Role of macrophages as modulators but not as autonomous accessory cells in primary antibody response

CELLULAR

IMMUNOLOGY

95,288-296 (1985)

Role of Macrophages as Modulators but not as Autonomous Accessory Cells in Primary Antibody Response’ SHIN KOMATSUBARA,’ YOSHITAKA HIRAYAMA, KAYO INABA, KOJI NAITO, KEIICHIRO YOSHIDA,~ JUN KAWAI, AND SHIGERU MURAMATS~ Department of Zoology, Faculty of Science, Kyoto University, Kyoto 606. Japan Received April Il. 1985; acceptedMay 30, I985

(M$) in the in vitro primary antibody responseof murine lymphocytes The role of macrophages to sheeperythrocyteswasinvestigated.Peritoneal Mb were activated to expressIa antigens either in vitro or in vivo. NonactivatedIa- M$ werealsoexamined.We ohservedthat not only Id Mb but also Ia+ M$ failed to trigger the antibody response, in contrast with splenic dendritic cells (DC) which served as potent and autonomous accessorycells, but that M$ modulated the level of response whichwasdependentprimarily on theDC contentof cnlture.Themodulationappeared to incline to suppressionrather than enhancement, when M$ were allowed to remain throughout the culture period for 4 days. A highly enhancing capacity of Mg, however, could be revealed by removing M@ 2 days after the initiation of culture, indicating that Mb exerted their suppressive effectmorestronglyin the late phasethan in the early phaseof in vitro antibody response.The modulatory activity seemed higher in Ia+ M# than in Ia- Mg. Q 1985 Academic FTCS,IDE.

INTRODUCTION

Steinman dendritic cells (DC)5 have been elucidated to be a potent and autonomous initiator of the T-cell response, such as primary mixed leukocyte reaction (MLR) (l-4) or antibody response (5-7). On the other hand, there seems no convincing evidence for the comparableness of the role of macrophages (M4), even though they are Ia-bearing (Ia+) M4, to that of DC (3,5, 8). We have demonstrated, however, that MI$ modulate the level of MLR (3) and enhance that of antibody response (5, 9). In the antibody response of murine spleen cells, we have so far examined the negatively selected M& populations prepared by treating splenic adherent cells with DC-specific or anti-Ia antibody plus complement, so that they comprise the mixture of Ia+ M~#J ’ This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science,and Culture of Japan, the Naito Foundation ResearchGrant for 1983,and the Shimizu Foundation ResearchGrant for 1984. * Presentaddress:Institute of Immunology,University of Toronto, Toronto, Ontario M5S lA8, Canada. 3 Present address:Basic ResearchDepartment, Central ResearchLaboratories, Ajinomoto Co., Inc., Yo kohama, 244 Japan. 4 To whom correspondence should be addressed: Department of Zoology, Faculty of Science, Kyoto University, Kitashirakawa-Oiwakecho, Sakyo-ku, Kyoto, 606 Japan. s Abbreviations used: A cell(s), accessorycell(s); CP, Corynebacterium parvum; CS, supematant of spleen cell culture with concanavalin A; DC, dendritic cell(s); MLR, mixed leukocyte reaction; M$, macrophage(s); PEC, peritoneal exudate cell% PFC, plaque-forming cell(s); SRBC, sheeperythrocyte@.);TGC, thioglycollate medium. 288 0008-8749/85 $3.00 Copyright 0 1985 by Academic Pxs, Inc. All right.3of trpmduction in any form iwend

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and Ia- M4 or Ia- M4 alone and other cells than M& On the other hand, the M$ modulation of MLR was investigated mainly by the use of pure populations of Ia+ or Ia- peritoneal M4 (3). Thus, it should be an indispensably important task for us to check whether MLR-modulatable peritoneal M4 can also modulate the primary antibody response. MATERIALS

