CELLULAR
IMMUNOLOGY
Accessory
93, 520-53 1 (1985)
Cell Function of Thoracic Duct Nonlymphoid Dendritic Cells, and Splenic Adherent Cells in the Brown-Norway Rat
Cells,
LEX NAGELKERKEN,'MIEKE HENFLING,AND PETERVAN BREDA VRIESMAN Department of Immunology, University of Limburg, P.O. Box 616, 6200 MD Maastricht, The Netherlands Received January 17, 1985; accepted February 21, 1985
Thoracic duct lymph of lymphadenectomized Brown-Norway (BN) rats is highly enriched for nonlymphoid cells (NLC) which share several characteristics with splenic dendritic cells (DC), e.g., the binding of monoclonal antibody 0X2. The accessorycell activity of NLC was analyzed by comparing these cells with DC and splenic adherent cells (SAC). In concanavalin A (Con A)-induced T-cell proliferation NLC, like DC, were very effective accessorycells at low cell numbers, as a consequenceof an efficient induction of interleukin 2 (IL-2) production and IL-2 responsiveness.Responsesin the presence of SAC were poor, even after the addition of excess IL-2. A fourfold enhancement of accessory cell activity of SAC was achieved by the depletion of FcR-positive cells, which were responsible for suppression of the Con A response. Low responsivenessof BN rats with respect to Lewis rats can in part be explained by a higher suppressive activity of macrophages in the BN rat. 0 1985 Academic press, IIIC.
INTRODUCTION It is well known that immune responses in Brown-Norway (BN)* rats are less than those of other rat strains, e.g., Lewis (LEW) rats. This has been demonstrated for mitogen-induced as well as antigen-specific lymphocyte responses in vitro (l-3) and in viva (4-6). The magnitude of a particular immune response may depend on the characteristics of the accessorycells involved. In general, accessorycell function has been attributed to Ia-positive macrophages, and it has been suggestedthat these macrophages exert their role in mitogen responses by the secretion of IL-l (7). However, in the past few years it has been demonstrated in several studies that DC (for review see (8)) are more effective in terms of accessory cell function. In fact, several cells that lack characteristics of macrophages but express Ia antigens are able to function as accessory cells, e.g., Langerhans’ cells (9) and B cells (10). Also, the nonlymphoid cells (NLC) that can be isolated from thoracic duct lymph lack characteristics of macrophages and are strongly Ia positive (11). This cell population is highly enriched for cells with dendritic morphology. ’ To whom correspondence should be addressed. * Abbreviations used: BN, Brown-Norway; BSA, bovine serum albumin; Con A, concanavalin A; DC, dendritic cells; FcR, receptor for the Fc-part of IgG, FCS, fetal calf serum; FITC, fluorescein isothiocyanate; IL-l, interleukin 1; IL-2, interleukin 2; LEW, Lewis; NLC, nonlymphoid cells from thoracic duct lymph; PBS, phosphate-buffered saline; PEG, polyethylene glycol; SAC, splenic adherent cells. 520 0008-8749/85 $3.00 Copyright 0 1985 by Academic F’res$ Inc. All rigbe, of reproduction in any form reserved
ACCESSORY
CELL
FUNCTION
IN THE
RAT
521
In the present study, we have compared the accessorycell activity of NLC, splenic DC, and splenic adherent cells (SAC) from BN and LEW rats in concanavalin A (Con A)-induced T-cell proliferation. Moreover, cells from the BN rat were analyzed for their capacity to induce IL-2 production and to induce IL-2 responsiveness, which are two accessory-cell-dependent steps that proceed T-cell proliferation ( 12-14). The results show that especially NLC are very efficient in this respect. Comparison of various accessory cells from BN rats and from LEW rats suggests that the low immune responsivenessof the BN rat is at least in part due to a higher suppressive activity of macrophages, in spite of the presence of very effective DC. MATERIALS
AND METHODS
