Immunopharmacology, 21 ( 1991 ) 73-82 Elsevier
73
IMPHAR 00534
Modulation of granuloma formation in vitro by endogenous mediators Ikuyo Y. Sato, Kazuo Kobayashi, Noriko Yamagata, Yusuke Shikama, Tsuyoshi Kasama, Keita K a s a h a r a and Terumi Takahashi The First Department of Internal Medicine, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan (Received 4 September 1990; accepted 29 November 1990)
Abstract: We have reported previously that in vitro granulomas are inducible by culturing murine spleen cells in the presence of artificial microparticles, dextran beads, and that macrophages and macrophage-derived cytokines (monokines) including interleukin 1 (IL-1) and tumor necrosis factor-e (TNF-c~) play a critical role in the initiation of bead-induced granulomas in vitro. To investigate regulatory mechanisms of granuloma formation, we examined the modulatory effects of various mediators such as IL-1, TNF-cq interferon-? (IFN-?), IL-4, IL-6, transforming growth factor-//(TGF-/~), dexamethasone and prostaglandin E2 (PGE2) on the development of lesions, because these mediators are known to play a pivotal role in inflammatory responses. The lesions were suppressed by the addition ofdexamethasone, PGE 2 or certain T cell-derived lymphokines such as IL-4 and IFN-?. These results suggest that suppressive signals are different from granulomatogenic cytokines including IL-1 and TNF-c¢ and that granulomas are regulated by multi-factor dependent mechanisms.
Key words:
Granuloma in vitro; Pharmacological modulation; Cytokine; Prostaglandin; Corticosteroid
Introduction
Granulomas are focal, predominantly mononuclear tissue inflammations evoked by persistent irritants (Adams, 1976; Boros, 1978). Monokine activities including interleukin 1 (IL-1) and tumor necrosis factor-c~ (TNF-c0 in the lesions are a common feature in hypersensitivity (Kobayashi etal., 1985b), BCG (Kindler etal., 1989; Kobayashi et al.,1985a; Sato et al., 1990) and Correspondence: Kazuo Kobayashi, The First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Abbreviations: IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; PG, prostaglandin; TGF, transforming growth factor. 0162-3109/91/$03.50 © 1991 Elsevier Science Publishers B.V.
foreign-body (dextran beads) granulomas (Kasahara et al., 1988; Shikama et al., 1989) in mice. Recent studies have shown that macrophages and monokines such as IL-1 and TNF-c~ play a critical role in the initiation/development of murine beadinduced or BCG granulomas in vivo (Kasahara et al., 1988, 1989; Kindler et al., 1989; Kobayashi et al., 1989) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Little information is available at this time concerning the regression mechanism of granulomas. To clarify the regulation mechanism of granulomas, we examined the effects of endogenous mediators such as IL-1, TNF-e, interferon-? (IFN-?), IL-2, IL-4, IL-6, transforming growth factor-// (TGF-/3), dexamethasone and prosta-
74 glandin E2 (PGE2) on murine granuloma formation in vitro, because these mediators are known to play a pivotal role in inflammatory responses. Evidence is presented suggesting that granuloma formation is inhibited by dexamethasone, PGE 2 and certain T cell-derived lymphokines including IL-4 and IFN- 7. Thus, suppressive signals of granulomas are different from granulomatogenic cytokines such as IL-1 and TNF-cc
Materials and Methods
Mice Female BALB/c mice were purchased from Charles River Japan Co. Ltd., Tokyo. All mice used were 10-20 weeks old. Culture medium Cells were cultured in RPMI 1640 supplemented with L-glutamine (2 mM), gentamycin (50/~g/ml, GIBCO, Grand Island, NY), 2-mercaptoethanol (5 x 10-SM), and 10~o heat-inactivated fetal bovine serum (GIBCO). This medium will be referred to as complete culture medium. Induction of granulomas in vitro In vitro granulomas were induced by culturing spleen cells in the presence of dextran beads according to a described method (Kobayashi et al., 1989; Shikama et al., 1989). Briefly, spleen cells were cultured in complete culture medium in the presence of dextran beads (70-100 #m in diameter, Sephadex G-50; Pharmacia Fine Chemicals, Piscataway, N J). Dextran beads were sterilized by autoclaving. The final culture contained 2 x 105 cells and 2 x 102 beads in 0.2 ml. The cells were cultured at 37 °C in 5~o CO2. This dose had previously been shown to result in optimal responses. Cell to bead interactions were classified according to the methods described previously (Bentley et al., 1985; Kobayashi etal., 1989; Shikama etal., 1989): 1, no reaction; 2, one to five cells adhering to the bead; 3, more than five cells adhering to the
bead; 4, more than five cells adhering the bead, with one or more cells demonstrating the morphologic changes of blast formation either directly adhering to the bead or being in the immediate vicinity of the bead, and being accompanied by mononuclear cell migration toward the bead; 5, one adherent layer of cells surrounding the entire bead associated with increased mononuclear cell migration; and 6, multiple layers of adherent cells about the bead accompanied by mononuclear cell migration. Beads 70-100 ~tm in diameter were selected for measurement. Determinations were made utilizing an inverted phasecontrast microscope (IMT-2, Olympus Co. Ltd., Tokyo, Japan). After classification, each bead reaction received the accompanying numerical score. The mean reaction for each experimental group was determined by multiplying the number of cell to bead reactions assigned to each of 6 categories by the number of that particular classification and then summing the total. One hundred determinations were made for each culture well. The results are expressed as the resultant mean value + SD of five separate experiments and has been called the granuloma index (GI) as described previously (Bentley et al., 1985; Kobayashi et al., 1989; Shikama et al., 1989).
Reagents and cytokines Recombinant human IL-1/?with a specific activity of 2 x 10 7 U/mg was kindly provided by Dr. Y. Hirai, Immunological Products and Development, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan (March et al., 1985). Recombinant murine TNF-~ (specific activity: 4 x 10vU/mg), recombinant murine IL-4 (1 x 108 U/mg) and recombinant human IL-6 (1 X 107 U/mg) were purchased from Genzyme, Boston, MA (Hirano et al., 1986; Lee et al., 1986; Pennica et al., 1985). Recombinant human IL-2 (specific activity: 1 x 107U/mg) and recombinant murine IFN-~ (specific activity: 1 x 10v U/mg) were gifts from Shionogi Pharmaceutical Co. Ltd., Osaka, Japan (Nagata et al., 1987; Taniguchi et al., 1983). Recombinant human TGF-/? ( E D 5 0 : 0 . 1 1 ng/ml) was put-
75 chased from King Brewing Co. Ltd., Kakogawa, Hyogo, Japan (Derynck et al., 1985). Cytokines used in this study contained below 0.5 ng of endotoxin per mg protein by the Limulus amoebocyte lysate assay (Sigma Chemical Co., St. Louis, MO). The water soluble, preservative-free dexamethasone sodium phosphate was a gift from Merck, Sharp and Dohme Japan Co., Tokyo. Prostaglandin E2 was purchased from Sigma Chemical Co. Various concentrations of reagents and cytokines were added to the granulomagenerating culture system. The viability assessed by trypan blue dye exclusion of spleen cells in the presence of all reagents except dexamethasone after 1, 3 and 7 days of cultivation was 90-95~o, 90-80~o and 85-75 ~o, respectively. The viability of dexamethasone containing cultures was 85-75~o (day 1), 80-70~o (day 3) and 75-65~o (day 7), respectively. In these studies, no granuloma formation was induced in cell cultures by the addition of reagents and cytokines in the absence ofnidi (e.g. dextran beads) as described previously (Kobayashi et al., 1989; Shikama et al., 1989).
