- Email: [email protected]
Regulatory mechanisms of cutaneous delayed-type hypersensitivity
CELLULARIMMUNOLOGY
81, 134-143(1983)
Regulatory Mechanisms of Cutaneous Delayed-Type Hypersensitivity II. Suppression of Cutaneous Delayed-Type Hypersensitivity by Macrophage Disappearance Reaction TAKAHIRO OCHIYA,
TORU BABA,’ KIKUO ONOZAKI, HIDEO YAOITA,
KENICHI UYENO, AND TATSUICHIRO Department
of Dermatology, University
HASHIMOTO
Institute of Clinical Medicine, and Institute of Basic Medical of Tsukuba, Sakura-Mura, Ibaraki-ken 305. Japan Received
May
17, 1983; accepted
Sciences,
June 9, I983
Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 pg of EA was injected into these animals and the animals were divided into two groups. One group of animls was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine y-globulin (DNPBGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGC-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supematants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.
INTRODUCTION
The macrophage disappearance reaction (MDR) is an in vivo manifestation of cellular immunity (1, 2) as well as cutaneous delayed-type hypersensitivity (DTH). A positive MDR represents an antigen-induced reduction in the number of macrophages in the peritoneal cavity. Mediation of this reaction by lymphokines has been verified (3-5). As for the distinct entity of MDR in an in vivo manifestation of cellmediated immunity, Sonozaki and Cohen have suggested that MDR may be an in vivo manifestation of the macrophage migration inhibitory factor (MIF), whereas cutaneous DTH may be more dependent upon chemotactic activity (3). ’ To whom correspondence should be addressed. 134 0008~8749/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
SUPPRESSION
OF
CUTANEOUS
DTH
BY
MDR
135
In our previous studies, we demonstrated that intraperitoneal (ip) injection of the MIF fraction, which had been recovered from an immunoadsorbent column prepared with anti-guinea pig MIF antiserum (6-Q into guinea pigs with elicited peritoneal macrophages resulted in a reduction in the number of peritoneal macrophages (9). The MIF fraction did not contain macrophage chemotactic factor, neutrophil chemotactic factor, vascular permeability factor, and skin reactive factor (8). Then, we discussed that MDR had a distinct entity from cutaneous DTH and was an in vivo manifestation of MIF activity. Although MDR is a well-known phenomenon, its biological significance is yet unelucidated. In the present study, attempts were made to explore the behavior of cutaneous DTH in animals with MDR. We will describe data suggesting that MDR exerts a suppressive effect on cutaneous DTH. MATERIALS
AND
METHODS
Immunization of animals. Hartley albino female guinea pigs weighing 450 to 500 g were used in all experiments. Dinitrophenylated bovine y-globulin (DNP-BGG) and dinitrophenylated egg albumin (DNP-EA) were prepared by standard techniques. The conjugate contained approximately 5 1 molecules of DNP per molecule of protein in DNP-BGG and 12 molecules in DNP-EA. Guinea pigs were immunized by footpad injection of 100 pg of either DNP-BGG or DNP-EA, or both in Freund’s complete adjuvant (CFA; Difco, Detroit, Mich.). Macrophage disappearance reaction (MDR). The MDR was induced by the methods described by Sonozaki and Cohen (2). Briefly, immunized guinea pigs bearing 4-day oil-induced peritoneal exudates received either an ip injection of 20 pg of antigen in 5 ml of phosphate-buffered saline (PBS) or an ip injection of 5 ml of PBS alone as a control. Six hours after the injection, the peritoneal exudates were harvested with 80 ml of Hanks’ balanced salt solution containing 10 units/ml of heparin. The total number of cells was assessed with a hemacytometer. Differential cell counts were performed by examining both Giemsa-Lusung-stained smears and esterase-stained smears prepared by the methods of Wachstein and Wolf (10). Preparation of guinea pig peritoneal macrophages. Twenty milliliters of sterilized liquid paraffin was injected into the peritoneal cavity of guinea pigs. Four days later, the peritoneal exudates were harvested with 80 ml of Hanks’ balanced salt solution. After washing two times with Hanks’ balanced salt solution and one time with RPM1 1640 medium, the cell concentration was adjusted to 2 X 10’ cells/ml in RPM1 1640 culture medium. The peritoneal exudate cells were >96% macrophages by morphology after May-Giemsa staining and >95% esterase positive. Preparation of the immunoadsorbed MIFfraction. First, lymphokines were prepared as follows. Regional lymph nodes were removed from animals immunized 7 days previously with DNP-BGG in CFA. Lymphocyte suspensions were made from such lymph nodes. After several washings, the cell concentration was adjusted to 1 X IO’ cells/ml in RPM1 1640 culture medium. The cells were incubated overnight in the presence or absence of 100 pg/ml of DNP-BGG. After incubation, the supematants were collected by centrifugation at 400g for 20 min. Control culture supematants incubated in the absence of the antigen were reconstituted with the antigen before centrifugation. Then, the supematants were fractionated by ultrafiltration through Amicon Diaflo membrane XM 1OOA (average molecular weight cut: 100,000) and
136
OCHIYA
ET
AL.