Animals. Male BN rats (4 weeks and 10-l 2 weeks of age) and male LEW rats (lo-12 weeks of age) were specific pathogen free, and obtained from the breeding stock of the Department of Animal Services of this University. Isolation of cells. Lymph node T cells were purified from a cell suspension prepared from mesenteric, cecal, and submandibular lymph nodes. Lymph node cells were washed twice with 50 ml Hank’s balanced salt solution, supplemented with 1% newborn calf serum (Gibco, Paisley, U.K.) and centrifuged (30 min, 3808 at 4°C) on a discontinuous (p = 1.053/l. 100 g/cm3) Percoll gradient (Pharmacia, Uppsala, Sweden). Cells at the interface of the Percoll layers were washed three times and were rendered accessorycell dependent by two passagesover nylon wool, according to the method of Julius et al. (15). DC were isolated from spleen as described by Klinkert et al. (16). Briefly, lowdensity cells were obtained by centrifugation of spleen cells (10,OOOgfor 30 min at 4°C in a Beckman SW 40 rotor) on PBS containing about 30% (p = 1.084 g/cm3) bovine serum albumin (Sigma, St. Louis, MO.). Low-density cells were washed three times, resuspended in culture medium supplemented with 10% autologous serum, irradiated (1000 rad, X ray), and maintained in culture for 4 days in plastic culture flasks (Nunc, Roskilde, Denmark) to remove adherent cells. Nonadherent cells were depleted of Fc-receptor (FcR)-positive cells. To this end, sheep erythrocytes were opsonized with a subagglutinating dose of IgG from a rabbit anti-sheep-erythrocyte serum. Nonadherent cells were mixed with the opsonized erythrocytes at a ratio of 1:50, centrifuged (5 min, 1OOg)and allowed to form rosettes for 30 min on ice. FcR-negative cells were recovered by centrifugation on dense BSA (p = 1.084 g/cm3), washed, and resuspended in culture medium. These nonadherent, FcRnegative cells were highly enriched (>80%) for cells with dendritic morphology. About 10% of the cells were positive for nonspecific esterase(performed as described by Koski et al. (17)) and were capable of ingesting latex particles. DC were strongly Ia positive (0X6); in addition, DC bound 0X2, a monoclonal antibody reactive with follicular DC (18). NLC were isolated from thoracic duct lymph of lymphadenectomized rats, as suggestedby Mason et al. (19). Four-week-old BN rats were lymphadenectomized, i.e., mesenteric, cecal, and portal lymph nodes were removed. Rats were allowed to recover from lymphadenectomy for 6 weeks and then received 500 rad of total body irradiation from an X-ray source (Philips-Miiller MG 300; dose rate 55 rad/ min). Eighteen hours later thoracic duct cannulation was performed as described
522
NAGELKERKEN,
HENFLING,
AND
VRIESMAN
by Ford (20). Thoracic duct lymph was collected over a period of 40 to 44 hr. Cells thus collected were washed twice and centrifuged on dense BSA as described for DC. Low-density cells were washed and resuspendedin culture medium supplemented with 10% rat serum. These cells represented about 10% of the total number of leukocytes in thoracic duct lymph of lymphadenectomized and irradiated rats. After overnight culture in plastic culture flasks, nonadherent cells were collected and centrifuged (30 min, 380g at 4°C) on a discontinuous Percoll gradient (p = 1.04/ 1.07 g/cm3) to remove nonviable cells. Cells at the interface of the Percoll layers, further indicated as NLC, were washed and resuspended in culture medium. NLC did not express receptors for the Fc-part of IgG or for C3, and did not ingest latex particles. About 50% of the cells were weakly to strongly positive for nonspecific esterase.More than 97% of the cells bound OX6 as well as 0X2. SAC were isolated by adherence of spleen cells (5 X 106/ml) to plastic culture flasks (20 ml cell suspension/70 cm2) for 2 hr at 37°C. Adherent cells were extensively washed and recovered by trypsin-EDTA treatment (5 to 10 min, 37°C (21)). Cells were washed and resuspended in culture medium. About 75% of the SAC were positive for nonspecific esterase.Contaminating cells were predominantly lymphocytes, i.e., less than 5% B cells (OX12 positive) and less than 15% T cells (W3/13 positive). Ten to twenty percent of the LEW-SAC and 20 to 30% of the BN-SAC bound 0X6; SAC were essentially OX2 negative. Separation of SAC into FcR-positive