Data analysis Analysis of variance (ANOVA) for factorial measure models and the Mann-Whitney U test for the analysis of non-parametric data were used; p values less than 0.05 were considered significant. All statistical analyses were performed using StatView II (Abacus Concepts, Berkeley, CA) run on a Macintosh II PC (Apple, Cupertino, CA).
Results
In vitro granulomas by artificial microparticles In vitro granulomas were induced by culturing spleen cells in the presence of artificial microparticles, dextran beads, as described (Kobayashi et al., 1989; Shikama et al., 1989). Briefly, in vitro granulomas induced by dextran beads were conspicuous by 1 day (GI: 3-4), reached maximum size (GI: 5-6) by 3 days and then gradually decreased in size thereafter (Figs. 1A-C and
2A-C). Histologically, lesions were composed predominantly of macrophages. A summary of the time course of in vitro granuloma formation is shown in Fig. 3. These results are consonant with our previous reports of in vitro granulomas by culturing spleen cells in the presence of dextran beads (Kobayashi et al., 1989; Shikama et al., 1989).
Suppression of in vitro granulomas by IL-4, IFN- 7, dexamethasone and PGEe Various concentrations of reagents and cytokines were added into the granuloma-generating culture system which consisted of spleen cells and dextran beads. Formation of in vitro granulomas was inhibited by the presence of IL-4 (Figs. 1D-F), IFN-7 (Figs. 1G-I), dexamethasone (Figs. 2 D - F ) or PGE z (Figs. 2G-I) when compared with controls. As shown in Fig. 3, the time-course study revealed two distinct patterns of suppression. Significant suppression by IL-4, IFN-7 or dexamethasone was observed from early to late stages (day 1 through 7) of granuloma formation, whereas inhibition by PGE2 was found only at the late stage (day 7). The suppression of in vitro granulomas by IL-4 (102-103U/ml), IFN-7 (1-103 U/ml), dexamethasone (10- 8-10-4 M) and PGE 2 (10-9-10 .5 M) was observed in a dose-related fashion. No significant alteration by IL-1, TNF-~, IL-2, IL-6, and TGF-fl To determine the effects of IL-1, TNF-~, IL-2, IL-6, and TGF-/? on in vitro granuloma formation, various concentrations of cytokines (IL-1, TNF-~, IL-2, and IL-6; 1-103 U/ml and TGF-/?; 10 - 1-10 ng/ml) were added into the culture. As seen in Fig. 4, no significant alteration of in vitro granulomas was seen by the addition of these cytokines from the results of histology and time-kinetic study when compared to controls, although slight augmentation of granuloma formation was seen by the addition of granulomatogenic monokines such as IL-1 and TNF-~. Also, no significant modulation of the lesions was observed in the presence of IL-2 (data not shown).
76
Fig. 1. Suppression ofgranulomas in vitro by IL-4 and IFN-7. (A-C) Representative in vitro granulomas seen in 1 day (A), 3 days (B) and 7 days (C) after culture of spleen cells and dextran beads without the addition of exogenous cytokines. Large granulomas are observed, and the cellular components are mainly macrophages ( x 150). (D-I) Representative in vitro granulomas seen in 1 day (D, G), 3 days (E, H) and 7 days (F, I) after culture in the presence of 103 U/ml of IL-4 (D-F) and t 03 U/ml of IFN-y (G-I). Development of lesions (day 1-7) is inhibited by the addition of IL-4 and IFN-7 ( x 150).
Discussion
In the present study, we have demonstrated that in vitro granuloma formation can be inhibited by endogenous mediators including certain T cellderived lymphokines such as IL-4 and IFN-7, dexamethasone and PGE2. Two distinct patterns of suppression emerged. IL-4, IFN- 7 and dexamethasone were classified as early to late effectors, whereas PGE 2 was typed as a late effector. Granuloma formation is the expression of a
series of complex inflammatory events. Generally, foreign-body granuloma-inducing agents persist in the tissue because they are insoluble or poorly degradable (Adams, 1976; Boros, 1978). We therefore used dextran beads as the nidi for both experimental foreign-body granulomas in vivo (Kasahara et al., 1988; Sato etal., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Evidence has accumulated in recent years, indicating that macrophages and cytokines, especially monokines such as IL-1 and TNF-~, play
77
Fig. 2. Inhibition of in vitro granulomas by dexamethasone and PGE2. (A-C) Representative in vitro granulomas seen in 1 day (A), 3 days (B) and 7 days (C) after culture in the absence of reagents ( x 150). (D-I) In vitro granulomas seen in 1 day (D, G), 3 days (E,H) and 7 days (F, I) after culture in the presence of dexamethasone (D-F) and PGE 2 (G-I). Development of lesions is inhibited by the addition of 10- 6 M of dexamethasone (day 1-7) and 10- 7 M of PGE 2 (day 7) ( × 150).
an essential role in the initiation and maintenance of murine granuloma formation in vivo (Chensue et al., 1989; Kasahara et al., 1988, 1989; Kindler et al., 1989; Kobayashi et al., 1985a; Kobayashi et al., 1985b; Sato et al., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Collectively, the nidi, monokines and cells, especially macrophages, play crucial roles in dextran bead-induced granuloma formation in vivo and in vitro. However, little information is available at this time concerning the regulatory/suppressive mechanism of granuloma formation. From the
aspects that monokines play an essential role in the development of granulomas, the suppression of lesions by IL-4, dexamethasone and PGE2 is easy to envisage, because these agents such as IL-4 (Hart etal., 1989; Weiss etal., 1989), dexamethasone (Beutler and Cerami, 1989; Kern et al., 1988; Lew et al., 1988; Narumi et al., 1990; Poubelle et al., 1990) and PGE 2 (Kunkel et al., 1986, 1989 ;) are known to inhibit the production and/or activity of monokines including IL-1 and TNF-~. We might postulate, therefore, that suppression of monokine production and/or activity
78 6
6
IL--4
IFN-¥
+1
~ o
c c o o 3
3
cr
0T 10 102103
10 10210"
day 1
10 102103
day 3
1 10 10210~
U/rn~
0
day 1
day 7
l 10 10210 ~
day 3
Dexamethasone
e
0
I 10 10210 ~
U,'m~
day 7
PGE2
~
0T
0T 10-'10"10"610 - ' day
1
0
10-at0-'10-'10 "' day 3
0
10-'I0"10-'10"
M
day 7
?0-'10"10-e day
1
0
10"IITT10-* day 3
10"110"710""
M
day 7
Fig. 3. Time-kinetic studies of suppression of in vitro granuloma formation by the addition of various concentrations of mediators. One hundred determinations were made for each culture well. W e performed five independent experiments of three mice per each condition. Thus, the mean GI + SD was derived from the determinations of 500 granulomas. Statistic differences (*) relative to controls (0.001 < p < 0.05).
by these agents resulted in inhibition ofgranuloma formation. Although the exact mechanism of the late suppressive effect of PGE2 is unclear, in vivo administration of PGE caused a significant reduction in granulomatous inflammation induced by dextran beads and Schistosoma mansoni (Chensue et al., 1983, 1986). The suppression of granulomas by IFN-7 is difficult to interpret, because the effects of IFN-7 on monokine production have been controversial. The amplifying
effect of IFN-7 on monokine production has been demonstrated (Arenzana-Seisdenos et al., 1985; Gerrard et al., 1987). In contrast, it has been reported that IFN-7 inhibits monokine production (Ghezzi and Dinarello, 1988; Ruschen etal., 1989), and they suggest that IFN- 7 is a regulatory molecule to abrogate self-perpetuating monokine production. Also, there have been reported antagonistic roles for IL-1 and IFN-v (Browning and Ribolini, 1987). Macrophages are the major corn-
79 6
6
I L-I/3
"
TNF--a
!
