan, M. R., and Duncan, G. R. J. Steroid Biochem. 10, 245, 1979. Leu, R. W., Eddleston, W. F., Hadden, J. W., and Good, R. A., J. Exp. Med. 136, 589, 1972. Bray, M. A., Gordon, D., and Morley, J., Prostaglandins Med. 1, 183, 1978. Neifeld, J. R. Lippman, M. E., and Tormey, D. C., J. Biol. Chem. 252, 2972, 1977. Haynes, R. C., and Larner, J., In “The Pharmacological Basis of Therapeutics” (L. S. Goodman and A. Gilman, Eds.), pp. 1472-1506. MacMillan, New York, 1975. Munck, A., and Leung, K., In “Receptors and Mechanism of Action of Steroid Hormones, Part II” (J. R. Pasqualini, Ed.), pp. 31 l-397. Dekker, New York, 1977. Werb, Z., Foley, R., and Munck, A., J. Exp. Med. 147, 1684, 1978. Hong, S. -C. L., and Levine, L., Proc. Nut. Acud. Sci. USA 73, 1730, 1976. Spector, W. G., and Mariano, M., In “Mononuclear Phagocytes in Immunity, Infection, and Pathology” (R. VanFurth, Ed.), pp. 927-938, Blackwell, Oxford, 1975.
36
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AND HADDEN
20. North, R. J., J. Exp. Med. 130, 299, 1969. 21. North, R. J., J. Exp. Med. 130, 315, 1969. 22. Wahl, S. M., Altman, L. C., Oppenheim, J. J., and Mergenhagen, S. E., Int. Arch. Allergy 46, 786, 1974. 23. Bennett, B., and Bloom, B. R., Proc. Nat. Acad. Sci. USA 59, 756, 1968. 24. Wynne, K. M., Spector, W. G., and Willoughby, D. A., Nature (London) 253, 636, 1975. 25. Smith, K. A., Munck, A., and Lachman, L., In “Abstracts of 4th International Congress of Immunology” (J. L. Preud’homme and V. A. L. Hawken, Eds.), Abstract 14.3.54, 1980. 26. Smith, K. A., Baker, P. E., Gillis, S., and Ruscetti, F. W., Mol. Immunol. 17, 579, 1980. 27. Wahl, S. M., Wilton, J. M., Rosenstreich, D. L., and Oppenheim, J. J., J. Immunol. 114, 1296, 1975. 28. Epstein, L. B., Cline, M. J., and Merigan, T. C., J. Clin. Invest. 50, 744, 1971. 29. Toffaletti, D., Yoshida, T., and Cohen, S., Cell. Immunol. 56, 487, 1980. 30. Bell, P. A., and Munck, A., Eiockem. J. 136, 97, 1973. 31. Gillis, S., Crabtree, G. R., and Smith, K. A., J. Zmmunol. 123, 1624, 1979. 32. Webel, M. L., and Ritts, Jr., R. E., Cell. Immunol. 32, 287, 1977. 33. Hadden, J. W., Hadden, E. M., Sadlik, J. R., and Coffey, R. G., Proc. Nat. Acad. Sci. USA 73, 1717, 1976. 34. Bray, M. A., and Gordon, D., Brit. J. Pkarmacol. 63, 635, 1978.
IMMUNOLOGY
67, 23-36 (1982)
Glucocorticoid
Modulation of Lymphokine-Induced Macrophage Proliferation’
MATTHEW R. DUNCAN,JOHN R. SADLIK,ANDJOHN Laboratory
of lmmunopharmacology, New Received
October
York,
Sloan-Kettering Institute New York 10021
7, 1981:
accepted
December
for
W. HADDEN Cancer
Research,
1. 1981
The ability of glucocorticoids to modify lymphokine-induced macrophage proliferation, an correlate of cellular immunity in the guinea pig, was investigated. Lymphocyte production of macrophage mitogenic factor (MMF) was decreased in the presence of physiological concentrations of glucocorticoids. Inhibition was concentration dependent (IC,, of triamcinolone acetonide (TA): 2 X 10m9M), glucocorticoid specific, and reversed by cortexolone. In contrast, pharmacological concentrations of glucocorticoids were necessary to inhibit macrophage proliferation induced by suboptimal dilutions of MMF. This inhibition was concentration dependent (I&, of TA: 4 X lo-’ M), glucocorticoid specific, and reversed by cortexolone. At supraoptimal dilutions of MMF, glucocorticoids caused a twofold potentiation of MMF-induced macrophage proliferation. Potentiation was concentration dependent (EC,, of TA: 3 X 10-s M), glucocorticoid specific, reversed by glucocorticoid antagonists, and occurred in the presence of indomethacin. Thus, glucocorticoids regulate both the initiation and effector phases of this in vitro model of delayed hypersensitivity. However, the results indicate that the major mechanism of glucocorticoid-mediated anti-inflammatory action occurs at the level of the MMF-producing lymphocyte rather than at the effector macrophage, as MMF-induced proliferation is likely controlled by opposing glucocorticoid-sensitive mechanisms. in vitro