and FcR-negative cells was achieved by EA rosetting (as described for DC) and centrifugation (30 min, 380g at 4°C) on Percoll (p = 1.09 g/cm3). FcR-positive cells in the pellet and FcR-negative cells at the interface were washed and treated (10 min at O’C) with 155 mM NH&l in 10 mM KHC03, 0.1 rniV EDTA, pH 7.4. Cells were washed and resuspended in culture medium. Culture conditions. Cells were cultured in RPM1 1640 (Flow Laboratories, Irvine, U.K.) containing penicillin (100 U/ml), streptomycin (100 pg/ml), L-glutamine (500 pg/ml), and 2-mercaptoethanol (5 X 10e5 M). For rat lymphocyte cultures, this medium was supplemented with 1% fresh, heat-inactivated rat serum. Murine cells were cultured in medium supplemented with 5% FCS (Gibco). The response of nylon-wool-purified T cells to Con A was measured by culturing 100,000 cells in 0.2 ml medium with Con A (0.5 pg/ml; Pharmacia) and various numbers of irradiated accessory cells (total in vitro dose, 2000 rad) in flat-bottom microtiter plates (Nunc). After 90 hr, cells were pulsed with 0.25 &i [methyl3H]thymidine ([ 3H] TdR; sp act, 5 Ci/mmol; Radiochemical Centre, Amersham, U.K.) and harvested 6 hr later. Interleukins. IL-2 was prepared by stimulating LEW spleen cells (5 X 106/ml) with Con A (2 pg/ml) and phorbol my&ate acetate (1 rig/ml; Sigma) under serumfree conditions in the presence of 0.0 1% PEG 4000. After 2 days of culture, cellfree supernatant was obtained by centrifugation. Partial purification of IL-2 was achieved by concentrating the supernatant on an Amicon YM-5 membrane and by gel filtration on Ultrogel ACA-44 (LKB, Bromma, Sweden), equilibrated with PBS/ 0.0 1% PEG 4000. IL-2 containing fractions (MW 12,000 to 18,000) were pooled. For the measurement of IL-l and IL-2 we used EL-4, an IL-2-secreting murine thymoma cell line (22) and CTLL, an IL-a-dependent murine T-cell line. IL-I was measured indirectly by enhancement of IL-2 production by EL-4 cells. To this end, lo5 EL-4 cells were cultured with Con A (5 pg/ml) in 0.2 ml medium in flat-bottom
ACCESSORY
CELL
FUNCTION
IN THE
RAT
523
microtiter plates in the presence of the supernatant to be tested (final concentration, 25%). After 40 hr, cell-free supernatant was harvested and assayedfor IL-2 activity in the CTLL assay. IL-2 was measured by culturing 5000 CTLL in 0.2 ml medium in flat-bottom microtiter plates in the presence of sample to be tested. Cells were pulsed with 0.2 &i [3H]TdR after 20 hr of culture and harvested 4 hr later. Results are expressed either in units/ml or in cpm; partially purified IL-2 was used as a standard. One unit is defined as the amount of IL-2 that results in half-maximal proliferation of the CTLL (23). Zmmunofuorescence. Cells (2 X 105) were incubated (30 min at 0°C) with 20 ~1 of antibody, diluted in PBS containing BSA (2% w/v), EDTA (0.1% w/v), and NaN3 (0.1% w/v). Ascites was diluted 1 to 50, supernatant 1 to 20. After incubation, cells were washed three times and incubated (30 min at 0°C) with 20 ~1 (dilution 1:20) FITC-labeled goat (Fab’)* anti-mouse IgG (rat absorbed; Cappel, Cochranville, Pa.). Cells were washed and the percentage of positive cells was determined with a fluorescence-activated cell sorter (FACS IV). Monoclonal antibodies used-W3/13, 0X8, and OX12 (in ascites form), and OX2 and OX6 (supernatants)-were purchased from Sera-Lab (Crawley Down, U.K.). RESULTS The eficiency of various accessory cells in Con A-induced T-cell proliferation. In the absence of accessory cells, BN and LEW T cells failed to respond to Con A. Addition of SAC to the cultures showed that Con A-induced T-cell proliferation required the presence of accessory cells. In Fig. 1, it can be seen that a [3H]TdR incorporation by LEW T cells of about 60,000 cpm was obtained in the presence of 20,000 SAC. In contrast, a relatively low response (about 20,000 cpm) was found when the Con A response of BN T cells in the presence of 10,000 BN-SAC was studied; equal or even lower responseswere found when 20,000 SAC were added. By decreasing the number of SAC per culture the proliferative response was