c
o
~3
3
1
~
s
1
day 1
1
1 21
day 3
a
1 210~
u,~
0
day 7
l
I0 102103
day 1
0
1
10 102103
day 3
0
1
I0 102103
U/m~
day 7
6
6
IL--6
TGF--p
+
I! c II o
o II
cr 1
II
TO 1 0 ~ 1 0 3
day 1
0
10 I0210 ~
day 3
I0 10210 ~ U/m~ day 7
0 0.1 1 10
0 0.1 1 10
0 0.1 1 10
day 1
day 3
day 7
ng/m~
Fig. 4. Effects of IL-1, TNF-c~, IL-6 and TGF-~. One hundred determinations were made for each culture well. We performed five independent experiments of three mice per each condition. Thus, the mean GI + SD was derived from the determinations of 500 granulomas. No significant difference relative to controls.
ponents of any granulomatous inflammation (Adams, 1976; Boros, 1978). IFN- 7 can modulate macrophage function and delayed-type hypersensitivity/cell-mediated responses (Trinchieri and Perussia, 1985; Balkwill, 1989). The modulating effects of IFN-7 may be responsible for the suppression of in vitro granuloma formation. Indeed, IFN-7 has therapeutic effects on certain human granulomatous diseases such as lepromatous leprosy (Nathan et al., 1986) and chronic
granulomatous disease (Sechler etal., 1988; Ezekowitz et al., 1988). However, further study of the effects of IFN-7 on cytokine production/action will be required to confirm the results. In addition, the suppression by IFN- 7 and IL-4 will be supported by studies demonstrating abrogation of the effects of these cytokines on granulomas by using specific anti-cytokine antibodies. In contrast, no significant modulation ofgranuloma formation in vitro was observed by the
80 addition of IL-1, TNF-c~, IL-6 and TGF-fl (Fig. 4). This may reflect that maximum granulomatous responses are inducible by spleen cells and dextran beads themselves (Kobayashi et al., 1989; Shikama et al., 1989). Therefore, it is difficult to assess the enhanced responses by the morphological method. However, granulomatogenic monokines such as IL-1 and TNF-~ caused a slight increase of granulomatous responses (Fig. 4). This may be due to monokine induction by monokines (Beutler and Cerami, 1989; di Giovine and Duff, 1990). Recent studies have shown that IL-6 suppresses IL- 1 and TNF-e production by human peripheral blood mononuclear cells stimulated with lipopolysaccharide or phytohemagglutinin (Schindler et al., 1990). They have reported that the suppression of monokine production (approximately 30~o inhibition) by IL-6 requires a large amount of IL-6 (100 ng/ml; more than 103 U/ml). In contrast, we added 1-103 U/ml of IL-6 into the in vitro granuloma system. Thus, it is unlikely to suppress monokine production in the in vitro system. Therefore, we could not find the suppression of in vitro granulomas by IL-6 (Fig. 4). It has been reported that TGF-fl can induce IL-1 production (Wahl, 1989) and deactivate macrophages (Tsunawaki et al., 1988). TGF-fl has pleiotropic effects on inflammatory processes (Wahl, 1989). The exact effects of TGF-fl on granulomas in vitro by dextran beads await further investigation, although we could not find the significant effect morphologically. Our previous studies have demonstrated that T cell-derived lymphokines such as IL-2 and IFN- 7 play little role in initiation of murine dextran bead-induced granulomas in vivo (Kasahara et al., 1988, 1989; Sato et al., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989), because these lymphokines were undetectable in the induction stage of granulomas and were unable to induce the lesions. Also, IL-2 did not possess modulatory effects on granulomatous responses (data not shown). Taken together with the present results of suppression of granulomas in vitro by certain T cell-derived lymphokines in-
cluding IL-4 and IFN-7, these lymphokines may participate in regression of lesions. These data may explain previous results that larger granulomas induced by dextran beads developed in athymic nude mice rather than in euthymic mice (Allred et al., 1985) and that schistosome-induced granulomas were suppressed by the adoptive transfer T lymphocytes (Chensue and Boros, 1979; Chensue et al., 1980). We have demonstrated previously that the histological features, time-kinetics and cytokine profiles of in vitro granulomas are consistent with in vivo lesions (Kobayashi et al., 1989; Shikama et al., 1989). Thus, regulatory mechanisms of both in vivo and in vitro models may be very similar, suggesting that data described in here are useful for investigating the regulation of in vivo granulomas. Therefore, the in vitro model may be a tool for screening granuloma-inhibiting agents.
Acknowledgements We would like to thank Dr. Takeshi Yoshida for helpful suggestions, and Ms. Hiroko T. Takeuchi for her excellent technical assistance. Also we would like to gratefully thank Dr. Yoshikatsu Hirai, Immunological Products and Development, Otsuka Pharmaceutical Co. Ltd., for providing recombinant human IL-lfl, and Shionogi Pharmaceutical Co. Ltd., for supplying recombinant human IL-2 and recombinant murine IFN-7. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, the Showa Medical Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan.
References Adams DO. The granulomatous inflammatory response. Am J Pathol 1976; 84: 164-191. Allred C, Kobayashi K, Yoshida T. Anergy-like immunosuppression in mice bearing pulmonary foreign-body granulomatous inflammation. Am J Pathol 1985; 121: 466-473. Arenzana-Seisdenos F, Virelizier IL, Fiers W. Interferons as
81 macrophage-activating factors. III. Preferential effects of interferon-7 on the interleukin 1 secretory potential of fresh or aged human monocytes. J Immunol 1985; 134: 2444-2448. Balkwill FR. Peptide regulatory factors: interferons. Lancet 1989; i: 1060-1063. Bentley AG, Phillips SM, Kaner RJ, Theodorides VJ, Linette GP, Doughty BL. In vitro delayed hypersensitivity granuloma formation: Development of an antigen-coated bead model. J Immunol 1985; 134: 4163-4169. Beutler B, Cerami A. The biology of cachectin/TNF. A primary mediator of the host response. Ann Rev Immunol 1989; 7: 625-655. Boros DL. Granulomatous inflammation. Prog Allergy 1978; 24: 184-243. Browning JL, RiboliniA. Interferon blocks interleukin 1-induced prostaglandin release from human peripheral monocytes. J Immunol 1987; 138: 2857-2863. Chensue SW, Boros DL. Modulation of granulomatous hypersensitivity. I. Characterization of Tlymphocytes involved in the adoptive suppression of granuloma formation in Schistosoma mansoni-infectedmice. J Immunol 1979; 123: 1409-1414. Chensue SW, Boros DL, David CS. Regulation ofgranulomatous inflammation in murine schistosomiasis. In vitro characterization of T lymphocyte subsets involved in the production and suppression of migration inhibition factor. J Exp Med 1980; 151: 1398-1412. Chensue SW, Kunkel SL, Ward PA, Higashi GI, Exogenously administered prostaglandins modulate pulmonary granulomas induced by Schistosoma mansoni eggs. Am J Pathol 1983; 111: 78-87. Chensue SW, Remick DG, Higashi GI, Boros DL, Kunkel SL. Modulation ofmurine schistosomiasis by exogenously administered prostaglandins. Am J Pathol 1986; 125: 28-34. Chensue SW, Otterness IG, Higashi GI, Forsch CS, Kunkel SL. Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor. J Immunol 1989; 142: 1281-1286. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV. Human transforming growth factor-beta c D N A sequence and expression in tumor cell lines. Nature 1985; 316: 701-705. Ezekowitz RAB, Dinauer MC, Jaffe HS, Orkin SH, Newburger PE. Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon gamma. N Engl J Med 1988; 319: 146-151. Gerrard TL, Siegel JP, Dyer DR, Zoon KC. Differential effects of interferon-~ and interferon--/ on interleukin 1 secretion by monocytes. J Immunol 1987; 138: 2535-2540.