INTRODUCTION Glucocorticoids play a major role in the treatment of inflammatory and immunologically mediated diseases. The precise mechanism by which glucocorticoids attenuate immunologic inflammatory responses is still unresolved but the functions of the mononuclear phagocyte system are known to be sensitive to glucocorticoid action. In vivo the anti-inflammatory effects of glucocorticoids are associated with decreases in the number of macrophages present in the inflammatory site and with changes in their states of activation (l-3). The anti-inflammatory action of glucocorticoids could occur at any stage of macrophage involvement in inflammation, from the initial recruitment of cells in the bone marrow to the final effector stage. Recent studies in vitro indicate the regulation of macrophage function by soluble T-lymphocyte products, called lymphokines, to be a major locus of glucocorticoid action (4-7). However, controversy exists as to whether glucocorticoids act to suppress the induction phase of the immune response necessary for lymphokine synthesis or the effector phase induced by lymphokine action. Glucocorticoids have ’ This work was supported by grants from the National Institutes of Health (CA-08748, CA-20178). 23 0008-8749/82/030023-14$02.00/O Copyright Q 1982 by Academic Press, Inc. All rights of reproduction in any form rescrvcd.
24
DUNCAN,
SADLIK,
AND HADDEN
in various reports been shown either to solely prevent lymphokine synthesis by activated lymphocytes (6, 7), to only inhibit the interaction between lymphokine and effector macrophage (4, 5), or to inhibit both processes (6, 7). The previously described macrophage mitogenic factor (MMF)’ assay of lymphokine-induced macrophage proliferation in the guinea pig is an in vitro correlate of the macrophage proliferation observed during inflammation (8), as well as being an immune equivalent of the cytokine-induced macrophage proliferation produced by fibroblast macrophage growth factor (9). It provides a model system for the analysis of immunopharmacologic agents acting on the macrophage. We therefore attempted to clarify further the inhibitory mechanism of glucocorticoids on delayed hypersensitivity reactions using the MMF assay. Studies using the guinea pig system have particular relevance to human physiology as lymphocytes from both species are, unlike lymphocytes from rats and mice, resistant to direct cytolysis by glucocorticoids ( 10). Our studies indicate that physiological concentrations of glucocorticoids inhibit lymphokine synthesis by immune lymphocytes but that pharmacological concentrations are necessary to inhibit MMF-induced macrophage proliferation. In additional experiments, glucocorticoids were shown to further control macrophage proliferation by potentiating the action of high concentrations of MMF via a mechanism unrelated to prostaglandin synthesis. MATERIALS Production of MMF-Containing phocytes
AND METHODS
Supernatants by Guinea Pig Lymph Node Lym-
Hartley strain guinea pigs (200 to 400 g) were immunized with bovine y-globulin (BGG) in Freund’s complete adjuvant supplemented with heat-killed tubercle bacilli (H,,Rv) as previously described (8). Regional lymph nodes were harvested 14 to 17 days later and the cells were collected and cultured at a concentration of 1.5 X lO’/ml in Eagle’s minimal essential medium with 100 units/ml penicillin and 100 pg/ml streptomycin (referred hereafter as medium) without serum, with or without BGG (1 mg/ml). After incubation at 37°C in a 5% CO2 and air atmosphere for 36 hr the cell-free supernatants were collected and antigen was added to the control supernatant. In the subsequent experiments, supernatants preincubated with antigen (P) are compared to those reconstituted with antigen after culture (R). In other experiments molar concentrations of steroid hormones and/or indomethacin (Sigma Chemical Co., St. Louis, MO.) were added to the lymphocyte incubation medium. In these cases, harvested supernatants were extensively dialyzed against fresh medium to remove the steroids and/or indomethacin before testing for MMF activity. The antiglucocorticoid, 17a-methylprogesterone, was synthesized as described previously ( 11). * Abbreviations used: MMF, macrophage mitogenic factor; BGG, bovine y-globulin; P supernatant, supernatants preincubated with antigen; R control, supernatants reconstituted with antigen after incubation; HBSS, Hanks’ balanced salt solution; FCS, fetal calf serum; MIF, macrophage migration inhibitory factor; TA, triamcinolone acetonide; MCF, monocyte chemotactic factor; I(E)&,, concentration giving half-maximum inhibition (effect); IL.1, interleukin 1; IL.2, interleukin 2; MAgF, macrophage aggregation factor; MAF, macrophage-activating factor; CAMP, adenosine 3’:5’-cyclic monophosphate.