Amicon DiatIo membrane DM 5 (average molecular weight cut: 5000). The fraction with molecular weights from 100,000 to 5000 was used for lymphokine preparation. Immunoadsorbed MIF and control fractions were prepared as described previously (6-8). Briefly, anti-guinea pig MIF antiserum was produced by immunization of rabbits with guinea pig MIF-containing fraction obtained by polyacrylamide gel electrophoresis. The immunoglobulin fraction was precipitated with 40% saturated ammonium sulfate and dialyzed against PBS. Anti-MIF antibody was coupled to BrCNactivated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). The lymphokine preparation was applied to the immunoadsorbent column and eluted with PBS after 1 hr incubation. The nonadsorbed fraction was eluted with PBS after 1 hr incubation. The nonadsorbed fraction was eluted with 50 ml of 1.O A4 NaCl-0.0 1 M phosphate buffer (pH 7.4). Then, specifically adsorbed fraction was eluted with 50 ml of 0.1 M acetic acid solution. The final fraction was dialyzed against PBS and concentrated to original volume on Amicon diaflo membrane PM 10 (average molecular weight: 10,000). As a control fraction, a preparation of lymphocyte culture supernatants treated in the same manner as that described above was used. Skin tests. Antigen solutions were prepared in PBS at a concentration of 50 pg/ ml. One-tenth milliliter of the antigen solution was intradermally injected into the preshaved skin of the back. Twenty-four hours later, the lesions were estimated as induration. The area of the lesion was calculated as the product of the smallest diameter and largest one. The solution of phytohemagglutinin (PHA) (Difco) was prepared with 0.0 1 M PBS (pH 7.4) to contain 50 ~1 of original solution per milliliter. One-tenth milliliter of such solution was injected intradermally into the preshaved skin of the back, and 24 hr after injection measurements of induration were made. Skin reactive factor (SRF) was prepared by 10 times concentration of both lymphokine and control preparations by ultrafiltration through an Amicon membrane PM 10 (average molecular weight cut: 10,000). One-tenth milliliter of SRF was injected intradermally into the preshaved skin of the back and the resulting skin reactions were estimated as induration 6 hr after injection. The area of skin reactions induced by SRF was 120.3 mm2 on the average and the area of that by control preparation was less than 10 mm*. Analysis of data. Means and standard errors of experimental and control groups were calculated and analysis by t test was performed. RESULTS Suppression of Cutaneous DTH
in the Animals
in Which MDR
Was Induced
The guinea pigs were immunized with DNP-EA and 3 days later the animals received an ip injection of 20 ml of liquid paralhn in order to elicit peritoneal macrophages. Further, 4 days later, the guinea pigs with macrophage-rich peritoneal exudates were subsequently given an ip injection of 20 rg of EA in 5 ml of PBS for induction of MDR and the animals divided into two groups; one group of animals (6 animals) was sacrificed for estimation of MDR at 6 hr after the subsequent ip injection and the other group (6 animals) received a skin test by EA (5 pg) at the time of the subsequent ip injection. A group of the immunized animals with macrophage-rich peritoneal exudates (12 animals) was given a subsequent ip injection of PBS alone as a control. These animals were equally divided into two groups and studied in a similar manner. The number of peritoneal macrophages in the animals
SUPPRESSION OF CUTANEOUS
137
DTH BY MDR
given the ip injection of EA was significantly reduced in comparison with that in animals that received ip injection of PBS alone (54.0% loss). As shown in Table 1. skin reactions induced by EA were markedly suppressed in the animals given ip injection of EA compared with those in control animals that received ip injection of PBS alone (P < 0.001). The immunized animals that did not receive the ip injection of liquid paraffin were also prepared. These animals were divided into two groups (6 animals each) in a similar manner at the time of the ip injection; one was given an ip injection of 20 pg of EA and the other received PBS alone. All animals in two groups received a skin test by EA at the time of the ip injection. The EA-induced skin reactions were not suppressed in the animals given ip injection of EA in comparison with those in animals given ip injection of PBS alone. These results seems to indicate that induction of MDR may result in suppression of cutaneous DTH. Antigen-Nonspecljic Induced
Suppression of Cutaneous DTH in Animals
in Which A4DR Was
In this experiment, guinea pigs were doubly immunized with DNP-EA and DNPBGG, and similar experiments were performed. Animals with macrophage-rich peritoneal exudates were prepared and subsequently given an ip injection of 20 pg of BGG in 5 ml of PBS for the induction of MDR. These animals were divided into two groups; one group (6 animals) was sacrificed for estimation of MDR and the other group (6 animals) received skin tests by EA and by BGG at the time of subsequent the ip injection. As a control, a group of the immunized animals with macrophagerich peritoneal exudates (12 animals) was given 5 ml of PBS alone and similarly divided into two groups. The number of peritoneal macrophages in the animals given ip injection of BGG was markedly reduced in comparison with that in control animals (60.3% loss). Not only BGG-induced skin reactions but also EA-induced skin reactions were suppressed in animals with macrophage-rich peritoneal exudates given an ip injection of BGG compared with those in control animals (P -C 0.001) (Table 2). A similar suppression of skin reactions by ip injection of the same dose of BGG could not be obtained by the use of the similarly immunized animals that did not receive the ip injection of liquid paraffin. These results seem to indicate that induction of MDR may lead to antigen-nonspecific suppression of cutaneous DTH. Suppression of PHA-Induced
and SRF-Induced
Skin Reactions by MDR
First, the effect of MDR on PHA-induced skin reactions was studied. Guinea pigs were immunized with DNP-EA and received an ip injection of liquid paraffin as TABLE 1 Suppression of Cutaneous DTH in Animals in Which MDR Was Induced Induration (mm’) Material injected ip 20 wg EA/5 ml PBS 5 ml PBS
With par&n
injected
Without paraffin injected
86.3 f 10.4” 161.0 + 8.2
’ P < 0.001, n = 12 in each experiment. Skin tests were performed by EA.
166.1 + 2.6 165.6 f 7.8
138
OCHIYA
ET AL.
TABLE 2 Antigen-Nonspecific Suppression of Cutaneous DTH in Animals in Which MDR Was Induced Induration (mm’) With paraffin injected
Without paraffin injected
Material injected ip
BGG
EA
BGG
EA
20 pg BGG/S ml PBS 5 ml PBS
32.5 f 9.4” 114.3 f 11.7
81.1 + 13.0’ 153.4 + 9.5
114.4 * 9.0 111.8 + 7.1
142.9 f 7.1 146.9 zk 5.4
a.’ aP < 0.001, bP < 0.001, n = 12 in each experiment. The guinea pigs were doubly immunized with DNP-BGG and DNP-EA.
described above. After the subsequent ip injection of 20 pg of EA in 5 ml of PBS, the animals were divided into two groups; one group (6 animals) was used for estimation of MDR and the other group (6 animals) received a skin test by PHA at the time of subsequent ip injection. As a control, animals with macrophage-rich peritoneal exudates subsequently given an ip injection of PBS alone (12 animals) were also prepared and treated in a similar manner. The number of peritoneal macrophages in the animals given ip injection of EA was markedly reduced in comparison with that in control animals (58.7% loss). As shown in Table 3, skin reactions induced by PHA were suppressed in animals with macrophage-rich peritoneal exudates subsequently given an ip injection of EA in comparison with those in control animals (P < 0.00 1). Using immunized animals that did not receive an ip injection of liquid paraffin, ip injection of the same dose of EA did not affect the reactivity to PHA. SRF is defined as lymphokine activity which induces a skin reaction histologically similar to a typical cutaneous DTH. In order to study the reactivity of effector cells to lymphokines in animals in which MDR was induced, reactivity to SRP was examined at 24 hr after the subsequent ip injection (at the time of estimation of cutaneous DTH). As shown in Table 3, SRF-induced skin reactions were suppressed in the animals with macrophage-rich peritoneal exudate subsequently given ip injection of EA in comparison with those in control animals (P < 0.001). In the animals that did not receive an ip injection of liquid pa&in, suppression of SRF-induced skin reactions was not obtained by ip injection of the same dose of EA. TABLE 3 Suppression of PHA-Induced Shin Reactions and SRP-Induced Skin Reactions in Animals in Which MDR Was Induced Induration (mm’) With paraffin
Without paraffin
Material injected ip
PHA
SRF
PI-IA
SRF
20 pegEA/5 ml PBS 5 ml PBS
61.4 + 13.0” 108.0 k 27.5
54.9 + 10.3b 85.0 f 10.0
116.0 + 2.3 116.1 + 2.1
88.3 + 4.3 88.3 + 1.5
““P
< 0.001, bP < 0.001, n = 12 in each experiment.