AND METHODS

Mice. Male and female C3H/HeSlc mice (Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka) were used at the age of 2-4 months. Medium. Hanks’ balanced salt solution (Nissui Seiyaku Co., Tokyo) was used for washing cells during cell preparation. For incubating or culturing cells, the following mediums were used. Medium 1: Eagle’s minimum essential medium containing 60 &ml kanamycin (Nissui) supplemented with 2 mM glutamine (Gln), 5 mM 4-(2hydroxyethyl)- 1-piperazineethane-sulfonic acid (Hepes), and 5 X 1Oe5A4 2-mercaptoethanol (2-ME). Medium 2: RPMI- 1640 medium (Nissui) supplemented with 2 mM Gln, 10 mM Hepes, 5 X lo-’ M 2-ME, 100 U/ml penicillin, and 100 &ml streptomycin. The pH of the mediums were adjusted to 7.2 by NaHCOs. G-IO-passed spleen cells. Dispersed spleen cells were treated with hemolytic Gey’s solution to eliminate erythrocytes, and suspended in Medium 1 containing 5% fetal bovine serum (FBS). In order to remove adherent cells,the cell suspensionthus prepared waspassedonce or twice through SephadexG- 10(Pharmacia Fine Chemicals, Uppsala) columns as described (9). More than 97% of cells in this population were lymphocytes, and M4 were virtually undetectable by morphology and phagocytosis test. Dendritic cells. DC were prepared from collagenase-dispersedspleen cells according to the method of Steinman and colleagues (10, 11) with minor modifications as described previously (3). Since no Fc receptor-bearing cells to form rosettes with and to phagocytoseopsonized sheeperythrocytes were detectable,the DC preparation seemed to be practically free from M4. Ia+ cell content and morphologically identifiable DC content were more than 90% and 60-90%, respectively. Macruphuges. Procedures for preparing M4 were similar to those described previously (3). C’S+A@ and CS- M$. Peritoneal exudate cells (PEC) harvested from mice injected intraperitoneally (ip) with 2 ml of thioglycollate medium (TGC, Brewer’s medium; Difco Labolatories, Detroit, Mich.) 4 days previously were incubated with Medium 1 containing 5% FBS on 16-mm dishes in 24-well plates (A/S Nunc, Kamstrup, Roskilde) for l-2 hr, and nonadherent cells removed. Adherent cells were irradiated with 650 X ray to prevent the growth of fibroblasts, and cultured for 5 days in the presence or absenceof 10%CS (supernatant of spleen cell culture with concanavalin A) to prepare CS M4 and CS M+, respectively. Virtually no cells other than M4 were present in these M$I preparations. The M4 were washed three times just prior to the culture for antibody response.Yields of CS+ Mr$ and CS M$ were 5065% and 40-50% of initially plated PEC, respectively. Ia+ M4 were usually more than 90% in CS+ M4 and less than 1% in CS M& BCG-PEC and CP-PEC were prepared from PEC harvested from mice which had been injected ip with either 0.8 mg of lyophilized Bacillus Calmette-Guerin (BCG) or 1.5 mg of lyophilized Corynebacterium parvum (CP) 7-14 days previously. Whole PEC were treated with anti-Thy 1.2 monoclonal antibody plus complement, followed by irradiation with 1300 R. 2hAd PEC were adherent cells after incubation of the

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whole PEC for 2 hr. 20hAd PEC and 20hNAd PEC were prepared as adherent cells and nonadherent cells, respectively, by incubating 2hAd PEC for an additional 18 22 hr. The yields from whole PEC and the M# contents of 2hAd PEC, 20hAd PEC, and 20hNAd PEC preparations were 30-50% and 40-70%, 20-30% and 60-80%, and l-4% and 20-40%, respectively. More than 95% of M@in these preparations were Ia+ M& Non-Fc receptor-bearing cells of dendritic morphology were observed to amount to IO-30% of 20hNAd PEC. In the figures of this paper, the cell doses indicated on abscissasfor CS M4, CS’ M& 2hAd PEC, and 20hAd PEC represent the number of PEC initially plated for preparing these adherent cell preparations. Antigen. Sheep erythrocytes (SRBC) stored in Alsever’s solution at 4°C for 1-4 weeks after aseptic bleeding were washed three times before use. Antibody response.Accessory cell (A cell) activity of DC and M@ was assessedas the ability to elicit plaque-forming cell (PFC) response from G-lo-passed spleen cells. G- lo-passed cells (4-5 X 106) supplemented with DC and/or Md were cultured in the presence or absence of SRBC in 1 ml of Medium 2 containing 5% FBS. The culture was performed at 37°C in 24-well plates for 4 days in 6% C02-94% air atmosphere. Only the direct PFC method to assayIgM antibody-secreting cells was employed, since indirect PFC were seldom detected under this experimental condition. The number of PFC in the culture without antigen was subtracted from that with antigen; the former was usually less than 10% of the latter. RESULTS

DC are potent and autonomousA cells. Potent A-cell activity of DC without help from M#J was observed throughout these experiments (Figs. 1,4, and 5), conforming to the result of Inaba et al. (5,6). The level of PFC response of G-lo-passed cells was dependent on the dose of DC under examination. h-f@are inadequateasA cells.We examined CS+(Ia’) M$, CS (Ia-) Md, and three

No. of DC or M4 06 of culture)

FIG. 1. DC, but not CS- M# nor CS+ M#, are potent A-cells in the in vitro primaryanti-S= antibody response.G-IO-passed spleen cells (4 X 106)were cultured with 4 X 106SRBC in the presence or absence of graded dosesof DC (A), CS+ Mg (0), or CS- Mg (O), indicated on the abscissain percentage of culture. Each symbol and vertical bar representthe mean PFC number of triplicate cultures and the SEM, respectively.