Ghezzi P, Dinarello CA. Interleukin 1 induces interleukin 1. III. Specific inhibition of interleukin 1 production by gamma-interferon. J Immunol 1988; 140: 4238-4244. di Giovine FS, Duff GW. Interleukin 1: The first interleukin. Immunol Today 1990; 11: 13-20. Hart PH, Vitti GE, Burgess DR, Whitty GA, Piccoli DS, Hamilton JA. Potential anti-inflammatoryeffects of interleukin 4: Suppression of human monocyte tumor necrosis factor c~, interleukin 1, and prostaglandin E2. Proc Natl Acad Sci USA 1989; 86: 3803-3807. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Takahara Y, Taniguchi T, Kishimoto T. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.Nature 1986; 324: 73-76. Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Soezima K, Ide H, Takahashi T. Direct evidence for granuloma-inducing activity of interleukin-1: Induction of experimental pulmonary granuloma formation in mice by interleukin-l-coupled beads. Am J Pathol 1988; 130: 629-638. Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Kaga S, Hashimoto M, Odagiri T, Soejima K, Ide H, Takahashi T, Yoshida T. The role ofmonokines in granuloma formation in mice: The ability of interleukin-1 and tumor necrosis factor-c~ to induce lung granulomas. Clin Immunol Immunopathol 1989; 51: 419-425. Kern JA, Lamb RJ, Reed JC, Daniele RP, Nowell PC. Dexamethasone inhibitionofinterleukin I beta production by human monocytes. Post-transcriptional mechanisms. J Clin Invest 1988; 81: 237-244. Kindler V, Sappino A-P, Grau GE, Piguet P-F, Vassalli P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 1989; 56: 731-740. Kobayashi K, Allred C, Castriotta R, YoshidaT. Strain variation of Bacillus Calmette Guerin-induced pulmonary granuloma formation is correlated with anergy and the local production of migration inhibition factor and interleukin 1. Am J Pathol 1985a: 119: 223-235. Kobayashi K, Allred C, Cohen S, Yoshida T. Role of interleukin 1 in experimental pulmonary granuloma in mice. J Immunol 1985b; 134: 358-364. Kobayashi K, Kasahara K, Shikama Y, Yoneya I, Takahashi T, Yoshida T. Comparative studies on granuloma formation in vivo and in vitro. In: Yoshida T, Torisu M, eds. Basic mechanisms of granulomatous inflammation. Amsterdam: Elsevier, 1989; 163-183. Kunkel SL, Wiggins RC, Chensue SW, Larrick J. Regulation of tumor necrosis factor production by prostaglandin E2. Biochem Biophys Res Commun 1986; 137: 404-410. Kunkel SL, Remick DG, Larrick JW. Mechanisms that regulate the production and effects of tumor necrosis factor-~. CRC Crit Rev Immunol 1989; 9: 93-117.
82 Lee F, Yokota T, Otsuka T, Meyerson P, Villaret D, Coffman R, Mosmann T, Rennick D, Roehm N, Smith C, Zlotnik A, Arai K. Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc Natl Acad Sci USA 1986; 83: 2061-2065. Lew W, Oppenheim JJ, Matsushima K. Analysis of the suppression of IL-la and IL-lft production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J Immunol 1988; 140: 1895-1902. March CL, MosleyB, LarsenA, Ceretti DP, Braedt G, Price V, Gillis S, Henny CS, Kronheim SR, Grabstein K, Conlon PJ, Hopp TP, Cosman D. Cloning, sequence and expression of two distinct human interleukin 1 complementary DNAs. Nature 1985; 315: 641-647. Nagata K, Ohara O, Teraoka H, Yoshida N, Watanabe Y, Kawade Y. Production and purification of recombinant mouse interferon-7 from E. coli. In: Clemens M J, Morris AG, Gearing AJH, eds. Lymphokines and interferons. A practical approach. Oxford: IRL Press, 1987; 29-52. Narumi S, Hamilton TA. Dexamethasone selectively regulates LPS-inducible gene expression in murine peritoneal macrophages. Immunopharmacology 1990; 19: 93-101. Nathan CF, Kaplan G, Lewis WR, Nusrat A, Witmer MD, Sherwin SA, Job CK, Horowitz CR, Steinman RM, Cohn ZA. Local and systemic effects ofintradermal recombinant interferon-? in patients with lepromatous leprosy. N Engl J Med 1986; 315: 6-15. Pennica D, Hayflick JS, Bringman TS, Palladino MA, Goeddel DV. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci USA 1985; 82: 6060-6064. Poubelle PE, Grassi J, Pradelles P, Marceau F, Pharmacological modulation of interleukin 1 production by cultured endothelial cells from human umbilical veins. Immunopharmacology 1990; 19: 121-130. Ruschen S, Lemm G, Warnatz H. Spontaneous and LPSstimulated production of intracellular IL-lft by synovial macrophages in rheumatoid arthritis is inhibited by 1FN-y. Clin Exp Immunol 1989; 76: 246-251.
Sato IY, Kobayashi K, Kasama T, Kaga S, Kasahara K, Kanemitsu H, Nakatani K, Takahashi T, Nakamura RM, Skamene E, Yoshida T. Regulation ofMycobacterium bovis (BCG) and foreign body granulomas in mice by the Bcg gene. Infect Immun 1990; 58: 1210-1216. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlations and interactions in the production ofinterleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells; IL-6 suppresses IL-1 and TNF. Blood 1990; 75: 40-47. Sechler JMG, Malech HL, White J, Gallin JI. Recombinant human interferon-7 reconstitutes defective phagocyte function in patients with chronic granulomatous disease in childhood. Proc Natl Acad Sci USA 1988; 85: 4874-4878. Shikama Y, Kobayashi K, Kasahara K, Kaga S, Hashimoto M, Yoneya I, Hosoda S, Soejima K, Ide H, Takahashi T. Granuloma formation by artificial microparticles in vitro: Macrophages and monokines play a critical role in granuloma formation. Am J Pathol 1989; 134: 1189-1199. Taniguchi T, Matsui H, Fujita T, Takaoka C, Kashima N, Yoshimoto R, Hamuro J. Structure and expression of a cloned cDNA for human interleukin 2. Nature 1983; 302: 305-310. Trinchieri G, Perussia B. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol Today 1985; 6: 131-136. Tsunawaki S, Sporn M, Ding A, Nathan CF. Deactivation of macrophages by transforming growth factor-ft. Nature 1988; 334: 260-262. Wahl SM, McCartney-Francis N, Mergenhagen SE. Inflammatory and immunomodulatory roles of TGF-fl. Immunol Today 1989; 10: 258-261. Weiss L, Haeffner-Cavaillon N, Laude M, Cavaillon JM, Kazatchikine MD. Human T cells and interleukin 4 inhibit the release ofinterleukin 1 induced by lipopolysaccharide in serum-free cultures of autologous monocytes. Eur J Immunol 1989; 19: 1347-1350.
73
IMPHAR 00534
Modulation of granuloma formation in vitro by endogenous mediators Ikuyo Y. Sato, Kazuo Kobayashi, Noriko Yamagata, Yusuke Shikama, Tsuyoshi Kasama, Keita K a s a h a r a and Terumi Takahashi The First Department of Internal Medicine, Showa University School of Medicine, Shinagawa-ku, Tokyo, Japan (Received 4 September 1990; accepted 29 November 1990)
Abstract: We have reported previously that in vitro granulomas are inducible by culturing murine spleen cells in the presence of artificial microparticles, dextran beads, and that macrophages and macrophage-derived cytokines (monokines) including interleukin 1 (IL-1) and tumor necrosis factor-e (TNF-c~) play a critical role in the initiation of bead-induced granulomas in vitro. To investigate regulatory mechanisms of granuloma formation, we examined the modulatory effects of various mediators such as IL-1, TNF-cq interferon-? (IFN-?), IL-4, IL-6, transforming growth factor-//(TGF-/~), dexamethasone and prostaglandin E2 (PGE2) on the development of lesions, because these mediators are known to play a pivotal role in inflammatory responses. The lesions were suppressed by the addition ofdexamethasone, PGE 2 or certain T cell-derived lymphokines such as IL-4 and IFN-?. These results suggest that suppressive signals are different from granulomatogenic cytokines including IL-1 and TNF-c¢ and that granulomas are regulated by multi-factor dependent mechanisms.