shown to be dependent on the number of accessorycells. The response of BN T cells was about three times lower than that of LEW T cells throughout the entire doseresponse curve. That these differences were not due to intrinsic properties of BN T cells was shown by adding NLC or DC as accessory cells, instead of SAC (Fig. 1). For example, with 1000 NLC the [3H]TdR incorporation was about 95,000 cpm. With 60 NLC per culture, the [3H]TdR incorporation was still 15,000 cpm. BNNLC were about two times more effective than BN-DC and about 50 times more effective than BN-SAC. Similarly, LEW-DC were fifty times more effective than LEW-SAC. Addition of 60 LEW-DC per culture already resulted in a [3H]TdR incorporation by LEW T cells of about 30,000 cpm. At low numbers of accessory cells, LEW-DC were about twice as efficient as BN-NLC. These results suggestthat low mitogen responsivenessof the BN rat is at least in part related to accessorycell function, in particular to the poor accessorycell activity of macrophages. Therefore, we further compared NLC, DC, and SAC from BN rats in their ability to induce the production of IL-2, to secrete IL-l, and to induce IL-2 responsiveness. The ejiciency of various accessory cells to induce IL-2 production and to secrete IL-l. In order to examine the efficiency of NLC, DC, and SAC in the induction of
524
NAGELRERKEN,
HENFLING, AND VRIESMAN
number
of accessory
cells
FIG. I. The efficiency of various accessory cells in Con A-induced T-cell proliferation. Nylon-woolpurified T cells (lOO,OOO/welI)were stimulated with Con A (0.5 pglml) and cultured in the presence of various numbers of accessorycells. After 90 hr, cultures were pulsed with 0.25 r.Ki [‘H]TdR and harvested 6 hr later. [‘H]TdR incorporation is given as the mean of triplicate values. Standard deviations were always less than 15%. -, Responseof BN T cells with BN-NLC (O), BN-DC (A) and BN-SAC (m); . * *, response of LEW T cells with LEW-DC (A) and LEW-SAC (0); response in the absence of accessory cells, BN T cells (v), LEW T cells (V).
IL-2 production, in relation to their effect on T-cell proliferation, IL-2 was measured in supernatants of cultures performed under the same proliferative conditions, as used for the experiment shown in Fig. 1. Maximum levels of IL-2 were found after 2 days of culture. In Fig. 2, it is shown that NLC and DC are more effective inducers of IL-2 production than SAC. IL-2 was only detected when more than 250 NLC or DC were added per culture. The amount of IL-2 detected depended on the number of accessory cells that was added. NLC were slightly more effective than DC: the addition of 2000 NLC or DC resulted in the secretion of 70 and 50 units IL-2/ml, respectively. With SAC, no IL-2 could be detected in supernatants harvested at Days l-4. Therefore, we analyzed the supernatants for the presence of IL- 1. IL-l was measured by studying the effect of culture supernatants (harvested at Day 2) on the production of IL-2 by EL-4 cells. This assay was about 500 times more sensitive than the assay in which the direct effect of IL-1 on the proliferation of C3H/HeJ mouse thymocytes was studied (not shown). In Fig. 3, data are shown for supernatants that did not cause proliferation of CTLL, with the exception of supematants from Con A cultures performed in the presence of 500 NLC (700 cpm at a final concentration of 6.25%; background of CTLL only, 300 cpm). It can be seen that a significant enhancement of IL-2 production is only achieved with supematant from cultures performed in the presence of SAC. By increasing the
ACCESSORY CELL FUNCTION IN THE RAT
number
of accessory
525
cells
FIG. 2. The efficiency of various BN accessorycells to induce the production of IL-2. Con A cultures were performed under proliferative conditions (as in Fig. 1) in the presence of various numbers of different accessorycells (0, NLC, A, DC, n , SAC, V, none). Supernatants were harvested at Day 2. IL-2 activity in these supematants was determined with the CTLL assay(see Methods) and expressedin units/ ml. Means f SD of triplicate values are given.