GLUCOCORTICOIDS
Preparation of Partially
AND MACROPHAGE
PROLIFERATION
25
Puri$ed MMF
Partial purification of MMF was accomplished by concentration of supernatants by vacuum dialysis and gel filtration using Sephadex G-100 chromatography as previously described (8). These procedures yielded an active fraction in the range of 35,000 to 70,000 daltons. Active fraction volume was adjusted to one-fourth the volume of original crude supernatant applied to the column. Dilution values given for MMF are relative to the volume of the original supernatant. Preparation of Macrophages
Peritoneal exudate cells used for assay of MMF activity were collected from normal, nonsensitized guinea pigs 72 hr after ip injection of 20 ml of sterile light paraffin oil (Fisher Scientific Co., Fairlawn, N.J.). Animals were sacrificed by cervical dislocation. Exudate cells were collected in 150 ml of cold Hank’s balanced salt solution containing 100 units/ml of penicillin and 100 pg/ml of streptomycin (HBSS) and were washed twice in HBSS by centrifugation at 220g for 10 min at 4°C. Assay of MMF
Activity
The proliferative effect of MMF-rich supernatants or partially purified MMF was assayed by tritiated thymidine uptake into microcultures (Microtest II-Falcon 3042, Falcon Division, Beckton, Dickinson, Oxnard, Calif.) of guinea pig peritoneal macrophage monolayers as has been previously described (8). One-tenth milliliter of suspension of macrophages (2 X 106/ml) in HBSS was added to wells of a microtest plate. Attachment proceeded for only 15 min at 37°C in COZ incubator. Attachment for such a short time circumvented the contamination with fibroblasts that can be seen if longer times are used for attachment. Wells were washed three times with HBSS, containing 10% fetal calf serum (FCS) to yield a monolayer of >98% pure macrophage (8) and 0.2 ml medium containing 20% FCS (Microbiological Associates, Bethesda, Md.), and various dilutions of MMF preparations were added to each well. The culture plates were incubated at 37°C in a humidified atmosphere of 3% CO2 in air for 3 days. Thymidine incorporation was determined by a terminal 24-hr pulse of [3H]thymidine (2.50 &i/ml, sp act 20 Ci/mmol, New England Nuclear, Boston, Mass.). At termination of culture the plate was frozen at -70°C and thawed and refrozen twice before processing with a multiple automatic sample harvester (Otto Hiller Co., Madison, Wise.) and liquid scintillation spectrophotometry. Assay of MMF activity in the presence of steroids or indomethacin was done by adding dilutions in medium of 10e2 M ethanolic stock solutions directly to the microcultures. These low concentrations of ethanol had no effect on the assay. In those experiments where a comparative quantitative assay of MMF was performed, it was accomplished by comparing the counts per minute (cpm) caused by a l/32 dilution of a MMF supernatant prepared in the presence of steroids and/or indomethacin to a linearized dilution curve (l/16 to l/256) of a concomitantly prepared standard MMF supernatant. Assay of Macrophage Migration
Inhibitory
Factor (MZF) Activity
Inhibition of migration of guinea pig peritoneal macrophages by MMF containing supernatants was assayed and quantified as previously described ( 12).
26
DUNCAN,
SADLIK,
AND HADDEN
160-
140
$ E
-
120-
8 x
loo-
I 0
60-
2 tii ,”
60-
: 40
-
20-
I
lo-"
IdO
10-g
I 10-a
TRIAMCINOLONE
I 10-7
ACETONIDE
# 10-g
I lb-s
10-d
(molar)
FIG. 1. Effect of varying concentrations of triamcinolone acetonide on the production of MMF by guinea pig lymph node cells in the absence (-) or presence (- - -) of indomethacin (1 Om5M). Points and vertical bars represent the mean f SD of two separate experiments of five replicate determinations of [3H]thymidine incorporation by macrophage monolayers stimulated by a l/32 dilution of MMF-rich supernatants generated in the presence of molar concentrations of triamcinolone acetonide. MMF production was determined as described under Materials and Methods and is expressed as a percentage of MMF produced by cultures containing neither steroid nor indomethacin.