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different preparations from BCG-PEC or CP-PEC, and obtained no evidence that M$ served as potent A cells in the primary antibody response. Neither CS M4 nor CS M4 prepared from TGC-PEC elicited PFC responsefrom G- 1O-passedcells, while DC manifested strong A-cell activity under the samecondition (Figs. 1, 4, and 5). Similar was the case with CS’ M@ and CS M4 prepared from proteose peptone-elicited PEC (data not shown). Ia+ M4 activated in vivo also seemedto be incompetent asA cells. In the experiment of Fig. 2, BCG-PEC were examined. 20hAd PEC of which the great majority were Ia+ M4 did not show A-cell activity, though 2hAd PEC induced PFC response to a significant extent. On the other hand, 20hNAd PEC manifested an efficient A-cell activity, indicating that actual A cells in 2hAd PEC were temporarily adherent and became nonadherent during overnight culture. Figure 3 shows the result of experiment for CP-PEC. Similar to the case of BCGPEC, 20hAd PEC failed to induce PFC response,while 20hNAd PEC manifested high A-cell activity. 2hAd PEC, however, appeared almost incompetent as A cells, when they were cocultured with G-lo-passed spleen cells through the culture period for 4 days (Fig. 3A). This seemsto be causedby a suppressiveeffect of some cells in 2hAD PEC on the development of PFC in the late phase of 4-day culture, since a limited coculture of 2hAd PEC and G-lo-passed cells for the first 1.5 days resulted in the induction of PFC response(Fig. 3B). 2OhAd PEC were still inadequate as A cells even under the same condition. Taken together, it is surmisable that potent A cells in BCG-PEC and CP-PEC were some temporarily adherent cells other than typical Ia+ M& Modulation of antibody responseby M$. G-lo-passed spleen cells were cultured for 4 days with SRBC and graded dosesof DC in the presenceor absenceof 1- 10%CS+ or CS M& Results are depicted in Fig. 4. Similar to the result in Fig. 1, neither CS’ M+ nor CS M4 by themselves served as A cells, and DC manifested autonomous Acell activity. Addition of Md to the culture containing DC resulted in reduction, slight augmentation, or no significant modulation of anti-SRBC response,depending on the

No. of PEC (% of culture)

FIG.2. Comparison amongdifferent preparations of BCG-PEC in the A-cell activity. G-IO-passedspleen cells (5 X 106)were cultured for 4 days with 5 X IO6SRBC in the absence(@)or the presenceof 2hAd (O), 2OhAd (e), or 2OhNAd (A) BCG-PEC. Other indications arc the same as in the legend of Fig. 1.

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A

1000

500 al 2 2

0u

Y

6

16

0

0.3 0.6

1

2

No. of PEC (% of culture)

FIG. 3. Comparison among different preparations of CP-PEC in the A-cell activity. G-lo-passed spleen cells (5 X 106)were cultured with 5 X lo6 SRBC in the absence (0) or the presence of 2hAd (0), 2OhAd (O), or 2OhNAd (A) CP-PEC. Whole components were cultured for 4 days (A), or nonadherent ceils were transferred 1.5 days after the initiation of culture, and continued to culture for additional 2.5 days. Each symbol represents the mean of duplicate cultures.

dose of DC and M4. Mentioning only augmentation and suppression, the very low response in the presence of 0.03% DC was slightly augmented by 1% CS M9 and suppressedby 10%CS M4, while the low but somewhat higher responsein the presence of 0.1% DC slightly augmented by 1% CS- M4, and slightly or strongly suppressed by 3% or 10% CS- and CS+ M& On a high response in the presence of 0.3% DC,

609

t

0% DC

t

0.03% DC

No.of M@JW of culture) FIG. 4. The effect of M& on the PFC responsetriggered by DC. G-lo-passed spleen cells (4 X 106)were cultured with 4 X 106SRBC for 4 days in the presence of graded dosesof DC (A) and/or CS M6 (0) or CS- M$I (0). Other indications are the same as in the legend of Fig. 1.