number of SAC in the initial cultures of T cells with Con A, the ability of the supernatant to stimulate IL-2 production by EL-4 cells increased. Stimulation of IL-2 production is most clearly visible with supernatant from Con A-cultures
number
of accessory
cells
FIG. 3. Detection of IL-1 in culture supematants. Supematants of Con A cultures, performed with various numbers of BN accessory cells (0, NLC; A, DC, 0, SAC; V, none) were added to EL-4 cells (lOO,OOO/weh)to a final concentration of 25%. After 40 hr of culture in the presence of Con A (5 &ml), EL-4 supematants were harvested and assayedfor the presence of IL-2 in the CTLL assay (final concentration of EL-4 supematant, 25%). [‘H]TdR incorporation by CTLL is indicated. The results are expressed as mean cpm + SD of quadruplicate determinations (EL-4 cultures in duplicate, CTLL assay in duplicate).
526
NAGELKERKEN,
HENFLING, AND VRIESMAN
performed in the presence of 10,000 SAC. Addition of this supernatant (final concentration: 25%) to EL-4 cells resulted in a supernatant that caused significant proliferation of CTLL: with this EL-4 supernatant (final concentration of 25% in the CTLL-assay), a [3H]TdR incorporation of 9000 cpm was found. The background production of IL-2 (no accessorycells in the initial Con A culture) was low (about 700 cpm). Also supernatants from Con A cultures performed with low numbers of NLC or DC enhanced production of IL-2 by EL-4 cells. In this respect, 250 NLC or DC in the initial culture had the same effect as 3000 SAC. By comparing, for example, the efficiency of 500 NLC with that of 10,000 SAC it can be concluded that SAC are much less efficient accessory cells than NLC in T-cell proliferation (Fig. 1) and induction of IL-2 production (Fig. 2) but-at the same cell numbersmore effective in the secretion of IL-1 (Fig. 3). Accessory cells in the induction of IL-2 responsiveness. Since SAC were poor accessory cells in the induction of IL-2 production, we studied whether the poor T-cell responses observed with SAC could be restored by the addition of IL-2. Actually, by adding an excess of IL-2 (60 units/ml) the ability of various cells to induce IL-2 responsivenesswas studied. The results are shown in Table 1. It can be seen that, in the presence of excessIL-2 accessorycells were still required for T-cell proliferation. The extent of proliferation depended on the number of accessorycells that was added. Addition of IL-2 slightly enhanced T-cell proliferation with either of the accessory cells. The increase in [3H]TdR incorporation ranged from 15,000 to 25,000 cpm. Although addition of IL-2 also enhanced responseswith SAC, these were still poor compared to the responsesfound with NLC or DC as accessorycells. Thus, in contrast to NLC or DC, SAC are poor inducers of IL-2 responsiveness. Enhanced accessorycell activity of FcR-negative SAC. In order to optimize T-cell responsesin the presence of SAC, the latter were further enriched for FcR-positive TABLE 1 NLC, DC, and SAC in the Induction of IL-2 Responsiveness [‘H]TdR incorporation (cpm; mean f SD)
Accessory cell
Number of cells per culture
-IL-2
+1L-2 (60 units/ml)
NLC
250 125 60
63,292 +_7451 34,361 5 4252 22,296 + 3941
78,502 -c 6110 59,650 zk 3869 40,294 + 4783
DC
500 250 125
30,340 + 4862 15,275 f 410 10,038 f 1300
5 1,173 f 5602 38,037 f 3657 26,021 + 2246
10,000 5,ooo 2,500 l,ooO -
25,750 + 1204 13,878 + 1607 6,541 f 1424 3,046 +_ 740
35,487 f 36,349 + 27,521 f 18,206 +
3010 3445 3323 1713
670 + 200
5,200 f
621
SAC
None
ACCESSORY CELL FUNCTION IN THE RAT
527
cells. By separation of BN-SAC into FcR-positive cells and FcR-negative cells it was, however, found that accessory cell activity was largely confined to the FcRnegative cells (Fig. 4, left). FcR-positive cells were less efficient than unseparated SAC. It can be seen that addition of 10,000 FcR-negative cells, unseparated SAC or FcR-positive cells per culture resulted in a [3H]TdR incorporation of 50,000, 25,000, and 12,000 cpm, respectively. Addition of indomethacin (1 pg/ml) did not result in enhancement of responses supported by either of these SAC populations (not shown). Enhanced accessorycell activity of FcR-negative SAC was also found with cells from LEW rats (Fig. 4, right). Addition of 1250 FcR-negative SAC per culture already resulted in a [3H]TdR incorporation of about 40,000 cpm, compared to 8000 cpm obtained with the same number of unseparated SAC. Differences in accessorycell activity of FcR-negative SAC, unseparated