RESULTS Effect of Glucocorticoids on Production of MMF
by Sensitized Lymphocytes
To determine if glucocorticoid inhibition of cell-mediated immunity might in part be due to a suppression of MMF production during the induction phase, guinea pig lymph node lymphocytes were stimulated with BGG and cultured in the presence of triamcinolone acetonide (TA) (10-i’ to 10m4 M). After 36 hr of culture, supernatant samples were harvested, dialyzed against fresh medium, and assayed for MMF activity. BGG-stimulated lymph node cells were found to be extremely sensitive to the presence of triamcinolone acetonide (Fig. 1). A concentration-dependent triamcinolone acetonide-induced inhibition of MMF production was observed. Cultures containing 1 X lop4 M triamcinolone acetonide produced less than 10% of the MMF produced by control cultures, while the concentration inducing half-maximum inhibition (I&,) of MMF production was estimated as 2x 10-9M. Since prostaglandins are also potent inhibitors of several lymphocyte functions including the production of some lymphokines and prostaglandins are produced
GLUCOCORTICOIDS
AND MACROPHAGE TABLE
27
PROLIFERATION
1
Effect of Various Steroids on the Production of MMF and MIF by Guinea Pig Lymph Node Cells Steroid (1 Om6M) None Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone Cortisone Progesterone Testosterone 17@‘-estradiol Cortexolone ( 1O-5 M) TA ( 10m6M) + cortexolone ( 10e5 M)
MMF production” (% control) 100 + 29f 23+ 30 f 58f 49f 89 f 85k 92f 93f 91f 65 f
8* 8 5 12 7 9 13 11 8 9 9 11’
MIF production” (% control) 100 f 11 35 f 14 92+ 10 102 f 12’
’ MMF-rich supernatants were produced in the presence of steroids or combination of steroids listed and assayed at a l/32 dilution for macrophage monolayer proliferation and at a l/2 dilution for inhibition of macrophage migration as described under Materials and Methods. MMF and MIF production are expressed as a percentage of MMF or MIF produced by cultures without steroids. b Values are the means of six determinations for MMF and three determinations for MIF + SD. ‘Significantly different (P < 0.05) from triamcinolone acetonide (TA) 10e6 M by two-tailed Student’s t test.
during lymphokine generation (13), we examined the effect of triamcinolone acetonide on MMF production in the presence of indomethacin ( 10e5 M), an inhibitor of cyclooxygenase-mediated prostaglandin synthesis, to eliminate interference by endogenously generated prostaglandins. While indomethacin constantly augmented the BGG-induced production of MMF by 50 to 60%, a concentration-dependent triamcinolone acetonide-induced inhibiton of MMF production was observed even in the presence of indomethacin (Fig. 1). Moreover, the concentration-response curve paralleled that generated in the absence of indomethacin indicating that triamcinolone acetonide inhibition of MMF production and prostaglandin biosynthesis are likely unrelated processes. Evidence indicating that the inhibition of MMF production was truly a glucocorticoid-specific phenomenon is displayed in Table 1. In specificity experiments, sensitized lymph node lymphocytes were cultured with BGG in the presence of nine steroid hormones at a concentration of lop6 M. Only cultures treated with active glucocorticoids showed depressed MMF production, whereas an inactive glucocorticoid analog (cortisone) and the sex hormones (progesterone, testosterone, and estradiol) had no inhibitory effects. Moreover, the active glucocorticoids inhibited MMF production according to their relative potencies as inhibitors of lymphocyte activation ( 14) and inflammation (15). Studies with lymphocytes from several species have established that most effects of glucocorticoids are mediated by specific macromolecular binding proteins, referred to as receptors (16). Since we observed inhibition of MMF production at concentrations of triamcinolone acetonide reported to saturate receptor binding it is likely that glucocorticoid inhibition of MMF production occurs via a receptor-
28
DUNCAN,
SADLIK,
AND HADDEN
o----0%--...J \\ I
‘\ ‘\ \\I
0
\
\ :
\ \ !
0 t
,;.11,‘o-To ,;a ,;a ,;a TRIAMCINOLONE
,f-.
ACETONIDE
FIG. 2. Effect of varying concentrations of triamcinolone acetonide on guinea pig peritoneal macrophage proliferation stimulated by a suboptimal (l/ 16) dilution of partially purified MMF in the absence (p) or presence (- - -) of indomethacin ( 10e5 M). Points and bars represent the mean f SD of two separate experiments of six replicate determinations of [ ‘Hlthymidine incorporation. Monolayer proliferation in the absence of steroid was 74,918 cpm; in the presence of indomethacin, 110,735 cpm; R control was 3942 cpm.
mediated process. This theory is supported by the observation that cortexolone, an antagonist of glucocorticoid-receptor interaction (16), blocked the triamcinolone acetonide-induced inhibition of MMF production, although inactive alone (Table 1). Furthermore, assay of supernatants for concomitant production of MIF revealed that cortexolone alone had no effect, but in combination with triamcinolone acetonide blocked the glucocorticoid-induced inhibition of MIF production (Table 1). Effect of Triamcinolone liferation
Acetonide
(1O-’ M) on MMF-Induced
Macrophage
Pro-
Because glucocorticoid-induced inhibition of MMF production might not be solely responsible for previous reports demonstrating suppression of macrophage proliferation in inflammatory exudates (l-3), we also investigated if glucocorticoids
GLUCOCORTICOIDS
AND MACROPHAGE TABLE
PROLIFERATION
2
Relative Glucocorticoid Inhibition of Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Suboptimal (l/16) Dilution of MMF Steroid
Estimated I&O of) 4.0 4.1 4.2 7.5 9.2
Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone ’ ICso were estimated for each listed steroid from concentration-response in Fig. 2.