SUPPRESSION
OF CUTANEOUS
DTH BY MDR
Kinetic Study on Suppression of the Skin Reaction in the Animals Was Induced
139 in Which MDR
Guinea pigs were immunized with DNP-EA, followed by an ip injection of liquid paraffin. The animals with macrophage-rich peritoneal exudates were divided into two groups. One group of the animals (6 animals) received subsequent ip injection of 20 pg of EA in 5 ml of PBS. As a control, the other group of animals (6 animals) was given an ip injection of the same dose of PBS alone. Studies were done on the kinetics of the hyporeactivity to EA, PHA, and SRF. Skin tests were performed at 24-hr intervals. Skin reactivities were shown as the percentage of control values. As shown in Fig. 1, the reactivity to either EA or PHA statistically returned to a control level at 96 hr, whereas hyporeactivity to SRF statistically diminished at 72 hr (Fig. 2). Suppression of Cutaneous DTH by the Culture Supernatants of Macrophages Incubated with the MIF Fraction The macrophage suspension in RPM1 1640 culture medium (2 X 10’ cells/ml) was incubated for 24 hr at 37°C under a 5% CO*, 100% humidity atmosphere with an equal volume of the MIF fraction which had previously been dialyzed against RPM1 1640 culture medium at 4°C for 48 hr. After incubation, the supematants were collected by centrifugation at 400g for 10 min. Ten milliliters of these supematants was intraperitoneally injected into the six guinea pigs which had been immunized with DNP-EA 7 days previously. A skin test by EA was performed at the time of ip injection. As controls, the following culture supematants were prepared: the culture supematants of macrophages similarly incubated with the control fraction instead of the MIF fraction and the culture supematants of macrophages alone (1 X 10’ cells/ ml). The MIF fraction and the control fraction similarly incubated with an equal volume of RPM1 1640 culture medium were also prepared. Ten milliliters of these control supematants was injected intraperitoneally into each group (six animals) of immunized animals. The immunized animals that did not receive any ip injection were prepared as well. As shown in Table 4, EA-induced skin reactions were markedly suppressed in the animals receiving ip injection of the culture supematants of macrophages-MIF in comparison with those in control animals (P < 0.00 1). Intraperitoneal injection of the MIF fraction alone could not affect the reactivity of the animals to EA.
t i.P inj.
24
hour5
4s after
72
96
120
injection
FIG. 1. Kinetic study on EA-induced skin reactions or PHA-induced skin reactions in animals in which MDR was induced. First skin tests were given at the time of ip injection of an antigen.
140
OCHIYA
ET AL.
.-.
t
24
i.R inj. hours
48
72
after
injection
SRF
96
120
FIG. 2. Kinetic study on SRF-induced skin reactions in the animals in which MDR was induced. First shin tests were given 24 hr after the ip injection of an antigen.
Suppression of PHA-Induced or SRF-Induced natants of Macrophages-MIF
Skin Reactions by the Culture Super-
The culture supematants of macrophages incubated with the MIF fraction were prepared as described above. The nonimmunized normal guinea pigs (six animals) received an ip injection of 10 ml of these culture supematants and skin tests by PHA and by SRF were done at the time of ip injection. Control groups of the animals (six animals each) were prepared in a similar manner as in the above experiment. As shown in Table 5, PHA-induced skin reactions were suppressed in the animals given an ip injection of the macrophage-MIF culture supematants compared with those in control animals (P < 0.001). SRF-induced skin reactions were also suppressed in these animals (P < 0.001). Intraperitoneal injection of the MIF fraction alone could affect the reactivities of the animals neither to PHA nor to SRF. Kinetic Study on Suppression of Cutaneous DTH Guinea pigs (six animals) were immunized with DNP-EA and 7 days later they received an ip injection of 10 ml of the macrophages-MIF culture supematants. Studies were performed on the kinetics of the hyporeactivity to EA in these animals. Skin tests by EA were done at 24-hr intervals. As a control, similarly immunized guinea pigs (six animals) were given an ip injection of the same volume of the macTABLE 4 Suppression of Cutaneous DTH by the Culture Supematants of Macrophages Incubated with the MIF Fraction Supematant injected ip
Induration”
Macrophages + MIF fraction Macrophages + control fraction Macrophages alone MIF fraction alone Control fraction alone None
152.0 221.3 219.2 224.1 224.4 223.3
u Skin tests were performed by EA. b P < 0.001, n = 12 in each experiment.
+ f + + k f
(mm*) 23.66 9.5 4.5 6.1 1.1 9.1
SUPPRESSION OF CUTANEOUS
141
DTH BY MDR
TABLE 5 Suppression of PHA-Induced and SRF-Induced Skin Reactions by the Culture Supernatants of Macrophages Incubated with the MIF Fraction Induration (mm*) Supematant injected ip
SRF
PHA
Macrophages + MIF fraction Macrophages + control fraction Macrophages alone MIF fraction alone Control fraction alone None
55.6 120.1 113.3 112.5 118.4 121.3
+ f + f k +
46.8 141.8 135.2 138.5 129.8 124.2
26.3” 27.3 9.0 12.3 12.5 6.9
f f f f f +
8.0b 16.9 12.3 11.1 13.6 11.1
a P < 0.001, n = 12 in each experiment. b P < 0.001, n = 12 in each experiment.
rophage-control fraction culture supematants. Skin reactivities were shown as the percentage of control values. As shown in Fig. 3, the reactivity to EA statistically returned to a control level at 96 hr. Kinetic Study on Suppression of PHA-Induced or SRF-Induced Skin Reactions
A similar study was performed on PHA-induced and SRF-induced skin reactions. Nonimmunized normal guinea pigs were divided into two groups. One group (six animals) was given an ip injection of 10 ml of the macrophages-MIF culture supernatants and the other group (six animals) received an ip injection of the same volume of the macrophage-control fraction culture supematants as a control. Skin tests by PHA and by SRF were done at 24-hr intervals. As shown in Fig. 4, the reactivity to PHA also returned to control level at 96 hr, whereas the hyporeactivity to SRF diminished at 12 hr. DISCUSSION Cutaneous delayed hypersensitivity in animals previously sensitized with antigen in complete Freud’s adjuvant can be suppressed by systemic injection of that antigen. This requires that the antigen be administered intravenously, intraperitoneally, or
I t
24
4s
72
after
injection
96
120
i.P. inj. hours
FIG. 3. Kinetic study on cutaneous DTH to EA in animals given ip injection of the culture supematants of macrophages incubated with the MIF fraction in vitro.
142
OCHIYA
-100
ET AL.
,,,,,,,,
x;;i
-----;
p f
1 B x
I'
.cx, .;
k'
,' .-
------
PHA
.---.SRF -P Le
b-p
.5 'I
I t i.P.
inj.