SAC, and FcR-positive SAC from BN rats were not related to the percentage of Ia-positive cells. Although the percentage of 0X6-positive cells varied in different experiments (20 to 40%), these were always about the same for the three cell populations. Similarly, the three SAC populations from LEW rats were not different concerning the percentage of 0X6-positive cells (10 to 20% in different experiments). Enhanced suppressive activity of BN-SAC. Differences in accessorycell activity of the various SAC populations were reflected in the effect of the various cells on Con A responsessupported by NLC or DC. Unseparated SAC and FcR-positive SAC of BN rats strongly inhibited (60 and 70% inhibition, respectively) the Con A response supported by 1000 NLC (Fig. 5). FcR-negative SAC were less suppressive (30% inhibition). Suppression was also found at the level of IL-2 production (not shown). With cells from LEW rats, suppression was only observed when FcR-positive SAC were added to cultures supported by 1000 DC (30% inhibition). Unseparated
LEW
number
of accessory
cells
FIG. 4. Accessory cell activity of SAC subpopulations. SAC (w) were separated into FcR-negative cells (0) and FcR-positive cells (Cl) and compared with NLC (0) or DC (A). The effect of various numbers of these cells on Con A-induced T-cell proliferation is shown for BN rats (left) and for LEW rats (right): ‘1. no accessory cells. [3H]TdR incorporation shown is the mean of triplicate values. Standard deviations were less than 15%.
528
NAGELKERKEN,
HENFLING, AND VRIESMAN
120
100
0
I
FcR+
SAC
FcR-
FIG. 5. FcR-positive cells are responsible for suppression by SAC. Ten thousand SAC, FcR-positive cells, or FcR-negative cells of BN rats were added to Con A cultures supported by 1000 NLC (black columns). The same was done with cells from LEW rats; then, the effect on cultures supported by 1000 DC was studied (white columns). The results (means of triplicate values) are expressed as percentage of the response with NLC (BN) or DC (LEW) alone, which were 80,000 and 90,000 cpm, respectively. Standard deviations are indicated.
SAC and FcR-negative cells enhanced responses in the presence of DC. Thus, differences between accessory cell activity of BN-SAC and LEW-SAC are in part explained by a higher suppressive activity of BN-SAC. DISCUSSION The present study was performed to determine whether the low responsiveness of the BN rat is due to a low accessory cell activity. Therefore, we compared the accessory cell activity of NLC, DC, and SAC from BN rats, and of DC and SAC from LEW rats in Con A-induced T-cell responses.In particular, we were interested in NLC from thoracic duct lymph of lymphadenectomized rats, since this cell population is highly enriched for cells with dendritic morphology (11, 19, 24). We found that NLC and DC bound 0X2, a monoclonal antibody that binds to thymocytes, B cells, and follicular dendritic cells, but not to macrophages. This finding indicates that NLC from thoracic duct lymph are related to DC. To compare the various accessorycells we have chosen for the Con A response, since this system does not require antigen processing and allows the analysis of accessory cell function in several steps of T-cell activation. Compared to LEW, T cells of BN rats responded poorly to Con A in the presence of SAC. However, with DC and especially with NLC, profound responseswere found. This finding indicates that the ability of BN T cells to respond to mitogen is not impaired. At low accessory cell numbers, responsesof LEW T cells in the presence of DC were two times as high as responses of BN T cells in the presence of NLC. The possibility
ACCESSORY
CELL
FUNCTION
IN THE
RAT
529
that this result is due to a higher activity of T suppressor cells in the BN rat was not investigated. The relative efficiency of NLC, DC, and SAC to support T-cell proliferation was reflected in the ability of the cells to induce the production of IL-2. It was found that a detectable IL-2 production occurred in the presence of more than 250 NLC or DC, but not in the presence of low or high numbers of SAC. IL-1 was detected in supernatants of cultures performed in the presence of either of the accessory cells. In this respect, SAC were not impaired. Since IL-2 production was measured under proliferative conditions, IL-2 will be consumed by T cells and will be detected only, when produced in excess.This may suggestthat the IL-2 produced with low numbers of NLC or DC (<250/well) or with SAC is