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Effect of hydrocortisone on the in vitro human antibody response: Interaction with monocytes and prostaglandins
CLINICAL
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
29, 403-414 (1983)
Effect of Hydrocortisone on the in Vitro Human Antibody Response: Interaction with Monocytes and Prostaglandins PIERRE GALANAUD,
MARIE-CLAUDE CREVON, DOMINIQUE ANNIE ABELLA
EMILIE,
AND
Inserm U 131 and Universitk Paris-Sud 32, rue des Carnets, 92140 Clamart, France
The specificplaque-forming cell response inducedby trinitrophenylpolyacrylamide beadsin culturesof nonadherenthumanperipheralbloodmononuclear cells(PBM) is resistantto the inhibitory effect of hydrocortisone(HC). Previousresultsshowedthat low concentrations(up to lo-’ M) of HC inhibit the sameresponse in culturesof unfractionatedhumanPBM. It is shownthat the additionof 5-10%monocytesto nonadherentPBM renderstheir responseHC-sensitive.One mechanism of the interaction betweenHC andmonocytesis the potentiationof prostaglandin Er-mediatedsuppressionby HC, whichcanbe demonstrated in vitro andafter in vivo administration of HC. However,interactionwith a non-prostaglandin-mediated suppression by monocytes should be involved, particularlywith high(10e6M) concentrations of HC. INTRODUCTION
The effects of corticosteroids on the immune response vary according to the model used to induce this response in vivo (1) or in vitro (2-7). This may be due to different intrinsic susceptibilities of the precursor lymphocytes (8, 9) or to the multiplicity of the effects of steroids on the regulatory cells. Indeed, corticosteroids can inhibit suppressor T cells (10, 11) as well as the production of lymphokines (12, 13) and monokines (14). We have shown that the anti-TNP response of human peripheral blood mononuclear cells (PBM) to trinitrophenyl polyacrylamide beads (TNP-PAA) is steroid sensitive (7) unlike the nonspecific B-cell response induced by pokeweed mitogen (3-5). We now show that the suppression of the anti-TNP response by hydrocortisone (HC) requires a threshold proportion of monocytes in the cultures of human PBM. HC interacts with monocytes to suppress the anti-TNP response, suggesting that the different HC sensitivity of B-cell responses is due to a different susceptibility to monocyte-mediated suppression. In our system this suppression is at least partially mediated by prostaglandin E2 (PGE&. MATERIALS
AND METHODS
Reagents. Polyacrylamide beads (Bio-Gel P-30, 100-200 mesh) were obtained from Bio-Rad Laboratories (Richmond, Calif.). They were amino-ethylated as already reported (15) and reacted with excess trinitrobenzenesulfonic acid (Sigma, St. Louis, MO.) in order to prepare TNP-PAA. HC (Merck), indomethacin (Sigma), and PGE2 (Sigma) were dissolved in ethanol at 10e2, 3 x 10p3, and lop2 M, respectively. These stock solutions were further diluted in phosphate-buffered saline and culture medium. We verified that 1% ethanol (corresponding to seven 403 0090-1229/83 $1.50 Copyright @ 1983 by Academic Ress, Inc. All rights of reproduction in any form reserved
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GALANAUD
ET AL.
times the highest concentration used) had no effect on the response. E3H]PGE2 was obtained from New England Nuclear (Amersham) and anti-PGE, antibody from Institut Pasteur Paris. In vitro antibody response. Blood was drawn from 18- to 35year-old healthy volunteers. Different donors were used in each series of experiments. PBM were separated (see below) and cultured at a concentration of 5 x 106 cells in l-ml cultures as already described (15). The only minor modification was in the culture medium (RPM1 1640 supplemented with 5 x lO-5 M 2-mercaptoethanol). This does not modify the effect of HC on the response. When the effect of exogenous PGE2 was being tested, indomethacin (3 x 10d6 M) was introduced into the culture medium to inhibit the production of endogeneous prostaglandins. Assay for the anti-TNP response. The cells were collected on Day 7 and the anti-TNP response was measured using a Jerne-type plaque assay (15). The target erythrocytes were unconjugated sheep red blood cells (SRBC) and TNP-SRBC. When necessary the total nonspecific B-cell response was measured using a reverse hemolytic plaque assay (16). The results are expressed as the number of plaque-forming cells/IO6 collected cells (PFC/l06) in each culture assayed individually. At least three cultures were performed in each experimental group. The cell recovery and viability were identical in all groups. Unless otherwise stated the results are the mean (2 SE) responses from different experiments involving different donors. Student’s t test was used for statistical analysis. Cellfructionations. Two different preparations were used as responding cells: unfractionated PBM isolated on Ficoll-Hypaque and nonadherent PBM, additionally filtered on a nylon wool column (17, 18). To recover the nonadherent PBM, the nylon wood was rinsed with warm medium and pressed with the plug of the syringe. Among the cells recovered by this procedure (about 50% of the starting cell number) the proportions of T and B lymphocytes were not modified (33). In addition, we recently verified that the Td/Ts ratio (19) is not modified after nylon filtration (from 1.64 to 1.59). In contrast, the proportion of peroxidase-positive cells decreases from 15-30% to 3-5% after