x lo-’ x lo-’ X 1O-6 x lo+ X lo-’
curves generated as detailed
inhibited the effector phase of delayed hypersensitivity via a suppression of MMFinduced macrophage proliferation. In these experiments guinea pig peritoneal macrophage monolayers were stimulated with various dilutions of partially purified MMF and cultured in the presence or absence of a tentative receptor saturating concentration of triamcinolone acetonide (lo-’ M) (17). After 3 days of culture, monolayers were harvested and assayed for [ 3H]thymidine incorporation. Macrophage proliferation in response to MMF was concentration dependent and generated a bell-shaped concentration-response profile similar to that previously reported (8). Triamcinolone acetonide was found to be both a negative and a positive regulator of MMF-induced macrophage proliferation. Triamcinolone acetonide (IO-’ M) inhibited the macrophage proliferation induced by suboptimal dilutions (l/64 to l/16) of MMF, while potentiating supraoptimal dilutions (l/4 to l), resulting in a net shift of the concentration-response profile toward higher concentrations of MMF (data not shown). Effect of Glucocorticoids on Macrophage Proliferation timal (l/16) Dilution of MMF
Stimulated by a Subop-
To further investigate the observed triamcinolone acetonide (lo-’ M)-induced inhibition of suboptimal MMF-stimulated macrophage proliferation, macrophage monolayers were stimulated with 1/ 16 dilution of MMF in the presence of varying concentrations of triamcinolone acetonide. A concentration-dependent triamcinolone acetonide-induced inhibition of macrophage proliferation was observed (Fig. 2). Proliferation in cultures containing 1 X 10m4 M triamcinolone acetonide was only 25% of that occurring in control cultures, and the I& was estimated at 2 X lo-’ M. A similar concentration-dependent inhibition of MMF-induced macrophage proliferation was observed with triamcinolone acetonide at MMF dilutions of l/32 and l/64 (data not shown). Since exogenous prostaglandins are inhibitors of MMF action (8) and MMF preparations increase prostaglandin synthesis (9) the triamcinolone acetonide-induced inhibition of MMF action was investigated in the presence of indomethacin, to prevent interference by endogenously generated prostaglandins. As previously reported (8), indomethacin ( 10m5 M) consistently augmented the MMF-induced macrophage proliferation by increases of 50 to 60% (Fig. 2) while inhibiting the
30
DUNCAN,
SADLIK,
AND HADDEN
TABLE 3 Antagonism by Cortexolone of the Glucocorticoid Inhibition of Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Suboptimal (l/16) Dilution of MMF Steroid None Cortexolone ( 1O-’ M) Triamcinolone acetonide ( 10m6M) TA ( 1Oe6 M) + cortexolone (lo-’ M)
Macrophage proliferation’ (% control) 100 94 34 64
+ + f +
7b 5 8 8’
a Macrophage proliferation is expressed as a percentage of proliferation stimulated by MMF without steroid (75,825 cpm). b Values are the means of six determinations + SD. ’ Significantly different (P < 0.05) from triamcinolone acetonide (TA) 10m6M by two-tailed Student’s t test.
formation of cyclooxygenase products from labeled arachidonic acid (data not shown). However, a concentration-dependent triamcinolone acetonide-induced inhibition of MMF-stimulated proliferation was observed even in the presence of indomethacin, and the concentration-response curve paralleled that generated in the absence of indomethacin (Fig. 2). Thus triamcinolone acetonide inhibited MMF action through a mechanism unrelated to prostaglandin biosynthesis. As indicated by the results in Table 2, the inhibition of suboptimal MMF action was a glucocorticoid-specific phenomenon. Only active glucocorticoids inhibited MMF action, whereas cortisone and cultures containing lop5 M progesterone, testosterone, or estradiol had no inhibitory effect (data not shown). The active glucocorticoids inhibited MMF action according to their relative anti-inflammatory potencies. Although IC&‘s were about 100 times higher than the concentrations reported to give half-maximum saturation of glucocorticoid receptors in mouse and rabbit macrophages (17), it is likely that the inhibition was receptor mediated as the glucocorticoid antagonist, cortexolone, reversed the triamcinolone acetonideinduced suppression of proliferation (Table 3). Effect of Glucocorticoids on Macrophage Proliferation Stimulated by a Supraoptimal (I/2) Dilution of MMF A more detailed study of the observed triamcinolone acetonide ( 1Oe7M)-induced potentiation of macrophage proliferation stimulated by supraoptimal dilutions of MMF was also carried out. Macrophage monolayers were stimulated with a l/2 dilution of MMF in the presence of varying concentrations of triamcinolone acetonide. A concentration-dependent potentiation of macrophage proliferation was observed, although high concentrations of triamcinolone acetonide ( 10e5 to lop4 M) caused less potentiation than intermediate concentrations ( 10d7 to lop6 M) (Fig. 3). Proliferation in cultures containing 10e6 M triamcinolone acetonide was twofold that occurring in control cultures. An ECSo of 2 X 10e8 A4 was estimated from the ascending portion (lo-” to 10m6M) of the concentration-response profile. Glucocorticoids inhibit the production of prostaglandins in various cell types, including macrophages, by blocking the release of arachidonic acid from phospholipids (18). Since prostaglandins also inhibit MMF action (8) and MMF preparations increase prostaglandin synthesis (9) we investigated whether the triamci-
GLUCOCORTICOIDS
AND MACROPHAGE
31
PROLIFERATION
70.