completely consumed, and that the produced amount of IL-2 limits the extent of T-cell proliferation. That this is only partly true appeared from the finding that, in the presence of excess IL-2, responseswere still accessorycell dependent, a finding that is consistent with several reports ( 12- 14). Furthermore, our studies demonstrated that SAC were not only poor inducers of IL-2 production but also of IL-2 responsiveness. In this respect, NLC and DC were about fifty times more efficient than SAC. To find an explanation for the poor accessory cell function of BN-SAC, these cells were further analyzed. First, the poor accessory cell activity of SAC was not due to the trypsin treatment performed to obtain these cells, for similar treatment did not influence the efficiency of NLC. Also, the percentage of Ia-positive cells did not account for the finding that BN-SAC were less efficient than LEW-SAC: considerable responses with SAC from LEW rats were obtained although the percentage of Ia-positive cells (10 to 20%) was lower than that of BN-SAC (20 to 40%). Most likely, a lower accessory cell activity of BN-SAC is due to a higher suppressive activity of macrophages. Suppression by macrophages has been described as a result of in vivo activation (25, 26) but has also been reported for responseswith cells from healthy animals (27, 28). That suppression by BN-SAC occurred in our experiments, appeared from the finding that responsesin the presence of NLC and SAC were significantly lower than responses in the presence of NLC alone. It is likely that suppression by SAC is mediated by macrophages and not by T suppressor cells, since SAC did not contain 0X8-positive cells. Depletion of 0X8-positive cells from the responder cell population by “panning,” resulted in enhancement of responseswith NLC and with SAC. However, the relative increase found with either of the accessorycells was the same. Furthermore, enrichment for macrophages by Fc rosetting resulted in the finding that FcR-positive cells were even more suppressive than unseparated SAC or FcR-negative cells. Often, suppression by macrophages can be attributed to the secretion of prostaglandin Ez (29, 30). However, in our system, the addition of indomethacin to inhibit prostaglandin synthesis did not result in enhancement of responses supported by SAC, FcR-positive, or FcR-negative cells. The possibility that suppression by SAC is mediated through oxygen intermediates (31) was not investigated. FcR-negative SAC were much more effective accessory cells than unseparated SAC or FcR-positive SAC, although these cell populations were equal concerning the percentage of Ia-positive cells. This was found with cells from BN rats, but more clearly with cells from LEW rats. Recently it has been demonstrated that
530
NAGELKERKEN,
HENFLING,
AND
VRIESMAN
human monocytes can also be separatedinto an FcR-positive subsetwith suppressive activity and an FcR-negative subset with accessory cell activity (32); these subsets were comparable concerning the expression of HLA-DR antigen. Our findings suggest that lack or loss of the Fc-receptor might be beneficial for accessory cell function. In our experiments Fc-receptor loss may be a consequence of the trypsin treatment employed to obtain the SAC, because Fc receptors on rat macrophages are in part trypsin sensitive (33). This would also account for the finding that a relatively low and variable percentage (50 to 70%) of BN-SAC and LEW-SAC were FcR positive. It has been demonstrated that Fc receptors on mouse macrophages regulate the secretion of agents that are suppressive in lymphocyte responses (34, 35). If the reverse is also true this would explain the lower suppressive activity and higher accessorycell activity of FcR-negative SAC. An alternative explanation would be that cells other than FcR-negative macrophages are responsible for the higher accessorycell activity of FcR-negative SAC, e.g., DC. Low responsiveness of the BN rat, in viva may be a consequence of a low DC/macrophage ratio, together with a high suppressive activity of FcR-positive macrophages. More insight into this phenomenon and into the capacities of DC may be obtained, in vivo, by transfer studies. Nonlymphoid cells from thoracic duct lymph may be very useful to this purpose. ACKNOWLEDGMENTS We are grateful to Mr. Bert Schutte (Department of Internal Medicine) for assistancewith the FACS analysis. This manuscript was kindly prepared by Mrs. F. Teng.
REFERENCES 1. 2. 3. 4. 5.