nylon filtration. Cells strongly adherent to plastic (adherent cells) were prepared as follows: 6 x lo6 unfractionated cells were incubated on culture dishes for 90 min at 37°C in a culture medium containing 20% fetal bovine serum. Nonadherent cells were removed by vigorous washings and adherent cells were recovered by washing the dishes with cold phosphate-buffered saline and with the help of a rubber policeman They contained more than 85% peroxidase-positive cells. In some experiments 2500-R-irradiated PBM or PBM depleted of E-rosette-forming cells (Ecells) were used to prepare adherent cells. Dosage of PGE2. The amount of PGE;! produced after 48 hr in cultures of unfractionated PBM was measured in pooled supernatants from three cultures. Prostaglandins were extracted (20) and PGE2 concentrations were evaluated by radioimmunoassay (2 1). RESULTS
The Anti-TNP Response of Nonadherent PBM Is He-Resistant When nonadherent PBM are stimulated with TNP-PAA in the presence of BP6 M HC the anti-TNP response is virtually unaffected (Table 1, line 1). In the
B-CELL-RESPONSE
SUPPRESSION TABLE
ADHERENT
CELL
REQUIREMENT
1 FOR SUPPRESSION
Anti-TNP Cells in culture Nonadherentb Nonadherent c Nonadherent + 10% adherentb Nonadherent + 10% adherentc
TNP-PAA” + + -
405
BY HYDROCORTISONE
BY
HC
responses when HC (10m6 M) -
+
322 r 133 10 f 3 220 2 45d 524
219 f 50 3+2 16 + Sd NDe
a TNP-PAA added or not added to the cultures. b Nine experiments. c Three experiments. d P < 0.001. p ND, not determined.
absence of antigen only few anti-TNP PFC develop regardless of the presence of HC (Table 1, line 2). This is also true when the non-cross-reactive antigen fluorescein-PAA (22) is present in the cultures (results not shown). In the same line we verified that HC does not markedly enhance the number of reverse PFC (16) in cultures of nonadherent PBM containing TNP PM (from 2892 + 355 to 3190 k 84 PFC/106). We previously showed that the anti-TNP response of unfractionated PBM is 80% inhibited by lop6 A4 HC (7). This suggested that an adherent cell is required for HC to suppress the in vitro antibody response. We used plastic adherence as a source of adherent cells (Table 1). The addition of 5 x ld adherent cells has some suppressive effect but this suppression is inconstant and not statistically significant (P > 0.45). However, in the presence of adherent cells HC is profoundly suppressive. We verified in two experiments that this suppression does not reflect a modification in the kinetics of the response. The active adherent cell is radio-resistant: HC suppressed the response of nonadherent PBM from 859 to 90 PFCAo6 in the presence of 10% irradiated monocytes (results from one out of three experiments). This celI is non-T: adherent cells isolated from E--cells also allowed HC to suppress the response of nonadherent PBM (from 671 to 25 PFC/ IO6 in one out of three experiments). Monocytes
and HC Interact
to Suppress the Anti-TNP
Response
The effect of various numbers of adherent cells on the response of a fixed concentration of HC is shown in Fig. 1. The results obtained with the highest number of monocytes (5 x 105) are in keeping with those presented in Table 1. HC has a comparable effect with 2.5 x 16 adherent cells (P < 0.02) and significantly suppresses the response (p < 0.05) when 1.25 x lo5 adherent cells are present. More importantly, for the two lower amounts of monocytes the effect of HC is directly related to the number of monocytes present in the cultures. In two experiments we measured the effect of three HC concentrations (10m6, 10p7, and lo-* M) on the response of nonadherent PBM cocultured with three concentrations of adherent cells. In these experiments even the highest number of adherent cells did not inhibit the response (see Fig. 2). In both experiments
GALANAUD
406 Percent
of controla
ET AL.
T
1
1 I I
1 1
T
.-
1 5.80 L
FIG. 1. Interaction between HC and various numbers of adherent cells. Response of nonadherent PBM cultured without increasing numbers of adherent cells without HC (Cl) or with 10” M HC (m). Results are the mean ( 2 SE) relative responses (compared to that of cultures containing neither HC nor adherent cells) in four to six experiments. Comparison between (0) and (m): P < 0.02 and P < 0.05 with 1.25 x lo5 and 2.5 x lo* adherent cells, respectively.
the inhibition was related to the concentration of HC and to the number of adherent cells. The results of one of these experiments are presented in Fig. 2. With the highest number of adherent cells (5 x 105), 3 log dilutions of HC (up to 10e8 A4) are inhibitory. With the lowest number (1.25 x 10’) HC has a linear doseeffect curve.
B-CELL-RESPONSE
SUPPRESSION
407
BY HYDROCORTISONE
Percent inhibition of controls A
100
15
50
25
I 10
I -6
I lo-'
I 10-1
tm
'
FIG. 2. Dose-effect curve of HC in the presence of various numbers of adherent cells. Inhibitory effects of three concentrations of HC on the response of 5 x lo6 nonadherent PBM containing 1.25 x lo5 (0). 2.5 x 10s (0), or 5 x 10s (Ir) adherent cells. The results (mean of three cultures in one experiment) are the percentage of inhibition by HC of the control response (without HC and with the same number of adherent cells). The absolute responses of the control cultures without additional adherent cells and with increasing numbers of adherent cells were, respectively, 443 f 53, 452 2 124, 330 f 48, and 877 2 264 PFC/106.