60
I: . ii :: : ii :: :. .1ii::::;:1 ;i ::ii1::.-ii:: J
ii :: :: ii ii :: :: :: :: :: ii
. i: :: :: :: :: ;; :::: :: .* :: / ::
i 10-l’ IO”0 TRIAMCINOLONE
1o-g
10-8 ACETONIDE
i; ii :: :: :: :: :: :: :: :: ::. . :: ii :: :: :: ii ::. . 1: :: ; ::
::. . ii :: ii i; ii I; :::: ::. . ii ii .::. .::. :::: :: f ..
I-’
lo-’
J
:: .. :: .. :: .. ii .. ii :: ii ::. . ii ii ii ; :: lo-)
i
:: :: :: :: :: :: ii :: :: :: .. ii :: ;I
lo-.
IMOLAR)
FIG. 3. Effect of varying concentrations of triamcinolone acetonide on guinea pig periotoneal macrophage proliferation stimulated by a supraoptimal (l/2) dilution of partially purified MMF in the absence (Ll) or presence (0) of indomethacin (lo-’ M). Columns and bars represent the mean f SD of three separate experiments of six replicate determination of [3H]thymidine incorporation. R control was 2985 cpm.
nolone acetonide-induced potentiation of macrophage proliferation was due to inhibition of macrophage prostaglandin synthesis. We found this not to be the case, as triamcinolone acetonide caused a concentration-dependent potentiation of MMFdriven proliferation even in the presence of a total blockade of prostaglandin synthesis by indomethacin (Fig. 3). Results indicated that the potentiation of the action of supraoptimal dilutions of MMF was a glucocorticoid-specific event (Table 4). Only cultures treated with active glucocorticoids showed increased proliferation, whereas, inactive analogs (cortisone) and sex steroids (progesterone, testosterone, estradiol) had no potentiating effects. The active glucocorticoids increased MMF-driven proliferation according to their relative potencies in other glucocorticoid-sensitive assays. As noted for glucocorticoid inhibitory actions on MMF production and action at suboptimal MMF concentrations, glucocorticoid antagonists also reversed this glucocorticoidinduced phenomenon. The antiglucocorticoids, cortexolone, progesterone, and 17 a-methylprogesterone, while inactive alone, all inhibited the triamcinolone acetonide-induced potentiation of MMF-driven proliferation (Table 4), suggesting that this glucocorticoid response is also receptor mediated. DISCUSSION Previous investigations have shown that great numbers of mononuclear phagocytes are present in sites of immunologic inflammation in viva ( 19-21). The pop-
32
DUNCAN,
SADLIK, TABLE
AND HADDEN 4
Effect of Various Steroids on Guinea Pig Peritoneal Macrophage Proliferation Stimulated by a Supraoptimal (l/2) dilution of MMF Steroid (lo-’
Macrophage proliferation” (W control)
M)
None Triamcinolone acetonide Dexamethasone Prednisolone Cortisol Corticosterone Cortisone Progesterone Testosterone 1‘IO-estradiol Cortexolone (lo-’ M) Progesterone (lo-’ M) 17a-methylprogesterone ( 1Oe5 M) TA (lo-’ M) + cortexolone ( 1Om5M) TA (lo-’ M) + progesterone ( low5 M) TA (lo-’ M) + 17a-methylprogesterone
( 10m5M)
100 f 126 251+ 8 230 3~ 15 174 + 10 147+ 9 113+ 4 95 + 14 97+ 13 103 * 10 102 f 12 94+ 4 72+ 16 79* 12 102 f 13’ 95 f 14’ 99+ 9
’ Macrophage proliferation is expressed as a percentage of proliferation induced by MMF without steroid (18,652 cpm). b Values are the means of three separate experiments of six determinations each + SD. ’ Significantly different (P < 0.05) from triamcinolone acetonide (TA) lo-’ M by two-tailed Student’s t test.