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however, M$ appeared to exert only suppressive effect. Thus, the responsewas moderately suppressedby 1% Mrj and almost completely by 3% and 10% Mb. The suppressive activity appeared to be slightly higher in CS+ M4 than in CS M$. The result in Fig. 4 may appear to indicate that M4 behave almost exclusively as a suppressor but not as an amplifier in antibody response. In Fig. 3, however, it was shown that 2hAd CP-PEC population revealed A-cell activity, when the presence of 2hAd CP-PEC in the culture was limited for the first 1.5 days in the total period for 4 days. Thus, we conducted an in vitro cell transfer experiment in which all other cells than adherent M~#J were transferred to other dishes at the end of the first 1 or 2 days, and continued to culture for another 3 or 2 days until PFC assay.The cultures kept untransferred for 4 days were also examined. Results are shown in Fig. 5. In the culture without M4, the PFC response became higher as the DC content increased. The cell transfer 1 or 2 days after the initiation of culture did not interfere with the progress of response.On the other hand, neither 0.3-3.0% CS+ M4 nor CS M4 were competent as A cells, even when cocultured with G-lo-passed cells only for the first 1 or 2 days. In the untransferred culture experiment, the effect of M4 on antiSRBC response of the culture containing 0.1 or 0.3% DC was essentially similar in tendency to that observed in the experiment shown in Fig. 4, except that l.O%, and also 0.3%, CS M4 augmented the response of culture containing 0.3% DC. Such a difference from the result in Fig. 4 may be caused by the fact that the level of PFC response in the presence of 0.3% DC without M4 in Fig. 5 was somewhat lower than that in Fig. 4. The enhancement of anti-SRBC response by the addition of M4 to the culture containing DC was disclosedin the cell transfer experiment. This was most conspicuous in the case in which the primary culture containing 0.3% DC and CS M~#Jor CS M4 at the optimal dose of 0.3% proceeded for 2 days. The response augmented by 0.3% M4 was suppressedby the further addition of M&J,and the suppressive activity was higher in CS+ M4 than in CS M& In the experiment in which the primary culture containing 0.3% DC and M4 proceeded only for 1 day, CS+ M+ were found to slightly enhance the response,and even excessdosesof M4 at 1% or more did not exert a strong suppressive effect. On the other hand, the cell transfer experiment in the culture containing 0.1% DC and M4 did not show any drastic enhancement or suppression of the response. This suggeststhat the synergistic effect of M@ with DC may be dependent on the basal level of antibody responsetriggered by the interaction between lymphocytes and DC. DISCUSSION Theseexperiments and our previous results (5) demonstrate that DC are autonomous A cells, and that M& whether splenic or peritoneal, are modulators in primary antibody response. This seems comparable to the role of M4 in MLR, in which DC trigger alloreactive T cells and M4 modulate their proliferation (3). Our argument that DC behave asautonomous A cells without help from any phagocytic cells seemsconsistent with the experimental result of Inaba et al, (5,6). One might doubt, however, if DC could manifest the A-cell activity by themselveswithout phagocytosing and processing such a particulate antigen as SRBC, since DC are known to be nonphagocytic cells (12). Though both DC and G-lo-passed spleen cell preparations used in our present experiments were actually free from M4, the G-IO-passed cell preparation usually contained O. l-3.0% polymorphs, and the DC preparation might contain an unde-

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Mti

0.3% DC + M$J

L

No. of DC or

M+ (% of culture)

FIG. 5. Highly enhancing effect of M$ revealed by cell transfer experiments. G-IO-passed spleen cells (4 X 106)were cultured with 4 X IO6SRBC in the presenceof graded dosesof DC (A) and/or CS+ M+ (0) or CS M& (0). Whole components were cultured for 4 days (A), or nonadherent cells were transferred 2 days (B) or 1 day (C) after the initiation of culture, and continued to culture for additional 2 days (B) or 3 days (C). Other indications are the same as in the legend of Fig. 1.