HC-Pretreated Nonadherent PBM Are Susceptible to the Suppressive Effect of Monocytes We had results indicating that when HC was present in the culture medium the inhibition could be obtained when the addition of adherent cells was delayed until Day 2 of the culture. We thus performed the following experiments. Nonadherent PBM were cultured for 48 hr without HC (control cells) or with low6 M HC (HC-
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treated cells). Adherent cells (2.5 x 105) from the same donor were cultured in parallel. On Day 2 nonadherent PBM were recovered, washed free of HC, and cultured either alone or with adherent cells (Fig. 3). Monocytes moderately suppressed the response of control cells and profoundly inhibited that of HC-treated cells. These results were reproduced in three different experiments: in each case the response of HC-treated cells was significantly (P < 0.05) lower than that of E?.f ,
lO(
50
n N
FIG. 3. HC-pretreated nonadherent PBM are susceptible to monocpte suppression. Nonadherent PBM were cultured from Day 0 to Day 2 with TNP-PAA and without NC or with 10M6 M HC. They were then washed and cultured without or with 2.5 x 16 adherent cells untit Day 7. Adherent cells had been prepared from the same donor on Day 0 and were incubated in parallel from Day 0 to Day 2. Results are the mean (+ SE) responses of three cultures in one representative experiment. The response of unstimulated cultures was 5 f 3 PFC/106.
B-CELL-RESPONSE
SUPPRESSION
BY HYDROCORTISONE
409
control cells when adherent cells were added. In two experiments, we verified that a 3-hr culture of nonadherent cells with HC had no effect on their subsequent response when cocultured with adherent cells. This showed that HC does not need to interact with the monocyte itself in order to suppress the in vitro antibody response. HC Is Suppressive in the Presence of Exogeneous PGE2
Nylon-nonadherent PBM were cultured in the presence of indomethacin and the interaction between 10e6 M HC and graded concentrations of exogeneous PGE;! (30 to 3000 nM) was examined (Fig. 4). HC is suppressive when 300 or 3000 nM PGE2 is present (P < 0.05 and P < 0.01, respectively). The presence of HC allows concentrations of PGE2 as low as 30 nA4 to significantly suppress the anti-TNP response (P < 0.05). We verified that the kinetics of the cultures con-
T
1 T
I_
-
~ 30
FIG. 4. HC is suppressive in the presence of exogeneous PGE2. Effects of PCiE2 (30-3000 nA4) on the response of nonadherent PBM cultured with 3 x 10m6M indomethacin and without (0) or with 10e6M of HC (m) are shown. Results were from six to nine experiments.
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taining PGE2 and lop6 A4 HC is not modified. In some of these experiments lO-7 M HC was also used and proved to be suppressive in the presence of 3000 nM (four experiments) or 300 nA4 (one experiment) PGE2. HC Potentiates the Suppressive Effect of PGE2
When exogeneous PGE2 is present during the whole culture period it apparently has no effect on the anti-TNP response (see Fig. 4). We recently showed (23) that this is the net result of a stimulatory and of an inhibitory effect. It was thus necessary to test whether HC negatively interacts with the former or positively interacts with the latter. We took advantage of the fact that PGE2 exerts its stimulatory effect in the early period of the response (before Day 2), whereas its suppressive effect is predominant after Day 2 (23). To test the first hypothesis nonadherent PBM were cultured with PGE2 (3000 nA4) and/or HC (lop6 M) from Day 0 to Day 2 or from Day 0 to Day 7 (Fig. 5). When the compounds are present during the whole culture period (full bars) the already-described effects are observed. When they are present during the first 48 Percent
oontrole A-
of -I-
200
I
100 1---------_ 50
i
T
_------
HC
F
ilIP-
FIG. 5. HC does not interact with the stimulatory efkct of PGE2. Nonadherent PBM were cultured in the presence of 3 x 10e6 M indomethacin. The cultures received no addition (control group) or PGEz (3000 nkf) and/or HC (10m6 M) on Day 0. In one experimental group (full bars) the cultures were untouched until Day 7. In the other group (empty bars) the cultures were collected on Day-2, washed, and then cultured without addition until Day 7. Results as means (k SE) of the percentage of corresponding controls in four experiments.