Key words:
Granuloma in vitro; Pharmacological modulation; Cytokine; Prostaglandin; Corticosteroid
Introduction
Granulomas are focal, predominantly mononuclear tissue inflammations evoked by persistent irritants (Adams, 1976; Boros, 1978). Monokine activities including interleukin 1 (IL-1) and tumor necrosis factor-c~ (TNF-c0 in the lesions are a common feature in hypersensitivity (Kobayashi etal., 1985b), BCG (Kindler etal., 1989; Kobayashi et al.,1985a; Sato et al., 1990) and Correspondence: Kazuo Kobayashi, The First Department of Internal Medicine, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142, Japan. Abbreviations: IL, interleukin; TNF, tumor necrosis factor; IFN, interferon; PG, prostaglandin; TGF, transforming growth factor. 0162-3109/91/$03.50 © 1991 Elsevier Science Publishers B.V.
foreign-body (dextran beads) granulomas (Kasahara et al., 1988; Shikama et al., 1989) in mice. Recent studies have shown that macrophages and monokines such as IL-1 and TNF-c~ play a critical role in the initiation/development of murine beadinduced or BCG granulomas in vivo (Kasahara et al., 1988, 1989; Kindler et al., 1989; Kobayashi et al., 1989) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Little information is available at this time concerning the regression mechanism of granulomas. To clarify the regulation mechanism of granulomas, we examined the effects of endogenous mediators such as IL-1, TNF-e, interferon-? (IFN-?), IL-2, IL-4, IL-6, transforming growth factor-// (TGF-/3), dexamethasone and prosta-
74 glandin E2 (PGE2) on murine granuloma formation in vitro, because these mediators are known to play a pivotal role in inflammatory responses. Evidence is presented suggesting that granuloma formation is inhibited by dexamethasone, PGE 2 and certain T cell-derived lymphokines including IL-4 and IFN- 7. Thus, suppressive signals of granulomas are different from granulomatogenic cytokines such as IL-1 and TNF-cc
Materials and Methods
Mice Female BALB/c mice were purchased from Charles River Japan Co. Ltd., Tokyo. All mice used were 10-20 weeks old. Culture medium Cells were cultured in RPMI 1640 supplemented with L-glutamine (2 mM), gentamycin (50/~g/ml, GIBCO, Grand Island, NY), 2-mercaptoethanol (5 x 10-SM), and 10~o heat-inactivated fetal bovine serum (GIBCO). This medium will be referred to as complete culture medium. Induction of granulomas in vitro In vitro granulomas were induced by culturing spleen cells in the presence of dextran beads according to a described method (Kobayashi et al., 1989; Shikama et al., 1989). Briefly, spleen cells were cultured in complete culture medium in the presence of dextran beads (70-100 #m in diameter, Sephadex G-50; Pharmacia Fine Chemicals, Piscataway, N J). Dextran beads were sterilized by autoclaving. The final culture contained 2 x 105 cells and 2 x 102 beads in 0.2 ml. The cells were cultured at 37 °C in 5~o CO2. This dose had previously been shown to result in optimal responses. Cell to bead interactions were classified according to the methods described previously (Bentley et al., 1985; Kobayashi etal., 1989; Shikama etal., 1989): 1, no reaction; 2, one to five cells adhering to the bead; 3, more than five cells adhering to the
bead; 4, more than five cells adhering the bead, with one or more cells demonstrating the morphologic changes of blast formation either directly adhering to the bead or being in the immediate vicinity of the bead, and being accompanied by mononuclear cell migration toward the bead; 5, one adherent layer of cells surrounding the entire bead associated with increased mononuclear cell migration; and 6, multiple layers of adherent cells about the bead accompanied by mononuclear cell migration. Beads 70-100 ~tm in diameter were selected for measurement. Determinations were made utilizing an inverted phasecontrast microscope (IMT-2, Olympus Co. Ltd., Tokyo, Japan). After classification, each bead reaction received the accompanying numerical score. The mean reaction for each experimental group was determined by multiplying the number of cell to bead reactions assigned to each of 6 categories by the number of that particular classification and then summing the total. One hundred determinations were made for each culture well. The results are expressed as the resultant mean value + SD of five separate experiments and has been called the granuloma index (GI) as described previously (Bentley et al., 1985; Kobayashi et al., 1989; Shikama et al., 1989).
Reagents and cytokines Recombinant human IL-1/?with a specific activity of 2 x 10 7 U/mg was kindly provided by Dr. Y. Hirai, Immunological Products and Development, Otsuka Pharmaceutical Co. Ltd., Tokushima, Japan (March et al., 1985). Recombinant murine TNF-~ (specific activity: 4 x 10vU/mg), recombinant murine IL-4 (1 x 108 U/mg) and recombinant human IL-6 (1 X 107 U/mg) were purchased from Genzyme, Boston, MA (Hirano et al., 1986; Lee et al., 1986; Pennica et al., 1985). Recombinant human IL-2 (specific activity: 1 x 107U/mg) and recombinant murine IFN-~ (specific activity: 1 x 10v U/mg) were gifts from Shionogi Pharmaceutical Co. Ltd., Osaka, Japan (Nagata et al., 1987; Taniguchi et al., 1983). Recombinant human TGF-/? ( E D 5 0 : 0 . 1 1 ng/ml) was put-
75 chased from King Brewing Co. Ltd., Kakogawa, Hyogo, Japan (Derynck et al., 1985). Cytokines used in this study contained below 0.5 ng of endotoxin per mg protein by the Limulus amoebocyte lysate assay (Sigma Chemical Co., St. Louis, MO). The water soluble, preservative-free dexamethasone sodium phosphate was a gift from Merck, Sharp and Dohme Japan Co., Tokyo. Prostaglandin E2 was purchased from Sigma Chemical Co. Various concentrations of reagents and cytokines were added to the granulomagenerating culture system. The viability assessed by trypan blue dye exclusion of spleen cells in the presence of all reagents except dexamethasone after 1, 3 and 7 days of cultivation was 90-95~o, 90-80~o and 85-75 ~o, respectively. The viability of dexamethasone containing cultures was 85-75~o (day 1), 80-70~o (day 3) and 75-65~o (day 7), respectively. In these studies, no granuloma formation was induced in cell cultures by the addition of reagents and cytokines in the absence ofnidi (e.g. dextran beads) as described previously (Kobayashi et al., 1989; Shikama et al., 1989).
Data analysis Analysis of variance (ANOVA) for factorial measure models and the Mann-Whitney U test for the analysis of non-parametric data were used; p values less than 0.05 were considered significant. All statistical analyses were performed using StatView II (Abacus Concepts, Berkeley, CA) run on a Macintosh II PC (Apple, Cupertino, CA).
Results
In vitro granulomas by artificial microparticles In vitro granulomas were induced by culturing spleen cells in the presence of artificial microparticles, dextran beads, as described (Kobayashi et al., 1989; Shikama et al., 1989). Briefly, in vitro granulomas induced by dextran beads were conspicuous by 1 day (GI: 3-4), reached maximum size (GI: 5-6) by 3 days and then gradually decreased in size thereafter (Figs. 1A-C and
2A-C). Histologically, lesions were composed predominantly of macrophages. A summary of the time course of in vitro granuloma formation is shown in Fig. 3. These results are consonant with our previous reports of in vitro granulomas by culturing spleen cells in the presence of dextran beads (Kobayashi et al., 1989; Shikama et al., 1989).