ulation of mononuclear phagocytes present in these inflammatory sites is controlled by both the immigration of circulating blood monocytes into the lesion and by the cell division of the recently immigrated monocytes and resident macrophages within the lesion (19-21). The number of macrophages undergoing replication varies with the site of inflammation, ranging from a limited 5% proliferation in cutaneous granuloma to much higher values in sites with numerous resident macrophages such as liver and peritoneum. Both the immigration and the proliferation of these mononuclear phagocytes are thought to be controlled by lymphokines. Combined in vivo-in vitro evidence has suggested that monocytes/macrophages are first attracted to sites of antigen T lymphocyte interaction by the lymphokine monocyte chemotactic factor (MCF) (22), retained in such sites by MIF (22), and then induced to proliferate by MMF (8, 9) or MMF-like factors (24). The anti-inflammatory action of glucocorticoids may be mediated through the macrophage as glucocorticoid administration has been shown to profoundly reduce the number of macrophages in inflammatory sites in vivo (l-3). Studies in vitro suggested that glucocorticoids reduce the population of inflammatory macrophages by suppressing lymphokine-mediated processes (4-7). However, it is not clear whether glucocorticoids exerted their major suppressive effect on lymphokine synthesis by lymphocytes or on the macrophage response to lymphokine. The results of experimentation detailed herein, using MMF-induced macrophage proliferation as in in vitro model, suggest two sites of glucocorticoid modulation in delayed
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hypersensitivity reactions. However, glucocorticoids had their major suppressive effect on lymphokine production by lymphocytes, inhibiting both MMF and MIF generation at physiological concentrations, while larger pharmacological concentrations were required to regulate MMF-driven macrophage proliferation. The postreceptor mechanism by which glucocorticoids inhibited MMF production by sensitized lymphocytes was investigated only insofar as to show cyclooxygenase prostaglandin products were not involved. However, since the conditions used to generate MMF also resulted in the production of other lymphokines, including MIF (Table 1) and MCF (data not shown), observations from other studies in which glucocorticoids inhibited lymphokine production permit some speculation. The production of both MCF and MIF has been reported suppressed by near physiological concentrations of hydrocortisone (6, 7). The observations that inhibition of MCF production occurred only if hydrocortisone was added early in culture and that hydrocortisone also caused a parallel concentration-dependent inhibition of lymphocyte proliferation suggest that glucocorticoids inhibit lymphokine generation by suppressing some early event involved in lymphocyte activation. One early event in lymphocyte activation known to be inhibited by physiological concentrations of glucocorticoids is the release of the monokine interleukin 1 (IL. 1) from antigen/mitogen-stimulated macrophages (25). IL. 1 is obligatory for stimulated T-lymphocyte production of the lymphokine, interleukin 2 (IL.2), whose action culminates in T-lymphocyte proliferation (26). Since the presence of viable macrophages is obligatory for the production of other lymphokines such as macrophage-activating factor (MAF) and MCF (27), it is possible that IL.1 may be necessary for the production of most lymphokines, including MMF, and that glucocorticoids universally inhibit T-cell lymphokine production by suppressing IL. 1 release. However, other studies indicate that nonviable macrophages can support the generation of some lymphokines, including interferon (28) and MIF (29) and thus the exact locus of glucocorticoid inhibition of lymphocyte activation remains uncertain. Whatever the mechanism, most glucocorticoid actions are believed to be initiated by glucocorticoid binding to specific cytosol receptors and mediated by the interaction of this activated steroid-receptor complex with selected chormosomal sites, resulting ultimately in control of the synthesis of specific protein products (16). Observations from three separate experiments suggest that the glucocorticoid-induced inhibition of MMF production was a receptor-mediated phenomenon. First, the ICsO for triamcinolone acetonide inhibition of MMF production was equivalent to the concentration of triamcinolone acetonide required for half-maximum saturation of lymphocyte receptor binding sites (30). Second, the inhibition of production caused by other glucocorticoids paralleled their relative receptor affinities ( 14, 15). Third, cortexolone, an antagonist which displaces glucocorticoid-receptor binding (16) also blocked the inhibition of production of both MMF and MIF by triamcinolone acetonide. While we must conclude that glucocorticoids exert their major suppressive effect on the initiation phase of immune responses, additional experiments described herein also indicate that glucocorticoids have a regulatory function at the level of the effector mechanisms. Glucocorticoids were found to exert both a negative and a positive effect on MMF-driven macrophage proliferation, depending on the concentration of MMF present.