B-CELL-RESPONSE
SUPPRESSION
411
BY HYDROCORTISONE
hr (empty bars) both PGE2 and HC are stimulatory and they do not seem to interact. We then examined whether the suppression by PGEz (added on Day 2) is potentiated by HC (added on Day 0). To approximate the situation where prostagladins are produced endogeneously (see below), we used 300 nM PGE2 and lo-’ M HC. In five experiments HC, which potentiated the response (189 + 64% of controls), increased the suppression by PGE2 (from 55 + 14 to 15 + 6% of controls; P < 0.05). Possible Interactions between HC and Endogeneous Prostaglandins We examined this point by two indirect approaches. The first was to determine whether indomethacin could prevent the effect of HC on the response of nonadherent cells cocultured with 5 x IO5 adherent cells (Table 2). Indomethacin inhibits the suppression by IO-’ M HC but does not reproducibly affect that exerted by 1O-6 M HC. The second approach was to determine to what extent HC inhibits the endogeneous production of PGE;! in our conditions. Cultures of 5 x lo6 unfractionated PBM were set up and after 48 hr, the supernatants were collected and PGE2 concentrations measured. In control cultures 393 ti PGE* was produced, and this was suppressed below 5 nM by indomethacin. When HC was present in the culture medium 10e6 M profoundly inhibited PGE2 production (41 n&Q but IO-’ M only partially suppressed it (105 nM) and 1O-8 M had no effect (372 nM). HC Administered in Vivo Potentiates the Suppressive Effect of PGE2 Six normal volunteers received 400 mg HC hemisuccinate intravenously. Nonadherent PBM were isolated from blood drawn immediately before (control PBM) or 4 hr after (treated PBM) the injection. They were cultured with indomethacin and the effects of 30 and 300 ti PGE;! on the response were determined (Table 3). After HC injection the anti-TNP response is decreased. More importantly, the effect of PGE2 is clearly different in control and in treated PBM. Whereas exogeneous PGE;! tends to stimulate the response of the former cells, the response of treated PBM is suppressed by PGE2 even at the 30 I&! concentration. DISCUSSION We have shown that the effect of HC on the in vitro specific human B-cell response depends on the nature of the cell preparations used in culture. The antiTABLE
2
INDOMETHACIN PARTIALLY PREVENTS THE EFFECT OF HC” Indomethacin (3 x 10 -6 M) HC 0 lo-‘M 1O-6 M
-
t
316 k 66b
435 2 24 354 t 596 98 k 28
73 f 24b 49 k 32
u Response as PFC/106 (mean k SE in four to five experiments). b P < 0.01.
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GALANAUD
ET AL.
TABLE HC in Vivo
POTENTIATES
THE in Vitro
Anti-TNP PGE2 in cultures 0
30 nM 3OOlLw
3 SUPPRESSION
BY EXOGENEOUS
PGEz
response of nonadherent PBM collected”
Before HC
After HC
1279 t 475 1547 -c 486 1681 Z!Y610
348 T 87h 139 2 54tJ 110 k 5hb
u Means f SE in six experiments involving different subjects. b P < 0.05 by the paired t test.
TNP response can be induced in cultures of PBM isolated by centrifugation over Ficoll-Hypaque (unfractionated PBM) (15) or in preparations of cells additionally filtered on nylon colunms (nonadherent PBM) (18). The B-cell response of nonadherent PBM is usually higher, in agreement with the original report of Luzzatti et al. (17). This response, like that of unfractionated PBM (24), is specific, T dependent, and monocyte dependent (22). In this respect the 3-5% monocytes present in nonadherent PBM are sufficient to support the response. Unlike the response of unfractionated PBM, which is 80% inhibited by low (IO-‘-BY7 h4) concentrations of HC (7), that of nonadherent PBM is resistant to HC. As HC may increase nonspecific human B-cell response (3, 4), this lack of suppression could reflect an enhancement of the anti-TNP background by HC. Our results exclude this hypothesis: the low or null anti-TNP background response observed in the absence of TNP-PAA is not increased by HC, and the total nonspecific Bcell response (measured by a reverse hemolytic plaque assay) is not affected. We thus looked for an adherent cell able to render nonadherent PBM response sensitive to HC. The addition of 5 or 10% plastic-adherent cells to cultures of nonadherent PBM allows 10e6 and IO-’ M HC to completely inhibit the antiTNP response. Thus, HC suppresses the anti-TNP response only in the presence of a threshold proportion of monocytes. The active cell can be identified as the monocyte based on the following arguments: the preparations of adherent cells contain more than 85% peroxydase-positive cells; the effect is radio resistant; and it can be obtained with adherent cells isolated from E- cells. Although we cannot exclude the possibility that HC interacts with other cells (T or B cells) lost on the nylon column, these results show that monocytes potentiate the suppressive effect of HC in our conditions. The most likely interpretation of our results is that HC positively interacts with the suppressive function of monocytes to inhibit the in vitro B-cell response. Two lines of evidence suggest that this suppression results from a real interaction and not from the addition of two suboptimal suppressions. In individual experiments the association of HC and monocytes could inhibit the response although both were stimulatory when introduced separately into cultures of nonadherent PBM (results not shown). More importantly, the dose-effect curves are suggestive of such an interaction. Two broad mechanisms can be envisaged for this phenomenon: (i) HC can modify the functional activity of monocytes, increasing their suppressive effect; and (ii)
B-CELL-RESPONSE
SUPPRESSION
BY HYDROCORTISONE
413