Suppression of in vitro granulomas by IL-4, IFN- 7, dexamethasone and PGEe Various concentrations of reagents and cytokines were added into the granuloma-generating culture system which consisted of spleen cells and dextran beads. Formation of in vitro granulomas was inhibited by the presence of IL-4 (Figs. 1D-F), IFN-7 (Figs. 1G-I), dexamethasone (Figs. 2 D - F ) or PGE z (Figs. 2G-I) when compared with controls. As shown in Fig. 3, the time-course study revealed two distinct patterns of suppression. Significant suppression by IL-4, IFN-7 or dexamethasone was observed from early to late stages (day 1 through 7) of granuloma formation, whereas inhibition by PGE2 was found only at the late stage (day 7). The suppression of in vitro granulomas by IL-4 (102-103U/ml), IFN-7 (1-103 U/ml), dexamethasone (10- 8-10-4 M) and PGE 2 (10-9-10 .5 M) was observed in a dose-related fashion. No significant alteration by IL-1, TNF-~, IL-2, IL-6, and TGF-fl To determine the effects of IL-1, TNF-~, IL-2, IL-6, and TGF-/? on in vitro granuloma formation, various concentrations of cytokines (IL-1, TNF-~, IL-2, and IL-6; 1-103 U/ml and TGF-/?; 10 - 1-10 ng/ml) were added into the culture. As seen in Fig. 4, no significant alteration of in vitro granulomas was seen by the addition of these cytokines from the results of histology and time-kinetic study when compared to controls, although slight augmentation of granuloma formation was seen by the addition of granulomatogenic monokines such as IL-1 and TNF-~. Also, no significant modulation of the lesions was observed in the presence of IL-2 (data not shown).
76
Fig. 1. Suppression ofgranulomas in vitro by IL-4 and IFN-7. (A-C) Representative in vitro granulomas seen in 1 day (A), 3 days (B) and 7 days (C) after culture of spleen cells and dextran beads without the addition of exogenous cytokines. Large granulomas are observed, and the cellular components are mainly macrophages ( x 150). (D-I) Representative in vitro granulomas seen in 1 day (D, G), 3 days (E, H) and 7 days (F, I) after culture in the presence of 103 U/ml of IL-4 (D-F) and t 03 U/ml of IFN-y (G-I). Development of lesions (day 1-7) is inhibited by the addition of IL-4 and IFN-7 ( x 150).
Discussion
In the present study, we have demonstrated that in vitro granuloma formation can be inhibited by endogenous mediators including certain T cellderived lymphokines such as IL-4 and IFN-7, dexamethasone and PGE2. Two distinct patterns of suppression emerged. IL-4, IFN- 7 and dexamethasone were classified as early to late effectors, whereas PGE 2 was typed as a late effector. Granuloma formation is the expression of a
series of complex inflammatory events. Generally, foreign-body granuloma-inducing agents persist in the tissue because they are insoluble or poorly degradable (Adams, 1976; Boros, 1978). We therefore used dextran beads as the nidi for both experimental foreign-body granulomas in vivo (Kasahara et al., 1988; Sato etal., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Evidence has accumulated in recent years, indicating that macrophages and cytokines, especially monokines such as IL-1 and TNF-~, play
77
Fig. 2. Inhibition of in vitro granulomas by dexamethasone and PGE2. (A-C) Representative in vitro granulomas seen in 1 day (A), 3 days (B) and 7 days (C) after culture in the absence of reagents ( x 150). (D-I) In vitro granulomas seen in 1 day (D, G), 3 days (E,H) and 7 days (F, I) after culture in the presence of dexamethasone (D-F) and PGE 2 (G-I). Development of lesions is inhibited by the addition of 10- 6 M of dexamethasone (day 1-7) and 10- 7 M of PGE 2 (day 7) ( × 150).
an essential role in the initiation and maintenance of murine granuloma formation in vivo (Chensue et al., 1989; Kasahara et al., 1988, 1989; Kindler et al., 1989; Kobayashi et al., 1985a; Kobayashi et al., 1985b; Sato et al., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989). Collectively, the nidi, monokines and cells, especially macrophages, play crucial roles in dextran bead-induced granuloma formation in vivo and in vitro. However, little information is available at this time concerning the regulatory/suppressive mechanism of granuloma formation. From the
aspects that monokines play an essential role in the development of granulomas, the suppression of lesions by IL-4, dexamethasone and PGE2 is easy to envisage, because these agents such as IL-4 (Hart etal., 1989; Weiss etal., 1989), dexamethasone (Beutler and Cerami, 1989; Kern et al., 1988; Lew et al., 1988; Narumi et al., 1990; Poubelle et al., 1990) and PGE 2 (Kunkel et al., 1986, 1989 ;) are known to inhibit the production and/or activity of monokines including IL-1 and TNF-~. We might postulate, therefore, that suppression of monokine production and/or activity
78 6
6
IL--4
IFN-¥
+1
~ o
c c o o 3
3
cr
0T 10 102103
10 10210"
day 1
10 102103
day 3
1 10 10210~
U/rn~
0
day 1
day 7
l 10 10210 ~
day 3
Dexamethasone
e
0
I 10 10210 ~
U,'m~
day 7
PGE2
~
0T
0T 10-'10"10"610 - ' day
1
0
10-at0-'10-'10 "' day 3
0
10-'I0"10-'10"
M
day 7
?0-'10"10-e day
1
0
10"IITT10-* day 3
10"110"710""
M
day 7
Fig. 3. Time-kinetic studies of suppression of in vitro granuloma formation by the addition of various concentrations of mediators. One hundred determinations were made for each culture well. W e performed five independent experiments of three mice per each condition. Thus, the mean GI + SD was derived from the determinations of 500 granulomas. Statistic differences (*) relative to controls (0.001 < p < 0.05).
by these agents resulted in inhibition ofgranuloma formation. Although the exact mechanism of the late suppressive effect of PGE2 is unclear, in vivo administration of PGE caused a significant reduction in granulomatous inflammation induced by dextran beads and Schistosoma mansoni (Chensue et al., 1983, 1986). The suppression of granulomas by IFN-7 is difficult to interpret, because the effects of IFN-7 on monokine production have been controversial. The amplifying
effect of IFN-7 on monokine production has been demonstrated (Arenzana-Seisdenos et al., 1985; Gerrard et al., 1987). In contrast, it has been reported that IFN-7 inhibits monokine production (Ghezzi and Dinarello, 1988; Ruschen etal., 1989), and they suggest that IFN- 7 is a regulatory molecule to abrogate self-perpetuating monokine production. Also, there have been reported antagonistic roles for IL-1 and IFN-v (Browning and Ribolini, 1987). Macrophages are the major corn-
79 6
6
I L-I/3
"
TNF--a
!
c
o
~3
3
1
~
s
1
day 1
1
1 21
day 3
a
1 210~
u,~
0
day 7
l
I0 102103
day 1
0
1
10 102103
day 3
0
1
I0 102103
U/m~
day 7
6
6
IL--6
TGF--p
+
I! c II o
o II
cr 1
II
TO 1 0 ~ 1 0 3
day 1
0
10 I0210 ~
day 3
I0 10210 ~ U/m~ day 7
0 0.1 1 10
0 0.1 1 10
0 0.1 1 10
day 1
day 3
day 7
ng/m~
Fig. 4. Effects of IL-1, TNF-c~, IL-6 and TGF-~. One hundred determinations were made for each culture well. We performed five independent experiments of three mice per each condition. Thus, the mean GI + SD was derived from the determinations of 500 granulomas. No significant difference relative to controls.