24 hwo
48 after
72
aa
120
injection
FIG. 4. Kinetic study on PHA-induced or SRF-induced skin reactions in animals given an ip injection of culture supernatants of macrophages incubated with the MIF fraction in vitro.
intramuscularly in large amounts (milligram) without adjuvant. The process leading to loss of reactivity is known as desensitization (11). The unresponsiveness involves the reactivity to an antigen different from the one used for the desensitization (3). In spite of potential importance of this phenomenon, for example, as an experimental model for clinical anergy state, its mechanism has never been fully investigated. In the present study, it was demonstrated that ip injection of 20 pg of an antigen into preimmunized guinea pigs with elicited peritoneal macrophages resulted in a transient reduction in the number of peritoneal macrophages as well as suppression of cutaneous DTH to the antigen, whereas the similar injection of the antigen into preimmunized animals in which peritoneal macrophages had not been elicited did not affect cutaneous DTH. Suppression of cutaneous DTH achieved by this treatment was antigen nonspecific and PHA-induced skin reactions were also suppressed. These results seem to indicate that induction of MDR leads to suppression of cutaneous DTH as systemic administration of large dose of an antigen. Sonozaki and Cohen have shown that MDR diminished by 20 hr (2) and we also obtained the similar result by ip injection of lymphokines into guinea pigs with elicited peritoneal macrophages (9). However, kinetic study showed that cutaneous DTH was still suppressed 24 hr after the induction of MDR. This seems to indicate that suppression of cutaneous DTH may not be due to a reduction in the number of peritoneal macrophages itself. MDR seems to be considered as in vivo manifestation of MIF activity, since in our previous study we demonstrated that ip injection of the MIF fraction into guinea pigs with elicited peritoneal macrophages led to a transient reduction in the number of peritoneal macrophages (9). Then, we assumed that the macrophages modulated by MIF may secrete the inhibitory substance(s) against cutaneous DTH. Next studies were conducted to examine whether the culture supernatants of the elicited peritoneal macrophages incubated with the MIF fraction in vitro have the ability to suppress cutaneous DTH in preimmunized animals. As shown in the results, ip injection of these culture supernatants into the immunized animals resulted in hyporeactivity to the antigen, whereas similar injection of the same amount of the MIF fraction alone did not affect delayed-hypersensitivity reactions in the animals. Reactivity to SRF was also suppressed in the animals in which MDR had been induced. Hyporeactivity to SRF was observed in the desensitized guinea pigs as well ( 12). Macrophages and polymorphonuclear cells obtained from these desensitized animals were deficient in their ability to respond for chemotactic stimuli in vitro (12).
SUPPRESSION
OF CUTANEOUS
DTH BY MDR
143
These results seem to indicate one of the possible mechanisms of suppression of effector cells to lymphokines. Hyporeactivity to SRF was also achieved by ip injection of the macrophage-MIF culture supematants into normal guinea pigs. Thus, the factor(s) which induces functional changes of effector cells may be secreted from the macrophages modulated by MIF. In the sera of desensitized animals, we found the inhibitory substance(s) against production of lymphokines by immune lymphocytes stimulated with specific antigen (12). Kinetic studies showed that reactivity to SRF returned to a control level 72 hr after the induction of MDR. Of interest was the fact that cutaneous DTH and PHAinduced skin reactions were still suppressed at 72 hr. This discrepancy seems to indicate that suppression of cutaneous DTH by induction of MDR may not be only due to functional changes in effector cells. This would suggest participation of the inhibitory substance(s) against lymphokine production in suppression of cutaneous DTH by induction of MDR. The similar discrepancy in suppression of skin reactions were obtained in the case of ip injection of the macrophage-MIF culture supematants. We also found the inhibitory substance(s) against lymphokine production in suppression of cutaneous DTH by induction of MDR. The similar discrepancy in suppression of skin reactions were obtained in the case of ip injection of the macrophage-MIF culture supematants. We also found inhibitory substance(s) against lymphokine production in the sera of the guinea pigs bearing elicited peritoneal macrophages given ip injection of the MIF fraction (submitted for publication). Thus, it seems conceivable that the inhibitory substance against lymphokine production may also be secreted from the macrophages modulated by MIF. Suppression of cutaneous DTH, in desensitized animals, to the specific antigen which had been used for the desensitization lasted longer than the one to nonspecific antigen (12), whereas dissociation was not observed, in MDR, between duration of suppression of cutaneous DTH to the antigen used for induction of MDR and that of PHA-induced skin reactions. Although MDR does not represent all of the events in desensitization, what should be stressed in this study is that suppression of cutaneous DTH can be achieved even by small amounts (microgram) of an antigen, when large amounts of elicited macrophages are present. Elicited macrophages are known to be more highly responsive to lymphokines than resident tissue macrophages (13) and this would be one of the explanations to our success. This is the first report describing suppressive effect of MDR on cutaneous DTH. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13.
Nelson, D. A., and Boyden, S. V., Immunology 6, 264, 1963. Sonozaki, H., and Cohen, S., J. Immunol. 106, 1404, 1971. Sonozaki, H., Papermaster, V., Yoshida, T., and Cohen, S., J. Immunol. 115, 1657, 1975. Baba, T., Yoshida, T., Yoshida, T., and Cohen, S., J. Immunol. 122, 838, 1979. Sonozaki, H., and Cohen, S., Cell. Immunol. 2, 341, 197 1. Onozaki, K., Haga, S., Miura, K., Ichikawa, M., and Hashimoto, T., Cell. Immunol. 48, 258, 1979. Onozaki, K., Haga, S., Miura, K., Homma, Y., and Hashimoto, T., Cell. Immunol. 55, 465, 1980. Onozaki, K., Haga, S., Ichikawa, M., Homma, Y., Miura, K., and Hashimoto, T., Cell. Immunol. 61, 165, 1981. Ochiya, T., Baba, T., Mizushima, A., Onozaki, K., and Yaoita, H., Cell. Immunol. 71, 346. 1982. Wachstein, M., and Wolf, G., J. Histochem. Cytochem. 6, 457, 1958. Uhr, J. W., and Papermaster, A. M., J. Exp. Med. 108, 891, 1958. Yoshida, T., Baba, T., Suko, M., and Cohen, S., In “Biochemical Characterization of Lymphokines” (A. L. deWeck, F. Kistensen, and M. Landy, Eds.), pp. 593-598. Academic Press, New York, 1980. North, R. J., J. Immunol. 121, 806, 1978.