HC can render the responding lymphocytes more sensitive to monocyte-mediated suppression. No argument could be found in favor of the former mechanism: neither monocytes preincubated with dexamethasone nor their dialyzed supernatants suppress the response (results not shown). In contrast, the latter mechanism is suggested by our demonstration that activation on nonadherent PBM for 48 hr in the presence of HC renders them sensitive to the inhibition by subsequent addition of adherent cells. A well-defined monocyte product (25) able to modulate the anti-TNP response of human PBM is PGEz (23). Moreover, prostaglandins can have a synergistic effect with corticosteroids in some models of lymphocyte activation (26, 27). We show that HC does not interfere with the stimulatory effect of PGE2 but potentiates its suppressive effect. This delineation provides an approach to define the target(s) of the synergistic effects of HC and PGE2. In addition, we show that, after a high intravenous dose of HC, PBM are more susceptible to the in vitro suppression by PGE2. This phenomenon could play a role in the influence of the stress on the prostaglandin sensitivity of lymphocyte response (28). Our in vitro results are obtained with concentrations of PGE,? (as low as 30 nM) which are present in the inflammatory sites (29) and concentrations of HC (1O-7-1O-6 A4) o bt ained with therapeutic regimens. The precise role of this interaction in the suppression of the B-cell response by HC and monocytes is more difficult to figure out. The following indirect elements suggest that the synergy between HC and PGE2 can be implicated in the suppressive effect of low concentrations (lo-‘M) of HC. (i) This concentration of HC only partially suppresses PGE2 production. (ii) Indomethacin counteracts the suppressive effect of IO-’ M HC. Although indomethacin may have several effects, the inhibition of cyclooxygenase is probably involved, as exogeneous PGE2 can render HC suppressive in the presence of indomethacin. However, other mechanisms should be operative and would be predominant when higher concentrations of HC-which inhibit PGE2 production-are used. HC could render the responding cells more susceptible to non-prostaglandin-mediated suppression by monocytes (30). Our results outline the different effects of corticosteroids on human and murine B-cell responses. In the latter case activated monocytes or their products protect from the inhibition by HC (6, 31). This suggests that HC acts essentially by inhibiting the production of interleukins, a mechanism apparently not involved in the human system. The fact that corticosteroids and monocytic cells can interact in two opposite ways may have an in vivo relevance. As outlined by Bradley and Mishell (31), the nonspecific stimulation of macrophages in infections could protect B cells from suppression by endogeneous corticosteroids. In contrast, an exaggeration of monocyte-mediated suppression may be present in situations such as systemic lupus erythematosus ((32) and our unpublished results) or aging (33). This could potentiate the immunosuppressive effect of endogeneous or therapeutically administered corticosteroids. ACKNOWLEDGMENTS This work was supported by grants from CNRS and from CRAMIE
414
GALANAUD
ET AL.
REFERENCES 1. Mantzouranis, E., and Borel, Y., Cell. Immunol. 43, 202, 1979. 2. Blomgren, H., J. Immunol. 3, 655, 1974. 3. Waldmann, T. A., Broder, S., Krakauer, R., Macdermott, R. P., Durm. M., Goldman, C., and Meade, B., Fed. Proc. 35, 2067, 1976. 4. Fauci, A. S., Pratt, R. K., and Whalen, G., J. Immunol. 119, 598, 1977. 5. Ilfeld, D. N., Krakauer, R. S., and Blaese, R. M., J. Immunol. 119, 428, 1977. 6. Mishell, R. I., Lucas, A., and Mishell, B. B., J. Zmmunol. 119, 118, 1977. 7. Galanaud, I?, Crevon, M. C., Hillion, D., and Delfraissy, J. E, Clin. Immunol. Immunopath. 18, 68, 1981. 8. Claman, H. N., N. Engl. J. Med 787, 388, 1972. 9. Fauci, A. S., and Dale, D. C., Blood, 46, 235, 1975. 10. Waldmann, T. A., Blaese, R. M., Broder, S., and Krakauer, R. S., Ann. Intern. Med. 88, 226. 1978. 11. Haynes, B. F., and Fauci, A. S., Cell. Immunol. 44, 157, 1979. 12. Gillis, S., Crabtree, G. R., and Smith, K. A., J. Zmmunol. 123, 1624, 1979. 13. Larsson, E. L., J. Immunol. 124, 2828, 1980. 14. Snyder, D. S., and Unanue, E. R., J. Immunol. 129, 1803, 1982. 15. Delfraissy, J. F., Galanaud, P., Dormont, J., and Wallon, C., J. Immunol. 118, 630, 1977. 16. Eby, W. C., Chong, C. A., Dray, S., and Molinaro, G. A., J. Immunol. 115, 1700, 1975. 17. Luzzatti, A. L., Heinzen, I., Hengartner, H., and Schreier, M. H., Clin. Exp. Zmmunot. 35, 405. 1979. 18. Galanaud, P., Delfraissy, J. F., Hillion, D., Segond, P., and Dormont, J., In “Human B-Lymphocyte Function. Activation and Immunoregulation” (A. S. Fauci and R. D. Ballieux, Eds.), pp. 169, Raven Press, New York, 1982. 19. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. G., Proc. Natl. Acad. Sci. USA 76, 4061, 1979. 20. Salomon, J. A., and Flower, R. J., In “Hormones in Blood” (C. H. Gray and V. H. T. James, Eds.), p. 237, Academic Press, New York, 1979. 21. Dray, E, Charbonnel, B., and Maclousf, J., Eur. J. Clin. Invest. 5, 311, 1975. 22. Vazquez, A., Charron, D., and Galanaud, P., Cell. Immunol. 71, 148, 1982. 23. Emilie, D., Crevon, M. C., and Galanaud, P., C/in. Immunol. immunopathol. 29, 415-423. 1983. 24. Galanaud, P., Immunol. Rev. 45, 141, 1979. 25. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 26. Mendelsohn, J., Multer, M. M., and Boone, R. E. J. Clin. Invest. 52, 2129, 1973. 27. Berenbaum, M. C., Cope, W. A., and Bundick, R. V., Glin. Exp. Immunol. 26, 534, 1976. 28. Goodwin, J. S., Bromberg, S., Staszak, C., Kaszubowski, P. A., Messner, R. E., and Neal, J. F., J. Immunol. 127, 518, 1981. 30. Gmeling-Meyling, R , and Waldmann, T. A., J. Immunol. 126, 529, 1981. 31. Bradley, L. M., and Mishell, R. I., Proc. Natl. Acad. Sci. USA 78, 3155, 1981. 32. Markenson, J. A., Lockshin, M. D.. Fuzesi, L., Warburg, M., Joachim, C., and Morgan, J. W.. J. Lab. Clin. Med. 95, 40, 1980. 33. Delfraissy, J. F., Galanaud, I?, Wallon, C., Balavoine, J. E, and Dormont, J.. Clin. Zmmunol. Immunopathol.