ponents of any granulomatous inflammation (Adams, 1976; Boros, 1978). IFN- 7 can modulate macrophage function and delayed-type hypersensitivity/cell-mediated responses (Trinchieri and Perussia, 1985; Balkwill, 1989). The modulating effects of IFN-7 may be responsible for the suppression of in vitro granuloma formation. Indeed, IFN-7 has therapeutic effects on certain human granulomatous diseases such as lepromatous leprosy (Nathan et al., 1986) and chronic
granulomatous disease (Sechler etal., 1988; Ezekowitz et al., 1988). However, further study of the effects of IFN-7 on cytokine production/action will be required to confirm the results. In addition, the suppression by IFN- 7 and IL-4 will be supported by studies demonstrating abrogation of the effects of these cytokines on granulomas by using specific anti-cytokine antibodies. In contrast, no significant modulation ofgranuloma formation in vitro was observed by the
80 addition of IL-1, TNF-c~, IL-6 and TGF-fl (Fig. 4). This may reflect that maximum granulomatous responses are inducible by spleen cells and dextran beads themselves (Kobayashi et al., 1989; Shikama et al., 1989). Therefore, it is difficult to assess the enhanced responses by the morphological method. However, granulomatogenic monokines such as IL-1 and TNF-~ caused a slight increase of granulomatous responses (Fig. 4). This may be due to monokine induction by monokines (Beutler and Cerami, 1989; di Giovine and Duff, 1990). Recent studies have shown that IL-6 suppresses IL- 1 and TNF-e production by human peripheral blood mononuclear cells stimulated with lipopolysaccharide or phytohemagglutinin (Schindler et al., 1990). They have reported that the suppression of monokine production (approximately 30~o inhibition) by IL-6 requires a large amount of IL-6 (100 ng/ml; more than 103 U/ml). In contrast, we added 1-103 U/ml of IL-6 into the in vitro granuloma system. Thus, it is unlikely to suppress monokine production in the in vitro system. Therefore, we could not find the suppression of in vitro granulomas by IL-6 (Fig. 4). It has been reported that TGF-fl can induce IL-1 production (Wahl, 1989) and deactivate macrophages (Tsunawaki et al., 1988). TGF-fl has pleiotropic effects on inflammatory processes (Wahl, 1989). The exact effects of TGF-fl on granulomas in vitro by dextran beads await further investigation, although we could not find the significant effect morphologically. Our previous studies have demonstrated that T cell-derived lymphokines such as IL-2 and IFN- 7 play little role in initiation of murine dextran bead-induced granulomas in vivo (Kasahara et al., 1988, 1989; Sato et al., 1990) and in vitro (Kobayashi et al., 1989; Shikama et al., 1989), because these lymphokines were undetectable in the induction stage of granulomas and were unable to induce the lesions. Also, IL-2 did not possess modulatory effects on granulomatous responses (data not shown). Taken together with the present results of suppression of granulomas in vitro by certain T cell-derived lymphokines in-
cluding IL-4 and IFN-7, these lymphokines may participate in regression of lesions. These data may explain previous results that larger granulomas induced by dextran beads developed in athymic nude mice rather than in euthymic mice (Allred et al., 1985) and that schistosome-induced granulomas were suppressed by the adoptive transfer T lymphocytes (Chensue and Boros, 1979; Chensue et al., 1980). We have demonstrated previously that the histological features, time-kinetics and cytokine profiles of in vitro granulomas are consistent with in vivo lesions (Kobayashi et al., 1989; Shikama et al., 1989). Thus, regulatory mechanisms of both in vivo and in vitro models may be very similar, suggesting that data described in here are useful for investigating the regulation of in vivo granulomas. Therefore, the in vitro model may be a tool for screening granuloma-inhibiting agents.
Acknowledgements We would like to thank Dr. Takeshi Yoshida for helpful suggestions, and Ms. Hiroko T. Takeuchi for her excellent technical assistance. Also we would like to gratefully thank Dr. Yoshikatsu Hirai, Immunological Products and Development, Otsuka Pharmaceutical Co. Ltd., for providing recombinant human IL-lfl, and Shionogi Pharmaceutical Co. Ltd., for supplying recombinant human IL-2 and recombinant murine IFN-7. This work was supported by grants from the Ministry of Education, Science, and Culture of Japan, the Showa Medical Foundation and the Mochida Memorial Foundation for Medical and Pharmaceutical Research, Tokyo, Japan.
References Adams DO. The granulomatous inflammatory response. Am J Pathol 1976; 84: 164-191. Allred C, Kobayashi K, Yoshida T. Anergy-like immunosuppression in mice bearing pulmonary foreign-body granulomatous inflammation. Am J Pathol 1985; 121: 466-473. Arenzana-Seisdenos F, Virelizier IL, Fiers W. Interferons as
81 macrophage-activating factors. III. Preferential effects of interferon-7 on the interleukin 1 secretory potential of fresh or aged human monocytes. J Immunol 1985; 134: 2444-2448. Balkwill FR. Peptide regulatory factors: interferons. Lancet 1989; i: 1060-1063. Bentley AG, Phillips SM, Kaner RJ, Theodorides VJ, Linette GP, Doughty BL. In vitro delayed hypersensitivity granuloma formation: Development of an antigen-coated bead model. J Immunol 1985; 134: 4163-4169. Beutler B, Cerami A. The biology of cachectin/TNF. A primary mediator of the host response. Ann Rev Immunol 1989; 7: 625-655. Boros DL. Granulomatous inflammation. Prog Allergy 1978; 24: 184-243. Browning JL, RiboliniA. Interferon blocks interleukin 1-induced prostaglandin release from human peripheral monocytes. J Immunol 1987; 138: 2857-2863. Chensue SW, Boros DL. Modulation of granulomatous hypersensitivity. I. Characterization of Tlymphocytes involved in the adoptive suppression of granuloma formation in Schistosoma mansoni-infectedmice. J Immunol 1979; 123: 1409-1414. Chensue SW, Boros DL, David CS. Regulation ofgranulomatous inflammation in murine schistosomiasis. In vitro characterization of T lymphocyte subsets involved in the production and suppression of migration inhibition factor. J Exp Med 1980; 151: 1398-1412. Chensue SW, Kunkel SL, Ward PA, Higashi GI, Exogenously administered prostaglandins modulate pulmonary granulomas induced by Schistosoma mansoni eggs. Am J Pathol 1983; 111: 78-87. Chensue SW, Remick DG, Higashi GI, Boros DL, Kunkel SL. Modulation ofmurine schistosomiasis by exogenously administered prostaglandins. Am J Pathol 1986; 125: 28-34. Chensue SW, Otterness IG, Higashi GI, Forsch CS, Kunkel SL. Monokine production by hypersensitivity (Schistosoma mansoni egg) and foreign body (sephadex bead)-type granuloma macrophages. Evidence for sequential production of IL-1 and tumor necrosis factor. J Immunol 1989; 142: 1281-1286. Derynck R, Jarrett JA, Chen EY, Eaton DH, Bell JR, Assoian RK, Roberts AB, Sporn MB, Goeddel DV. Human transforming growth factor-beta c D N A sequence and expression in tumor cell lines. Nature 1985; 316: 701-705. Ezekowitz RAB, Dinauer MC, Jaffe HS, Orkin SH, Newburger PE. Partial correction of the phagocyte defect in patients with X-linked chronic granulomatous disease by subcutaneous interferon gamma. N Engl J Med 1988; 319: 146-151. Gerrard TL, Siegel JP, Dyer DR, Zoon KC. Differential effects of interferon-~ and interferon--/ on interleukin 1 secretion by monocytes. J Immunol 1987; 138: 2535-2540.