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Macrophage proliferation stimulated by suboptimal dilutions of MMF was inhibited by glucocorticoids in a concentration-dependent and potency-related manner (Fig. 2 and Table 3). However, it is unlikely that the observed inhibition represents a physiological mechanism, as the estimated ICSO’s are 100 times the reported glucocorticoid concentrations at half-maximum receptor saturation in mouse and rabbit macrophages ( 17). Similarly, other investigators have shown that pharmacological concentrations of glucocorticoids were necessary to inhibit the action of several lymphokines including guinea pig macrophage aggregation factor (MAgF) and MIF (4-7) and rat IL.2 (31). Nevertheless, suppression of the effector phase of cell-mediated immunity by glucocorticoid inhibition of MMF-induced macrophage proliferation may still be partially responsible for the observed antiinflammatory effects of glucocorticoids as pharmacological doses of glucocorticoids can easily produce an in vivo concentration of 10e5 A4 (32). Even though pharmacological glucocorticoid concentrations were necessary to inhibit MMF-induced macrophage proliferation, the fact that the inhibition was concentration dependent, glucocorticoid specific, and blocked by a glucocorticoid antagonist suggests that it was receptor mediated. The events distal to receptorgenome interaction responsible for the glucocorticoid inhibition of MMF action are not known, but could involve an alteration in MMF receptors, resulting in a decreased MMF signal transmission. In contrast to the suppression observed with suboptimal dilutions of MMF, glucocorticoids potentiated the macrophage proliferation induced by supraoptimal dilutions of MMF. The observed potentiation was not only significant but also occurred at close to physiological concentrations (ECSO triamcinolone acetonide 3 X lo-* M). The potentiation exhibited all the characteristics of a receptor-mediated glucocorticoid action, being concentration dependent, potency related, and suppressed by glucocorticoid antagonists (Fig. 3, Table 4). The postreceptor mechanism responsible for the glucocorticoid potentiation of supraoptimal MMF action is unknown and may simply be due to a decrease in MMF signal transmission, However, the observation that high concentration of MMF stimulated only limited macrophage proliferation, resulting in a bell-shaped concentration response curve (8), suggests that macrophage proliferation induced by supraoptimal dilutions of MMF is subject to inhibitory controls not operative at lower MMF concentrations. This is true for mitogen-induced lymphocyte proliferation where high concentrations of mitogens induce elevated intracellular levels of adenosine 3’:5’-cyclic monophosphate (CAMP) which acts to limit lymphocyte proliferation (33). Similarly, it is possible that high concentrations of MMF may induce an antiproliferative factor that acts to limit MMF-driven macrophage proliferation. Alternatively, an antiproliferative factor could be a minor contaminant in partially purified MMF present in effective amounts only at higher MMF concentrations. In either case, glucocorticoids could potentiate the action of supraoptimal MMF by suppressing the action or production of the antiproliferative factor. Evidence for two separate factors controlling supraoptimal MMF action (i.e., MMF and an antiproliferative factor) is suggested by the fact that ECso of triamcinolone acetonide for the inhibition of suboptimal MMF action is a log greater than that for potentiation of supraoptimal MMF and by the observation that the highest concentrations of triamcinolone acetonide did not give maximum potentiation of supraoptimal MMF action.
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While the nature of the antiproliferative factor remains speculative, one class of substances known to inhibit macrophage proliferation are the prostaglandins (8). Furthermore, MMF preparations are known to increase prostaglandin production (9) and glucocorticoids have inhibited prostaglandin synthesis in starchelicited macrophages (34). However, the glucocorticoid potentiation of supraoptimal MMF-induced macrophage proliferation was not due to a suppression of prostaglandin synthesis, as triamcinolone acetone caused a concentration-dependent potentiation even in the presence of indomethacin. Thus, the exact nature of the antiproliferative factor remains unknown but additional preliminary experiments indicate that the factor is a heat-labile component present in both MMF-rich supernatants and partially purified MMF preparations. Heating MMF preparations at 70°C for 2 min ablated the effect of high concentrations of MMF to induce less than optimum proliferation and a plateau-shaped response curve resulted. Triamcinolone acetonide (lo-’ M) failed to potentiate the action of high concentrations of this heat-treated MMF but rather was inhibitory at all MMF concentrations. The relationship of the observed potentiation of supraoptimal MMF-induced macrophage proliferation to the regulation of delayed hypersensitivity reactions in vivo is not clear. However, if the heat-labile antiproliferative factor is a physiological product involved in the inflammatory response, then the observed potentiation may be operative in vivo. If such is the case, then the anti-inflammatory action of glucocorticoids might be entirely due to the suppression of lymphokine synthesis during the induction phase as the net effect of glucocorticoids on the lymphokinemediated effector phase would depend on the in vivo concentration of MMF, with glucocorticoid suppression occurring at low MMF levels but glucocorticoid augmentation occurring at higher MMF concentrations. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
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