81, 134-143(1983)
Regulatory Mechanisms of Cutaneous Delayed-Type Hypersensitivity II. Suppression of Cutaneous Delayed-Type Hypersensitivity by Macrophage Disappearance Reaction TAKAHIRO OCHIYA,
TORU BABA,’ KIKUO ONOZAKI, HIDEO YAOITA,
KENICHI UYENO, AND TATSUICHIRO Department
of Dermatology, University
HASHIMOTO
Institute of Clinical Medicine, and Institute of Basic Medical of Tsukuba, Sakura-Mura, Ibaraki-ken 305. Japan Received
May
17, 1983; accepted
Sciences,
June 9, I983
Studies were performed on the behavior of cutaneous delayed-type hypersensitivity (DTH) in guinea pigs in which macrophage disappearance reaction (MDR) was induced. Guinea pigs were immunized with dinitrophenylated egg albumin (DNP-EA), followed by intraperitoneal (ip) injection of liquid paraffin in order to elicit peritoneal macrophages. Subsequently 20 pg of EA was injected into these animals and the animals were divided into two groups. One group of animls was sacrificed for estimation of MDR 6 hr after the subsequent ip injection. The other group received a skin test by EA at the time of the subsequent ip injection. The first group of animals sacrificed for estimation of MDR exhibited a marked reduction in the number of peritoneal macrophages. The second group of animals that received skin tests revealed suppressed skin reactions 24 hr after the subsequent ip injection. A similar experiment was performed using the guinea pigs doubly immunized with DNP-EA and dinitrophenylated bovine y-globulin (DNPBGG). Induction of MDR was performed by ip injection of BGG and skin tests were done by both EA and BGG. As a result, suppression of not only BGC-induced skin reactions but also EA-induced skin reactions was observed in animals in which MDR had been induced by BGG. In addition, the guinea pigs in which MDR was induced showed hyporeactivity to phytohemagglutinin (PHA). Reactivity to skin reactive factor (SRF) was also suppressed in these animals. The culture supematants of macrophages incubated with the MIF fraction in vitro showed the ability to suppress skin reactions of cutaneous DTH, PHA and SRF.
INTRODUCTION
The macrophage disappearance reaction (MDR) is an in vivo manifestation of cellular immunity (1, 2) as well as cutaneous delayed-type hypersensitivity (DTH). A positive MDR represents an antigen-induced reduction in the number of macrophages in the peritoneal cavity. Mediation of this reaction by lymphokines has been verified (3-5). As for the distinct entity of MDR in an in vivo manifestation of cellmediated immunity, Sonozaki and Cohen have suggested that MDR may be an in vivo manifestation of the macrophage migration inhibitory factor (MIF), whereas cutaneous DTH may be more dependent upon chemotactic activity (3). ’ To whom correspondence should be addressed. 134 0008~8749/83 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved.
SUPPRESSION
OF
CUTANEOUS
DTH
BY
MDR
135
In our previous studies, we demonstrated that intraperitoneal (ip) injection of the MIF fraction, which had been recovered from an immunoadsorbent column prepared with anti-guinea pig MIF antiserum (6-Q into guinea pigs with elicited peritoneal macrophages resulted in a reduction in the number of peritoneal macrophages (9). The MIF fraction did not contain macrophage chemotactic factor, neutrophil chemotactic factor, vascular permeability factor, and skin reactive factor (8). Then, we discussed that MDR had a distinct entity from cutaneous DTH and was an in vivo manifestation of MIF activity. Although MDR is a well-known phenomenon, its biological significance is yet unelucidated. In the present study, attempts were made to explore the behavior of cutaneous DTH in animals with MDR. We will describe data suggesting that MDR exerts a suppressive effect on cutaneous DTH. MATERIALS
AND
METHODS
Immunization of animals. Hartley albino female guinea pigs weighing 450 to 500 g were used in all experiments. Dinitrophenylated bovine y-globulin (DNP-BGG) and dinitrophenylated egg albumin (DNP-EA) were prepared by standard techniques. The conjugate contained approximately 5 1 molecules of DNP per molecule of protein in DNP-BGG and 12 molecules in DNP-EA. Guinea pigs were immunized by footpad injection of 100 pg of either DNP-BGG or DNP-EA, or both in Freund’s complete adjuvant (CFA; Difco, Detroit, Mich.). Macrophage disappearance reaction (MDR). The MDR was induced by the methods described by Sonozaki and Cohen (2). Briefly, immunized guinea pigs bearing 4-day oil-induced peritoneal exudates received either an ip injection of 20 pg of antigen in 5 ml of phosphate-buffered saline (PBS) or an ip injection of 5 ml of PBS alone as a control. Six hours after the injection, the peritoneal exudates were harvested with 80 ml of Hanks’ balanced salt solution containing 10 units/ml of heparin. The total number of cells was assessed with a hemacytometer. Differential cell counts were performed by examining both Giemsa-Lusung-stained smears and esterase-stained smears prepared by the methods of Wachstein and Wolf (10). Preparation of guinea pig peritoneal macrophages. Twenty milliliters of sterilized liquid paraffin was injected into the peritoneal cavity of guinea pigs. Four days later, the peritoneal exudates were harvested with 80 ml of Hanks’ balanced salt solution. After washing two times with Hanks’ balanced salt solution and one time with RPM1 1640 medium, the cell concentration was adjusted to 2 X 10’ cells/ml in RPM1 1640 culture medium. The peritoneal exudate cells were >96% macrophages by morphology after May-Giemsa staining and >95% esterase positive. Preparation of the immunoadsorbed MIFfraction. First, lymphokines were prepared as follows. Regional lymph nodes were removed from animals immunized 7 days previously with DNP-BGG in CFA. Lymphocyte suspensions were made from such lymph nodes. After several washings, the cell concentration was adjusted to 1 X IO’ cells/ml in RPM1 1640 culture medium. The cells were incubated overnight in the presence or absence of 100 pg/ml of DNP-BGG. After incubation, the supematants were collected by centrifugation at 400g for 20 min. Control culture supematants incubated in the absence of the antigen were reconstituted with the antigen before centrifugation. Then, the supematants were fractionated by ultrafiltration through Amicon Diaflo membrane XM 1OOA (average molecular weight cut: 100,000) and
136
OCHIYA
ET
AL.