Ghezzi P, Dinarello CA. Interleukin 1 induces interleukin 1. III. Specific inhibition of interleukin 1 production by gamma-interferon. J Immunol 1988; 140: 4238-4244. di Giovine FS, Duff GW. Interleukin 1: The first interleukin. Immunol Today 1990; 11: 13-20. Hart PH, Vitti GE, Burgess DR, Whitty GA, Piccoli DS, Hamilton JA. Potential anti-inflammatoryeffects of interleukin 4: Suppression of human monocyte tumor necrosis factor c~, interleukin 1, and prostaglandin E2. Proc Natl Acad Sci USA 1989; 86: 3803-3807. Hirano T, Yasukawa K, Harada H, Taga T, Watanabe Y, Matsuda T, Kashiwamura S, Nakajima K, Koyama K, Iwamatsu A, Tsunasawa S, Sakiyama F, Matsui H, Takahara Y, Taniguchi T, Kishimoto T. Complementary DNA for a novel human interleukin (BSF-2) that induces B lymphocytes to produce immunoglobulin.Nature 1986; 324: 73-76. Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Soezima K, Ide H, Takahashi T. Direct evidence for granuloma-inducing activity of interleukin-1: Induction of experimental pulmonary granuloma formation in mice by interleukin-l-coupled beads. Am J Pathol 1988; 130: 629-638. Kasahara K, Kobayashi K, Shikama Y, Yoneya I, Kaga S, Hashimoto M, Odagiri T, Soejima K, Ide H, Takahashi T, Yoshida T. The role ofmonokines in granuloma formation in mice: The ability of interleukin-1 and tumor necrosis factor-c~ to induce lung granulomas. Clin Immunol Immunopathol 1989; 51: 419-425. Kern JA, Lamb RJ, Reed JC, Daniele RP, Nowell PC. Dexamethasone inhibitionofinterleukin I beta production by human monocytes. Post-transcriptional mechanisms. J Clin Invest 1988; 81: 237-244. Kindler V, Sappino A-P, Grau GE, Piguet P-F, Vassalli P. The inducing role of tumor necrosis factor in the development of bactericidal granulomas during BCG infection. Cell 1989; 56: 731-740. Kobayashi K, Allred C, Castriotta R, YoshidaT. Strain variation of Bacillus Calmette Guerin-induced pulmonary granuloma formation is correlated with anergy and the local production of migration inhibition factor and interleukin 1. Am J Pathol 1985a: 119: 223-235. Kobayashi K, Allred C, Cohen S, Yoshida T. Role of interleukin 1 in experimental pulmonary granuloma in mice. J Immunol 1985b; 134: 358-364. Kobayashi K, Kasahara K, Shikama Y, Yoneya I, Takahashi T, Yoshida T. Comparative studies on granuloma formation in vivo and in vitro. In: Yoshida T, Torisu M, eds. Basic mechanisms of granulomatous inflammation. Amsterdam: Elsevier, 1989; 163-183. Kunkel SL, Wiggins RC, Chensue SW, Larrick J. Regulation of tumor necrosis factor production by prostaglandin E2. Biochem Biophys Res Commun 1986; 137: 404-410. Kunkel SL, Remick DG, Larrick JW. Mechanisms that regulate the production and effects of tumor necrosis factor-~. CRC Crit Rev Immunol 1989; 9: 93-117.
82 Lee F, Yokota T, Otsuka T, Meyerson P, Villaret D, Coffman R, Mosmann T, Rennick D, Roehm N, Smith C, Zlotnik A, Arai K. Isolation and characterization of a mouse interleukin cDNA clone that expresses B-cell stimulatory factor 1 activities and T-cell- and mast-cell-stimulating activities. Proc Natl Acad Sci USA 1986; 83: 2061-2065. Lew W, Oppenheim JJ, Matsushima K. Analysis of the suppression of IL-la and IL-lft production in human peripheral blood mononuclear adherent cells by a glucocorticoid hormone. J Immunol 1988; 140: 1895-1902. March CL, MosleyB, LarsenA, Ceretti DP, Braedt G, Price V, Gillis S, Henny CS, Kronheim SR, Grabstein K, Conlon PJ, Hopp TP, Cosman D. Cloning, sequence and expression of two distinct human interleukin 1 complementary DNAs. Nature 1985; 315: 641-647. Nagata K, Ohara O, Teraoka H, Yoshida N, Watanabe Y, Kawade Y. Production and purification of recombinant mouse interferon-7 from E. coli. In: Clemens M J, Morris AG, Gearing AJH, eds. Lymphokines and interferons. A practical approach. Oxford: IRL Press, 1987; 29-52. Narumi S, Hamilton TA. Dexamethasone selectively regulates LPS-inducible gene expression in murine peritoneal macrophages. Immunopharmacology 1990; 19: 93-101. Nathan CF, Kaplan G, Lewis WR, Nusrat A, Witmer MD, Sherwin SA, Job CK, Horowitz CR, Steinman RM, Cohn ZA. Local and systemic effects ofintradermal recombinant interferon-? in patients with lepromatous leprosy. N Engl J Med 1986; 315: 6-15. Pennica D, Hayflick JS, Bringman TS, Palladino MA, Goeddel DV. Cloning and expression in Escherichia coli of the cDNA for murine tumor necrosis factor. Proc Natl Acad Sci USA 1985; 82: 6060-6064. Poubelle PE, Grassi J, Pradelles P, Marceau F, Pharmacological modulation of interleukin 1 production by cultured endothelial cells from human umbilical veins. Immunopharmacology 1990; 19: 121-130. Ruschen S, Lemm G, Warnatz H. Spontaneous and LPSstimulated production of intracellular IL-lft by synovial macrophages in rheumatoid arthritis is inhibited by 1FN-y. Clin Exp Immunol 1989; 76: 246-251.
Sato IY, Kobayashi K, Kasama T, Kaga S, Kasahara K, Kanemitsu H, Nakatani K, Takahashi T, Nakamura RM, Skamene E, Yoshida T. Regulation ofMycobacterium bovis (BCG) and foreign body granulomas in mice by the Bcg gene. Infect Immun 1990; 58: 1210-1216. Schindler R, Mancilla J, Endres S, Ghorbani R, Clark SC, Dinarello CA. Correlations and interactions in the production ofinterleukin-6 (IL-6), IL-1, and tumor necrosis factor (TNF) in human blood mononuclear cells; IL-6 suppresses IL-1 and TNF. Blood 1990; 75: 40-47. Sechler JMG, Malech HL, White J, Gallin JI. Recombinant human interferon-7 reconstitutes defective phagocyte function in patients with chronic granulomatous disease in childhood. Proc Natl Acad Sci USA 1988; 85: 4874-4878. Shikama Y, Kobayashi K, Kasahara K, Kaga S, Hashimoto M, Yoneya I, Hosoda S, Soejima K, Ide H, Takahashi T. Granuloma formation by artificial microparticles in vitro: Macrophages and monokines play a critical role in granuloma formation. Am J Pathol 1989; 134: 1189-1199. Taniguchi T, Matsui H, Fujita T, Takaoka C, Kashima N, Yoshimoto R, Hamuro J. Structure and expression of a cloned cDNA for human interleukin 2. Nature 1983; 302: 305-310. Trinchieri G, Perussia B. Immune interferon: a pleiotropic lymphokine with multiple effects. Immunol Today 1985; 6: 131-136. Tsunawaki S, Sporn M, Ding A, Nathan CF. Deactivation of macrophages by transforming growth factor-ft. Nature 1988; 334: 260-262. Wahl SM, McCartney-Francis N, Mergenhagen SE. Inflammatory and immunomodulatory roles of TGF-fl. Immunol Today 1989; 10: 258-261. Weiss L, Haeffner-Cavaillon N, Laude M, Cavaillon JM, Kazatchikine MD. Human T cells and interleukin 4 inhibit the release ofinterleukin 1 induced by lipopolysaccharide in serum-free cultures of autologous monocytes. Eur J Immunol 1989; 19: 1347-1350.