24, 377, 1982.
Received December 28, 1982; accepted with revision March 23, 1983.
IMMUNOLOGY
AND
IMMUNOPATHOLOGY
29, 403-414 (1983)
Effect of Hydrocortisone on the in Vitro Human Antibody Response: Interaction with Monocytes and Prostaglandins PIERRE GALANAUD,
MARIE-CLAUDE CREVON, DOMINIQUE ANNIE ABELLA
EMILIE,
AND
Inserm U 131 and Universitk Paris-Sud 32, rue des Carnets, 92140 Clamart, France
The specificplaque-forming cell response inducedby trinitrophenylpolyacrylamide beadsin culturesof nonadherenthumanperipheralbloodmononuclear cells(PBM) is resistantto the inhibitory effect of hydrocortisone(HC). Previousresultsshowedthat low concentrations(up to lo-’ M) of HC inhibit the sameresponse in culturesof unfractionatedhumanPBM. It is shownthat the additionof 5-10%monocytesto nonadherentPBM renderstheir responseHC-sensitive.One mechanism of the interaction betweenHC andmonocytesis the potentiationof prostaglandin Er-mediatedsuppressionby HC, whichcanbe demonstrated in vitro andafter in vivo administration of HC. However,interactionwith a non-prostaglandin-mediated suppression by monocytes should be involved, particularlywith high(10e6M) concentrations of HC. INTRODUCTION
The effects of corticosteroids on the immune response vary according to the model used to induce this response in vivo (1) or in vitro (2-7). This may be due to different intrinsic susceptibilities of the precursor lymphocytes (8, 9) or to the multiplicity of the effects of steroids on the regulatory cells. Indeed, corticosteroids can inhibit suppressor T cells (10, 11) as well as the production of lymphokines (12, 13) and monokines (14). We have shown that the anti-TNP response of human peripheral blood mononuclear cells (PBM) to trinitrophenyl polyacrylamide beads (TNP-PAA) is steroid sensitive (7) unlike the nonspecific B-cell response induced by pokeweed mitogen (3-5). We now show that the suppression of the anti-TNP response by hydrocortisone (HC) requires a threshold proportion of monocytes in the cultures of human PBM. HC interacts with monocytes to suppress the anti-TNP response, suggesting that the different HC sensitivity of B-cell responses is due to a different susceptibility to monocyte-mediated suppression. In our system this suppression is at least partially mediated by prostaglandin E2 (PGE&. MATERIALS
AND METHODS
Reagents. Polyacrylamide beads (Bio-Gel P-30, 100-200 mesh) were obtained from Bio-Rad Laboratories (Richmond, Calif.). They were amino-ethylated as already reported (15) and reacted with excess trinitrobenzenesulfonic acid (Sigma, St. Louis, MO.) in order to prepare TNP-PAA. HC (Merck), indomethacin (Sigma), and PGE2 (Sigma) were dissolved in ethanol at 10e2, 3 x 10p3, and lop2 M, respectively. These stock solutions were further diluted in phosphate-buffered saline and culture medium. We verified that 1% ethanol (corresponding to seven 403 0090-1229/83 $1.50 Copyright @ 1983 by Academic Ress, Inc. All rights of reproduction in any form reserved
404
GALANAUD
ET AL.