Amicon DiatIo membrane DM 5 (average molecular weight cut: 5000). The fraction with molecular weights from 100,000 to 5000 was used for lymphokine preparation. Immunoadsorbed MIF and control fractions were prepared as described previously (6-8). Briefly, anti-guinea pig MIF antiserum was produced by immunization of rabbits with guinea pig MIF-containing fraction obtained by polyacrylamide gel electrophoresis. The immunoglobulin fraction was precipitated with 40% saturated ammonium sulfate and dialyzed against PBS. Anti-MIF antibody was coupled to BrCNactivated Sepharose 4B (Pharmacia Fine Chemicals, Uppsala, Sweden). The lymphokine preparation was applied to the immunoadsorbent column and eluted with PBS after 1 hr incubation. The nonadsorbed fraction was eluted with PBS after 1 hr incubation. The nonadsorbed fraction was eluted with 50 ml of 1.O A4 NaCl-0.0 1 M phosphate buffer (pH 7.4). Then, specifically adsorbed fraction was eluted with 50 ml of 0.1 M acetic acid solution. The final fraction was dialyzed against PBS and concentrated to original volume on Amicon diaflo membrane PM 10 (average molecular weight: 10,000). As a control fraction, a preparation of lymphocyte culture supernatants treated in the same manner as that described above was used. Skin tests. Antigen solutions were prepared in PBS at a concentration of 50 pg/ ml. One-tenth milliliter of the antigen solution was intradermally injected into the preshaved skin of the back. Twenty-four hours later, the lesions were estimated as induration. The area of the lesion was calculated as the product of the smallest diameter and largest one. The solution of phytohemagglutinin (PHA) (Difco) was prepared with 0.0 1 M PBS (pH 7.4) to contain 50 ~1 of original solution per milliliter. One-tenth milliliter of such solution was injected intradermally into the preshaved skin of the back, and 24 hr after injection measurements of induration were made. Skin reactive factor (SRF) was prepared by 10 times concentration of both lymphokine and control preparations by ultrafiltration through an Amicon membrane PM 10 (average molecular weight cut: 10,000). One-tenth milliliter of SRF was injected intradermally into the preshaved skin of the back and the resulting skin reactions were estimated as induration 6 hr after injection. The area of skin reactions induced by SRF was 120.3 mm2 on the average and the area of that by control preparation was less than 10 mm*. Analysis of data. Means and standard errors of experimental and control groups were calculated and analysis by t test was performed. RESULTS Suppression of Cutaneous DTH
in the Animals
in Which MDR
Was Induced
The guinea pigs were immunized with DNP-EA and 3 days later the animals received an ip injection of 20 ml of liquid paralhn in order to elicit peritoneal macrophages. Further, 4 days later, the guinea pigs with macrophage-rich peritoneal exudates were subsequently given an ip injection of 20 rg of EA in 5 ml of PBS for induction of MDR and the animals divided into two groups; one group of animals (6 animals) was sacrificed for estimation of MDR at 6 hr after the subsequent ip injection and the other group (6 animals) received a skin test by EA (5 pg) at the time of the subsequent ip injection. A group of the immunized animals with macrophage-rich peritoneal exudates (12 animals) was given a subsequent ip injection of PBS alone as a control. These animals were equally divided into two groups and studied in a similar manner. The number of peritoneal macrophages in the animals
SUPPRESSION OF CUTANEOUS
137
DTH BY MDR
given the ip injection of EA was significantly reduced in comparison with that in animals that received ip injection of PBS alone (54.0% loss). As shown in Table 1. skin reactions induced by EA were markedly suppressed in the animals given ip injection of EA compared with those in control animals that received ip injection of PBS alone (P < 0.001). The immunized animals that did not receive the ip injection of liquid paraffin were also prepared. These animals were divided into two groups (6 animals each) in a similar manner at the time of the ip injection; one was given an ip injection of 20 pg of EA and the other received PBS alone. All animals in two groups received a skin test by EA at the time of the ip injection. The EA-induced skin reactions were not suppressed in the animals given ip injection of EA in comparison with those in animals given ip injection of PBS alone. These results seems to indicate that induction of MDR may result in suppression of cutaneous DTH. Antigen-Nonspecljic Induced
Suppression of Cutaneous DTH in Animals
in Which A4DR Was
In this experiment, guinea pigs were doubly immunized with DNP-EA and DNPBGG, and similar experiments were performed. Animals with macrophage-rich peritoneal exudates were prepared and subsequently given an ip injection of 20 pg of BGG in 5 ml of PBS for the induction of MDR. These animals were divided into two groups; one group (6 animals) was sacrificed for estimation of MDR and the other group (6 animals) received skin tests by EA and by BGG at the time of subsequent the ip injection. As a control, a group of the immunized animals with macrophagerich peritoneal exudates (12 animals) was given 5 ml of PBS alone and similarly divided into two groups. The number of peritoneal macrophages in the animals given ip injection of BGG was markedly reduced in comparison with that in control animals (60.3% loss). Not only BGG-induced skin reactions but also EA-induced skin reactions were suppressed in animals with macrophage-rich peritoneal exudates given an ip injection of BGG compared with those in control animals (P -C 0.001) (Table 2). A similar suppression of skin reactions by ip injection of the same dose of BGG could not be obtained by the use of the similarly immunized animals that did not receive the ip injection of liquid paraffin. These results seem to indicate that induction of MDR may lead to antigen-nonspecific suppression of cutaneous DTH. Suppression of PHA-Induced
and SRF-Induced
Skin Reactions by MDR
First, the effect of MDR on PHA-induced skin reactions was studied. Guinea pigs were immunized with DNP-EA and received an ip injection of liquid paraffin as TABLE 1 Suppression of Cutaneous DTH in Animals in Which MDR Was Induced Induration (mm’) Material injected ip 20 wg EA/5 ml PBS 5 ml PBS
With par&n
injected
Without paraffin injected
86.3 f 10.4” 161.0 + 8.2
’ P < 0.001, n = 12 in each experiment. Skin tests were performed by EA.
166.1 + 2.6 165.6 f 7.8
138
OCHIYA
ET AL.
TABLE 2 Antigen-Nonspecific Suppression of Cutaneous DTH in Animals in Which MDR Was Induced Induration (mm’) With paraffin injected
Without paraffin injected
Material injected ip
BGG
EA
BGG
EA
20 pg BGG/S ml PBS 5 ml PBS
32.5 f 9.4” 114.3 f 11.7
81.1 + 13.0’ 153.4 + 9.5
114.4 * 9.0 111.8 + 7.1
142.9 f 7.1 146.9 zk 5.4
a.’ aP < 0.001, bP < 0.001, n = 12 in each experiment. The guinea pigs were doubly immunized with DNP-BGG and DNP-EA.
described above. After the subsequent ip injection of 20 pg of EA in 5 ml of PBS, the animals were divided into two groups; one group (6 animals) was used for estimation of MDR and the other group (6 animals) received a skin test by PHA at the time of subsequent ip injection. As a control, animals with macrophage-rich peritoneal exudates subsequently given an ip injection of PBS alone (12 animals) were also prepared and treated in a similar manner. The number of peritoneal macrophages in the animals given ip injection of EA was markedly reduced in comparison with that in control animals (58.7% loss). As shown in Table 3, skin reactions induced by PHA were suppressed in animals with macrophage-rich peritoneal exudates subsequently given an ip injection of EA in comparison with those in control animals (P < 0.00 1). Using immunized animals that did not receive an ip injection of liquid paraffin, ip injection of the same dose of EA did not affect the reactivity to PHA. SRF is defined as lymphokine activity which induces a skin reaction histologically similar to a typical cutaneous DTH. In order to study the reactivity of effector cells to lymphokines in animals in which MDR was induced, reactivity to SRP was examined at 24 hr after the subsequent ip injection (at the time of estimation of cutaneous DTH). As shown in Table 3, SRF-induced skin reactions were suppressed in the animals with macrophage-rich peritoneal exudate subsequently given ip injection of EA in comparison with those in control animals (P < 0.001). In the animals that did not receive an ip injection of liquid pa&in, suppression of SRF-induced skin reactions was not obtained by ip injection of the same dose of EA. TABLE 3 Suppression of PHA-Induced Shin Reactions and SRP-Induced Skin Reactions in Animals in Which MDR Was Induced Induration (mm’) With paraffin
Without paraffin
Material injected ip
PHA
SRF
PI-IA
SRF
20 pegEA/5 ml PBS 5 ml PBS
61.4 + 13.0” 108.0 k 27.5
54.9 + 10.3b 85.0 f 10.0
116.0 + 2.3 116.1 + 2.1
88.3 + 4.3 88.3 + 1.5
““P
< 0.001, bP < 0.001, n = 12 in each experiment.