Newlin, C. M., and Gasser, D. L., J. Immunol. 110, 622, 1973. Williams, R. M., Moore, M. J., and Benacetraf, B., J. Immunol. 111, 1579, 1973. Raff, H. V., and Hinrichs, D. J., Cell. Immunol. 29, 109, 1977. Ruddle, N. H., and Waksman, B. H., J. Exp. Med. 128, 1255, 1968. Williams, R. M., and Moore, M. J., J. Exp. Med. 138, 775, 1973. 6. Koch, C., Stand. J. Immunol. 5, 1149, 1976. 7. Smith, K. A., Gilbride, K. J., and Favata, M. F., Nature (London) 287, 853, 1980. 8. Steinman, R. M., and Nussenzweig, M. C., Immunol. Rev. 53, 127, 1980. 9. Stingl, G., Katz, S. I., Clement, L., Grean, I., and Shevach, E. M., J. Immunol. 121, 2005, 1978. 10. Ashwell, J. D., DeFranco, A. L., Paul, W. E., and Schwartz, R. H., J. Exp. Med., 159, 881, 1984. I 1. Pugh, C. W., MacPherson, G. G., and Steer, H. W., J. Exp. Med. 157, 1758, 1983. 12. Habu, S., and Raff, M. C., Eur. J. Immunol. 7, 451, 1977. 13. Larsson, E. L., Iscove, N. N., and Coutinho, A., Nature (London) 283, 664, 1980. 14. Hiinig, T., Loos, M., and Schimpl, A., Eur. J. Immunol. 13, 1, 1983. 15. Julius, M. M., Simpson, E., and Herzenberg, L. A., Eur. J. Immunol. 3,645, 1973. 16. Khnkert, W. E. F., LaBadie, J. H., O’Brien, J. P., Beyer, C. F., and Bowers, W. E., Proc. Natl. Acad. Sci. USA 77, 5414, 1980. 17. Koski, I. R., Poplack, D. G., and Blaese, R. M., In “In Vitro Methods in Cell-Mediated and Tumor Immunity” (B. R. Bloom and J. R. David, Eds.), pp. 359-362, Academic Press, New York/ London, 1976. 18. Barclay, A. N., Immunology 44, 727, 1981. 19. Mason, D. W., Pugh, C. W., and Webb, M., Immunology 44, 75, 1981. 20. Ford, W. L., In “Handbook of Experimental Immunology” (D. M. Weir, Ed.), pp. 23.1-23.22, Blackwell Scientific, Oxford, 1978.
ACCESSORY
CELL
FUNCTION
IN THE
RAT
531
21. Rosenberg, J. S., Gilman, S. C., and Feldman, J. D., J. Immunol. 130, 1754, 1983. 22. Farrar, J. J., Fuller-Farrar, J., Simon. P. L., Hilfiker. M. L., Stadler, B. M., and Farrar, W. L., J Immunol.
125,2555,
1980.
23. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A., J Immunol. 120, 2027, 1978. 24. Fossum, S., &and. J. Immunol. 19, 49, 1984. 25. Kruisbeek, A. M., and van Hees, M., J. Natl. Cancer Inst. 58, 1653, 1977. 26. Lichtenstein, A., Murahata, R., Terpenning. M., Cantrell, J., and Zighelboim, J., Cell. Immunol. 64, 150, 1981. 27. Weiss, A., and Fitch, F. W., J. Immunol. 120, 357. 1978. 28. Oehler, J. R., Herberman, R. B., Campbell, D. A., and Djeu, J. Y., Cell. Immunol. 29, 238, 1977. 29. Baker, P. E., Fahey, J. V., and Munck, A., Cell. Immunol. 61, 52, 1981. 30. Rappaport, R. S., and Dodge, C. R., J. Exp. Med. 155, 943, 1982. 31. Metzger, Z., Hoffeld, J. T., and Oppcnheim, J. J., J. Immunol. 124, 983, 1980. 32. Zembala, M., Uracz, W., Ruggiero, I., Mytra, B., and Pryjma, J., J. Immunol. 133, 1293, 1984. 33. Boltz-Nitulescu, G., Bazin, H., and Spiegelberg, H. L., J. Exp. Med. 154, 374, 1981. 34. Passwell,J. H., Dayer, J., Gass, K., and Edelson, P. J., J. Immunol. 125, 910, 1980. 35. Ezekowitz, R. A. B., Bampton, M., and Gordon, S., J. Exp. Med. 157, 807, 1983.