times the highest concentration used) had no effect on the response. E3H]PGE2 was obtained from New England Nuclear (Amersham) and anti-PGE, antibody from Institut Pasteur Paris. In vitro antibody response. Blood was drawn from 18- to 35year-old healthy volunteers. Different donors were used in each series of experiments. PBM were separated (see below) and cultured at a concentration of 5 x 106 cells in l-ml cultures as already described (15). The only minor modification was in the culture medium (RPM1 1640 supplemented with 5 x lO-5 M 2-mercaptoethanol). This does not modify the effect of HC on the response. When the effect of exogenous PGE2 was being tested, indomethacin (3 x 10d6 M) was introduced into the culture medium to inhibit the production of endogeneous prostaglandins. Assay for the anti-TNP response. The cells were collected on Day 7 and the anti-TNP response was measured using a Jerne-type plaque assay (15). The target erythrocytes were unconjugated sheep red blood cells (SRBC) and TNP-SRBC. When necessary the total nonspecific B-cell response was measured using a reverse hemolytic plaque assay (16). The results are expressed as the number of plaque-forming cells/IO6 collected cells (PFC/l06) in each culture assayed individually. At least three cultures were performed in each experimental group. The cell recovery and viability were identical in all groups. Unless otherwise stated the results are the mean (2 SE) responses from different experiments involving different donors. Student’s t test was used for statistical analysis. Cellfructionations. Two different preparations were used as responding cells: unfractionated PBM isolated on Ficoll-Hypaque and nonadherent PBM, additionally filtered on a nylon wool column (17, 18). To recover the nonadherent PBM, the nylon wood was rinsed with warm medium and pressed with the plug of the syringe. Among the cells recovered by this procedure (about 50% of the starting cell number) the proportions of T and B lymphocytes were not modified (33). In addition, we recently verified that the Td/Ts ratio (19) is not modified after nylon filtration (from 1.64 to 1.59). In contrast, the proportion of peroxidase-positive cells decreases from 15-30% to 3-5% after nylon filtration. Cells strongly adherent to plastic (adherent cells) were prepared as follows: 6 x lo6 unfractionated cells were incubated on culture dishes for 90 min at 37°C in a culture medium containing 20% fetal bovine serum. Nonadherent cells were removed by vigorous washings and adherent cells were recovered by washing the dishes with cold phosphate-buffered saline and with the help of a rubber policeman They contained more than 85% peroxidase-positive cells. In some experiments 2500-R-irradiated PBM or PBM depleted of E-rosette-forming cells (Ecells) were used to prepare adherent cells. Dosage of PGE2. The amount of PGE;! produced after 48 hr in cultures of unfractionated PBM was measured in pooled supernatants from three cultures. Prostaglandins were extracted (20) and PGE2 concentrations were evaluated by radioimmunoassay (2 1). RESULTS
The Anti-TNP Response of Nonadherent PBM Is He-Resistant When nonadherent PBM are stimulated with TNP-PAA in the presence of BP6 M HC the anti-TNP response is virtually unaffected (Table 1, line 1). In the
B-CELL-RESPONSE
SUPPRESSION TABLE
ADHERENT
CELL
REQUIREMENT
1 FOR SUPPRESSION
Anti-TNP Cells in culture Nonadherentb Nonadherent c Nonadherent + 10% adherentb Nonadherent + 10% adherentc
TNP-PAA” + + -
405
BY HYDROCORTISONE
BY
HC
responses when HC (10m6 M) -
+
322 r 133 10 f 3 220 2 45d 524
219 f 50 3+2 16 + Sd NDe
a TNP-PAA added or not added to the cultures. b Nine experiments. c Three experiments. d P < 0.001. p ND, not determined.
absence of antigen only few anti-TNP PFC develop regardless of the presence of HC (Table 1, line 2). This is also true when the non-cross-reactive antigen fluorescein-PAA (22) is present in the cultures (results not shown). In the same line we verified that HC does not markedly enhance the number of reverse PFC (16) in cultures of nonadherent PBM containing TNP PM (from 2892 + 355 to 3190 k 84 PFC/106). We previously showed that the anti-TNP response of unfractionated PBM is 80% inhibited by lop6 A4 HC (7). This suggested that an adherent cell is required for HC to suppress the in vitro antibody response. We used plastic adherence as a source of adherent cells (Table 1). The addition of 5 x ld adherent cells has some suppressive effect but this suppression is inconstant and not statistically significant (P > 0.45). However, in the presence of adherent cells HC is profoundly suppressive. We verified in two experiments that this suppression does not reflect a modification in the kinetics of the response. The active adherent cell is radio-resistant: HC suppressed the response of nonadherent PBM from 859 to 90 PFCAo6 in the presence of 10% irradiated monocytes (results from one out of three experiments). This celI is non-T: adherent cells isolated from E--cells also allowed HC to suppress the response of nonadherent PBM (from 671 to 25 PFC/ IO6 in one out of three experiments). Monocytes
and HC Interact
to Suppress the Anti-TNP
Response
The effect of various numbers of adherent cells on the response of a fixed concentration of HC is shown in Fig. 1. The results obtained with the highest number of monocytes (5 x 105) are in keeping with those presented in Table 1. HC has a comparable effect with 2.5 x 16 adherent cells (P < 0.02) and significantly suppresses the response (p < 0.05) when 1.25 x lo5 adherent cells are present. More importantly, for the two lower amounts of monocytes the effect of HC is directly related to the number of monocytes present in the cultures. In two experiments we measured the effect of three HC concentrations (10m6, 10p7, and lo-* M) on the response of nonadherent PBM cocultured with three concentrations of adherent cells. In these experiments even the highest number of adherent cells did not inhibit the response (see Fig. 2). In both experiments
GALANAUD
406 Percent
of controla
ET AL.