tectably small number of Mvld,which might be indistinguishable from DC by morphological observation and by phagocytosis test at the beginning of culture. In several preliminary experiments, we entirely removed polymorphs from G- 1O-passedcells by incubating them for further 12-20 hr in petri dishes, followed by an additional passage of nonadherent cells over a Sephadex G- 10 column. In addition, DC preparations were treated with leucine methyl ester as a lysosomotropic agent to cause death of M4 ( 13). These treatments of G- 1O-passedcells and DC did not affect the anti-SRBC response (data not shown). Since the first discovery by Mosier in 1967 that the A-cell activity in antibody responseresidesin adherent cell population (14), the A cell has generally been believed to be Ia+ M4 [reviewed in Ref. (15)]. Similar to our previous documentation on splenic M4 (5), however, we obtained no evidence that Ia+ peritoneal M+ serve as potent A cells. Thus, G-lo-passed cells did not develop significant PFC response in the presence of not only CS (Ia-) but also CS (Ia’) M4 (Figs. 1, 4, and 5). This was also the case when M4 were removed from the culture in the first half of culture period (Fig. 5). M4 activated in vivo to express Ia by intraperitoneal inoculation of BCG or CP also seem unable to elicit antibody response. Among 2hAd PEC, 20hAd PEC, and 20hNAd PEC prepared from BCG-PEC or CP-PEC, Ia+ M4 content was highest in 20hAd PEC; nevertheless, they did not manifest A-cell activity in contrast with 2hAd PEC and 20hNAd PEC (Figs. 2 and 3). This seemsto argue that potent A

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cells in BCG-PEC and CP-PEC are not Ia+ M4 but some other cells which adhere temporarily to be contained in 2hAd PEC and become detaching during overnight culture from plastic surface to move into 20hNAd PEC. DC may be a candidate for such an A cell, since we frequently found DC-like cells in 20hNAd PEC, and since murine DC are temporarily adherent cells ( 10, 12). The reason that even Ia+ M$ fail to trigger the antibody response is still uncertain, as in the case of MLR in which they also incompetent as potent stimulators. There are several possibilities as discussedin a previous paper (3). One of the possibilities is that Ia antigens on M4 may be so highly sialylated, as well as those on B cells (16), that Ia+ M4 are difficult to interact with unprimed T lymphocytes. Treatment of CS+ M4 with neuraminidase, however, did not disclosetheir A-cell activity (data not shown). Another possibility is that some suppressiveagents,such as prostaglandins or hydrogen peroxide ( 17-19), which may be produced by Ia+ M$ and counteract their A-cell activity, if present. We observed no A-cell activity of M4, however, in the culture supplemented with indomethacin as an inhibitor of prostaglandin synthesis and/or catalase as a scavenger of hydrogen peroxide (data not shown). Thus, even if some factors may suppressa possible A-cell activity of Ia+ M4, they would be neither prostaglandins nor hydrogen peroxide. The modulatory role of M$Jin the antibody responsewas investigated in this study by using CS M4 and CS M4 as representatives of Ia+ M4 and Ia- M& but not by Ia+ M4 populations prepared from BCG-PEC or CP-PEC. The reason for this was that the formers comprised only M& while the latters contained non-M4 cells which may exert some influences upon the antibody response triggered by DC. Thus, we conducted experiments of which results are shown in Figs. 4 and 5. The presence of M4 and DC in the culture for antibody responsegave somewhat complicated results, depending on the dose of DC and M4, and on the duration of presence of M$J after the initiation of culture. When M4 were kept unremoved throughout the culture for 4 days, their effect appeared to almost incline to suppress the antibody response, especially in otherwise high response.Broadly speaking, the suppressiveeffect seemed higher in Ia+ M& than in Ia- Mb, since a slight enhancement by low percentagesof M4 (0. l- 1.O%)was seen more frequently in Ia- M$. Separation of nonadherent cells from the adherent M4 in the first half of the culture period seemssuccessfulto disclosethe enhancing effectof M4 on the antibody response. As shown in Fig. 5, the PFC response in the presence of 0.3% DC was remarkably augmented by the addition of 0.3% Ia+ or Ia- M4, when M4 were removed from the culture at the end of the first 2 days of culture. Further increase of the M$Jdose resulted in the reduction of response. This may be ascribable either to the aftereffect of excess dosesof M$ contained only in the primary culture or to the carry-over of a number of M4 to the second culture, or to both. At any rate, Ia+ Mb were more suppressive than Ia- M4, suggestingthat the suppressiveactivity is higher intrinsically in Ia+ M@ than in Ia- M4. The enhancing potential may be also higher in Ia+ M4, since the PFC number augmented by 0.3% Ia+ M$J and that by 0.3% Ia- M4 was apparently not different from each other. This seemsto be supported by another experiment in which M4 were allowed to be present only for 1 day from the initiation of culture. The low responseof the culture containing 0.1% DC was not so drastically modulated by the addition of M4, though slightly enhanced or suppressed by M& This may indicate that M4 modulation requires a certain degree of the basal level of response triggered by DC. In the case of MLR, however, we previously documented that M$, especially Ia+ M4, greatly enhanced an otherwise undetectably low level of T-cell