SUPPRESSION
OF CUTANEOUS
DTH BY MDR
Kinetic Study on Suppression of the Skin Reaction in the Animals Was Induced
139 in Which MDR
Guinea pigs were immunized with DNP-EA, followed by an ip injection of liquid paraffin. The animals with macrophage-rich peritoneal exudates were divided into two groups. One group of the animals (6 animals) received subsequent ip injection of 20 pg of EA in 5 ml of PBS. As a control, the other group of animals (6 animals) was given an ip injection of the same dose of PBS alone. Studies were done on the kinetics of the hyporeactivity to EA, PHA, and SRF. Skin tests were performed at 24-hr intervals. Skin reactivities were shown as the percentage of control values. As shown in Fig. 1, the reactivity to either EA or PHA statistically returned to a control level at 96 hr, whereas hyporeactivity to SRF statistically diminished at 72 hr (Fig. 2). Suppression of Cutaneous DTH by the Culture Supernatants of Macrophages Incubated with the MIF Fraction The macrophage suspension in RPM1 1640 culture medium (2 X 10’ cells/ml) was incubated for 24 hr at 37°C under a 5% CO*, 100% humidity atmosphere with an equal volume of the MIF fraction which had previously been dialyzed against RPM1 1640 culture medium at 4°C for 48 hr. After incubation, the supematants were collected by centrifugation at 400g for 10 min. Ten milliliters of these supematants was intraperitoneally injected into the six guinea pigs which had been immunized with DNP-EA 7 days previously. A skin test by EA was performed at the time of ip injection. As controls, the following culture supematants were prepared: the culture supematants of macrophages similarly incubated with the control fraction instead of the MIF fraction and the culture supematants of macrophages alone (1 X 10’ cells/ ml). The MIF fraction and the control fraction similarly incubated with an equal volume of RPM1 1640 culture medium were also prepared. Ten milliliters of these control supematants was injected intraperitoneally into each group (six animals) of immunized animals. The immunized animals that did not receive any ip injection were prepared as well. As shown in Table 4, EA-induced skin reactions were markedly suppressed in the animals receiving ip injection of the culture supematants of macrophages-MIF in comparison with those in control animals (P < 0.00 1). Intraperitoneal injection of the MIF fraction alone could not affect the reactivity of the animals to EA.
t i.P inj.
24
hour5
4s after
72
96
120
injection
FIG. 1. Kinetic study on EA-induced skin reactions or PHA-induced skin reactions in animals in which MDR was induced. First skin tests were given at the time of ip injection of an antigen.
140
OCHIYA
ET AL.
.-.
t
24
i.R inj. hours
48
72
after
injection
SRF
96
120
FIG. 2. Kinetic study on SRF-induced skin reactions in the animals in which MDR was induced. First shin tests were given 24 hr after the ip injection of an antigen.
Suppression of PHA-Induced or SRF-Induced natants of Macrophages-MIF
Skin Reactions by the Culture Super-
The culture supematants of macrophages incubated with the MIF fraction were prepared as described above. The nonimmunized normal guinea pigs (six animals) received an ip injection of 10 ml of these culture supematants and skin tests by PHA and by SRF were done at the time of ip injection. Control groups of the animals (six animals each) were prepared in a similar manner as in the above experiment. As shown in Table 5, PHA-induced skin reactions were suppressed in the animals given an ip injection of the macrophage-MIF culture supematants compared with those in control animals (P < 0.001). SRF-induced skin reactions were also suppressed in these animals (P < 0.001). Intraperitoneal injection of the MIF fraction alone could affect the reactivities of the animals neither to PHA nor to SRF. Kinetic Study on Suppression of Cutaneous DTH Guinea pigs (six animals) were immunized with DNP-EA and 7 days later they received an ip injection of 10 ml of the macrophages-MIF culture supematants. Studies were performed on the kinetics of the hyporeactivity to EA in these animals. Skin tests by EA were done at 24-hr intervals. As a control, similarly immunized guinea pigs (six animals) were given an ip injection of the same volume of the macTABLE 4 Suppression of Cutaneous DTH by the Culture Supematants of Macrophages Incubated with the MIF Fraction Supematant injected ip
Induration”
Macrophages + MIF fraction Macrophages + control fraction Macrophages alone MIF fraction alone Control fraction alone None
152.0 221.3 219.2 224.1 224.4 223.3
u Skin tests were performed by EA. b P < 0.001, n = 12 in each experiment.
+ f + + k f
(mm*) 23.66 9.5 4.5 6.1 1.1 9.1
SUPPRESSION OF CUTANEOUS
141
DTH BY MDR
TABLE 5 Suppression of PHA-Induced and SRF-Induced Skin Reactions by the Culture Supernatants of Macrophages Incubated with the MIF Fraction Induration (mm*) Supematant injected ip
SRF
PHA
Macrophages + MIF fraction Macrophages + control fraction Macrophages alone MIF fraction alone Control fraction alone None
55.6 120.1 113.3 112.5 118.4 121.3
+ f + f k +
46.8 141.8 135.2 138.5 129.8 124.2
26.3” 27.3 9.0 12.3 12.5 6.9
f f f f f +
8.0b 16.9 12.3 11.1 13.6 11.1
a P < 0.001, n = 12 in each experiment. b P < 0.001, n = 12 in each experiment.
rophage-control fraction culture supematants. Skin reactivities were shown as the percentage of control values. As shown in Fig. 3, the reactivity to EA statistically returned to a control level at 96 hr. Kinetic Study on Suppression of PHA-Induced or SRF-Induced Skin Reactions
A similar study was performed on PHA-induced and SRF-induced skin reactions. Nonimmunized normal guinea pigs were divided into two groups. One group (six animals) was given an ip injection of 10 ml of the macrophages-MIF culture supernatants and the other group (six animals) received an ip injection of the same volume of the macrophage-control fraction culture supematants as a control. Skin tests by PHA and by SRF were done at 24-hr intervals. As shown in Fig. 4, the reactivity to PHA also returned to control level at 96 hr, whereas the hyporeactivity to SRF diminished at 12 hr. DISCUSSION Cutaneous delayed hypersensitivity in animals previously sensitized with antigen in complete Freud’s adjuvant can be suppressed by systemic injection of that antigen. This requires that the antigen be administered intravenously, intraperitoneally, or
I t
24
4s
72
after
injection
96
120
i.P. inj. hours
FIG. 3. Kinetic study on cutaneous DTH to EA in animals given ip injection of the culture supematants of macrophages incubated with the MIF fraction in vitro.
142
OCHIYA
-100
ET AL.
,,,,,,,,
x;;i
-----;
p f
1 B x
I'
.cx, .;
k'
,' .-
------
PHA
.---.SRF -P Le
b-p
.5 'I
I t i.P.
inj.
24 hwo
48 after
72
aa
120
injection
FIG. 4. Kinetic study on PHA-induced or SRF-induced skin reactions in animals given an ip injection of culture supernatants of macrophages incubated with the MIF fraction in vitro.
intramuscularly in large amounts (milligram) without adjuvant. The process leading to loss of reactivity is known as desensitization (11). The unresponsiveness involves the reactivity to an antigen different from the one used for the desensitization (3). In spite of potential importance of this phenomenon, for example, as an experimental model for clinical anergy state, its mechanism has never been fully investigated. In the present study, it was demonstrated that ip injection of 20 pg of an antigen into preimmunized guinea pigs with elicited peritoneal macrophages resulted in a transient reduction in the number of peritoneal macrophages as well as suppression of cutaneous DTH to the antigen, whereas the similar injection of the antigen into preimmunized animals in which peritoneal macrophages had not been elicited did not affect cutaneous DTH. Suppression of cutaneous DTH achieved by this treatment was antigen nonspecific and PHA-induced skin reactions were also suppressed. These results seem to indicate that induction of MDR leads to suppression of cutaneous DTH as systemic administration of large dose of an antigen. Sonozaki and Cohen have shown that MDR diminished by 20 hr (2) and we also obtained the similar result by ip injection of lymphokines into guinea pigs with elicited peritoneal macrophages (9). However, kinetic study showed that cutaneous DTH was still suppressed 24 hr after the induction of MDR. This seems to indicate that suppression of cutaneous DTH may not be due to a reduction in the number of peritoneal macrophages itself. MDR seems to be considered as in vivo manifestation of MIF activity, since in our previous study we demonstrated that ip injection of the MIF fraction into guinea pigs with elicited peritoneal macrophages led to a transient reduction in the number of peritoneal macrophages (9). Then, we assumed that the macrophages modulated by MIF may secrete the inhibitory substance(s) against cutaneous DTH. Next studies were conducted to examine whether the culture supernatants of the elicited peritoneal macrophages incubated with the MIF fraction in vitro have the ability to suppress cutaneous DTH in preimmunized animals. As shown in the results, ip injection of these culture supernatants into the immunized animals resulted in hyporeactivity to the antigen, whereas similar injection of the same amount of the MIF fraction alone did not affect delayed-hypersensitivity reactions in the animals. Reactivity to SRF was also suppressed in the animals in which MDR had been induced. Hyporeactivity to SRF was observed in the desensitized guinea pigs as well ( 12). Macrophages and polymorphonuclear cells obtained from these desensitized animals were deficient in their ability to respond for chemotactic stimuli in vitro (12).
SUPPRESSION
OF CUTANEOUS
DTH BY MDR
143
These results seem to indicate one of the possible mechanisms of suppression of effector cells to lymphokines. Hyporeactivity to SRF was also achieved by ip injection of the macrophage-MIF culture supematants into normal guinea pigs. Thus, the factor(s) which induces functional changes of effector cells may be secreted from the macrophages modulated by MIF. In the sera of desensitized animals, we found the inhibitory substance(s) against production of lymphokines by immune lymphocytes stimulated with specific antigen (12). Kinetic studies showed that reactivity to SRF returned to a control level 72 hr after the induction of MDR. Of interest was the fact that cutaneous DTH and PHAinduced skin reactions were still suppressed at 72 hr. This discrepancy seems to indicate that suppression of cutaneous DTH by induction of MDR may not be only due to functional changes in effector cells. This would suggest participation of the inhibitory substance(s) against lymphokine production in suppression of cutaneous DTH by induction of MDR. The similar discrepancy in suppression of skin reactions were obtained in the case of ip injection of the macrophage-MIF culture supematants. We also found the inhibitory substance(s) against lymphokine production in suppression of cutaneous DTH by induction of MDR. The similar discrepancy in suppression of skin reactions were obtained in the case of ip injection of the macrophage-MIF culture supematants. We also found inhibitory substance(s) against lymphokine production in the sera of the guinea pigs bearing elicited peritoneal macrophages given ip injection of the MIF fraction (submitted for publication). Thus, it seems conceivable that the inhibitory substance against lymphokine production may also be secreted from the macrophages modulated by MIF. Suppression of cutaneous DTH, in desensitized animals, to the specific antigen which had been used for the desensitization lasted longer than the one to nonspecific antigen (12), whereas dissociation was not observed, in MDR, between duration of suppression of cutaneous DTH to the antigen used for induction of MDR and that of PHA-induced skin reactions. Although MDR does not represent all of the events in desensitization, what should be stressed in this study is that suppression of cutaneous DTH can be achieved even by small amounts (microgram) of an antigen, when large amounts of elicited macrophages are present. Elicited macrophages are known to be more highly responsive to lymphokines than resident tissue macrophages (13) and this would be one of the explanations to our success. This is the first report describing suppressive effect of MDR on cutaneous DTH. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13.
Nelson, D. A., and Boyden, S. V., Immunology 6, 264, 1963. Sonozaki, H., and Cohen, S., J. Immunol. 106, 1404, 1971. Sonozaki, H., Papermaster, V., Yoshida, T., and Cohen, S., J. Immunol. 115, 1657, 1975. Baba, T., Yoshida, T., Yoshida, T., and Cohen, S., J. Immunol. 122, 838, 1979. Sonozaki, H., and Cohen, S., Cell. Immunol. 2, 341, 197 1. Onozaki, K., Haga, S., Miura, K., Ichikawa, M., and Hashimoto, T., Cell. Immunol. 48, 258, 1979. Onozaki, K., Haga, S., Miura, K., Homma, Y., and Hashimoto, T., Cell. Immunol. 55, 465, 1980. Onozaki, K., Haga, S., Ichikawa, M., Homma, Y., Miura, K., and Hashimoto, T., Cell. Immunol. 61, 165, 1981. Ochiya, T., Baba, T., Mizushima, A., Onozaki, K., and Yaoita, H., Cell. Immunol. 71, 346. 1982. Wachstein, M., and Wolf, G., J. Histochem. Cytochem. 6, 457, 1958. Uhr, J. W., and Papermaster, A. M., J. Exp. Med. 108, 891, 1958. Yoshida, T., Baba, T., Suko, M., and Cohen, S., In “Biochemical Characterization of Lymphokines” (A. L. deWeck, F. Kistensen, and M. Landy, Eds.), pp. 593-598. Academic Press, New York, 1980. North, R. J., J. Immunol. 121, 806, 1978.