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proliferation in response to low dose allogeneic DC (3). Such a discrepancy in the requirement of M4 modulation for the basal level of response between MLR and antibody response would be explained by assuming that the effect of M4 is much more complicated in the latter than in the former because of the difference in the number of variables between them. Results obtained by the in vitro cell transfer experiments mentioned above seemto indicate that M4 exert a suppressive effect more strongly in the late phase than in the early phase of the culture for antibody response. This would explain the result of an experiment shown in Fig. 3B in which the A-cell activity of 2hAd CP-PEC was revealed when adherent M$I were removed from the culture in the early phase of antibody response. The 2hAd PEC population comprised Ia+ M4 in the majority and DC-like cells in the minority, and its A-cell activity may probably be manifested by the synergism between these two cell types. The retention of Ia+ M4 still in the late phase of culture could counteract the A-cell activity which otherwise be manifested by such a synergism. Considering that the retention of M4 even throughout the culture period results in the drastic enhancement of the low level MLR (3), it seemssurmisable that the enhancing effectsof Md contribute mainly to the induction of helper T cells in antibody response, and M4 activated by the interaction with the induced T cells exert a suppressive effect on the B-cell response. This assumption may be relevant to the present result that the suppressiveeffect seemshigher in Ia+ Mq5than in Ia- M~#J. More detailed investigations on the mechanism of cellular and molecular interactions among DC, Md, T cells, and B cells are under way. ACKNOWLEDGMENTS We gratefully acknowledge the advice of Dr. Ralph M. Steinman of the Rockefeller University in the course of manuscript preparation. Thanks are also due to all other members of our laboratory for their fruitful discussionsand technical assistance.

REFERENCES I. Steinman, R. M., and Witmer, M. D., Proc. Natl. Acad. Sci. USA 75, 5132, 1978. 2. Steinman, R. M., Gutchinov, B., Witmer, M. D., and Nussenzweig, M. C., J. Exp. Med. 157, 613, 1983. 3. Naito, K., Komatsubara, S., Kawai, J., Mori, K., and Muramatsu, S., Cell. Zmmunol. 88, 361, 1984. 4. Inaba, K., and Steinman, R. M., J. Exp. Med. 160, 1717, 1984. 5. Inaba, K., Steinman, R. M., Van Voorhis, W. C., and Muramatsu, S., Proc. Natl. Acad. Sci. USA 80, 6041, 1983. 6. Inaba, K., Granelli-Pipemo, A., and Steinman, R. M., J. Exp. Med. 158, 2040, 1983. 7. Inaba, K., Witmer, M. D., and Steinman, R. M., J. Exp. Med. 160, 858, 1984. 8. Steinman, R. M., Nogueira, N., Witmer, M. D., Tydings, J. D., and Mellman, I. S., J. Exp. Med. 152, 1248, 1980. 9. Inaba, K., Nakano, K., and Muramatsu, S., J. Immunol. 127,452, 1981. 10. Steinman, R. M., Kaplan, G., Witmer, M. D., and Cohn, Z. A., J. Exp. Med. 149, 1, 1979. 11. Nussenzweig, M. C., and Steinman, R. M., J. Exp. Med. 151, 1196, 1980. 12. Steinman, R. M., andcohn, Z. A., J. Exp. Med. 137, 1142, 1973. 13. Thiele, D. L., Kurosaka, M., and Lipsky, P. E., J. Immunol. 131, 2282, 1983. 14. Mosier, D. E., Science (Washington, DC.) 158, 1573, 1967. 15. Unanue, E. R., Adv. Immunol. 31, 1, 1981. 16. Cowing, C., and Chapdelaine, J. M., Proc. Nati. Acad. Sci. USA 80,6000, 1983. 17. Schultz, R. M., Pavlidis, N. A., Stylos, W. A., and Chirigos, M. A., Science (Washington, D.C.) 202, 320, 1978. 18. Nathan, C. F., Silverstein, S. C., Brukner, L. H., and Cohn, Z. A., J. Exp. Med. 149, 100, 1979. 19. Metzger, Z., Hoffeld, J. T., and Gppenheim, J. J., J. Immunol. 124,983, 1980.