T
1
1 I I
1 1
T
.-
1 5.80 L
FIG. 1. Interaction between HC and various numbers of adherent cells. Response of nonadherent PBM cultured without increasing numbers of adherent cells without HC (Cl) or with 10” M HC (m). Results are the mean ( 2 SE) relative responses (compared to that of cultures containing neither HC nor adherent cells) in four to six experiments. Comparison between (0) and (m): P < 0.02 and P < 0.05 with 1.25 x lo5 and 2.5 x lo* adherent cells, respectively.
the inhibition was related to the concentration of HC and to the number of adherent cells. The results of one of these experiments are presented in Fig. 2. With the highest number of adherent cells (5 x 105), 3 log dilutions of HC (up to 10e8 A4) are inhibitory. With the lowest number (1.25 x 10’) HC has a linear doseeffect curve.
B-CELL-RESPONSE
SUPPRESSION
407
BY HYDROCORTISONE
Percent inhibition of controls A
100
15
50
25
I 10
I -6
I lo-'
I 10-1
tm
'
FIG. 2. Dose-effect curve of HC in the presence of various numbers of adherent cells. Inhibitory effects of three concentrations of HC on the response of 5 x lo6 nonadherent PBM containing 1.25 x lo5 (0). 2.5 x 10s (0), or 5 x 10s (Ir) adherent cells. The results (mean of three cultures in one experiment) are the percentage of inhibition by HC of the control response (without HC and with the same number of adherent cells). The absolute responses of the control cultures without additional adherent cells and with increasing numbers of adherent cells were, respectively, 443 f 53, 452 2 124, 330 f 48, and 877 2 264 PFC/106.
HC-Pretreated Nonadherent PBM Are Susceptible to the Suppressive Effect of Monocytes We had results indicating that when HC was present in the culture medium the inhibition could be obtained when the addition of adherent cells was delayed until Day 2 of the culture. We thus performed the following experiments. Nonadherent PBM were cultured for 48 hr without HC (control cells) or with low6 M HC (HC-
408
GALANAUD
ET AL.
treated cells). Adherent cells (2.5 x 105) from the same donor were cultured in parallel. On Day 2 nonadherent PBM were recovered, washed free of HC, and cultured either alone or with adherent cells (Fig. 3). Monocytes moderately suppressed the response of control cells and profoundly inhibited that of HC-treated cells. These results were reproduced in three different experiments: in each case the response of HC-treated cells was significantly (P < 0.05) lower than that of E?.f ,
lO(
50
n N
FIG. 3. HC-pretreated nonadherent PBM are susceptible to monocpte suppression. Nonadherent PBM were cultured from Day 0 to Day 2 with TNP-PAA and without NC or with 10M6 M HC. They were then washed and cultured without or with 2.5 x 16 adherent cells untit Day 7. Adherent cells had been prepared from the same donor on Day 0 and were incubated in parallel from Day 0 to Day 2. Results are the mean (+ SE) responses of three cultures in one representative experiment. The response of unstimulated cultures was 5 f 3 PFC/106.
B-CELL-RESPONSE
SUPPRESSION
BY HYDROCORTISONE
409
control cells when adherent cells were added. In two experiments, we verified that a 3-hr culture of nonadherent cells with HC had no effect on their subsequent response when cocultured with adherent cells. This showed that HC does not need to interact with the monocyte itself in order to suppress the in vitro antibody response. HC Is Suppressive in the Presence of Exogeneous PGE2
Nylon-nonadherent PBM were cultured in the presence of indomethacin and the interaction between 10e6 M HC and graded concentrations of exogeneous PGE;! (30 to 3000 nM) was examined (Fig. 4). HC is suppressive when 300 or 3000 nM PGE2 is present (P < 0.05 and P < 0.01, respectively). The presence of HC allows concentrations of PGE2 as low as 30 nA4 to significantly suppress the anti-TNP response (P < 0.05). We verified that the kinetics of the cultures con-
T
1 T
I_
-
~ 30
FIG. 4. HC is suppressive in the presence of exogeneous PGE2. Effects of PCiE2 (30-3000 nA4) on the response of nonadherent PBM cultured with 3 x 10m6M indomethacin and without (0) or with 10e6M of HC (m) are shown. Results were from six to nine experiments.
410
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ET AL.
taining PGE2 and lop6 A4 HC is not modified. In some of these experiments lO-7 M HC was also used and proved to be suppressive in the presence of 3000 nM (four experiments) or 300 nA4 (one experiment) PGE2. HC Potentiates the Suppressive Effect of PGE2
When exogeneous PGE2 is present during the whole culture period it apparently has no effect on the anti-TNP response (see Fig. 4). We recently showed (23) that this is the net result of a stimulatory and of an inhibitory effect. It was thus necessary to test whether HC negatively interacts with the former or positively interacts with the latter. We took advantage of the fact that PGE2 exerts its stimulatory effect in the early period of the response (before Day 2), whereas its suppressive effect is predominant after Day 2 (23). To test the first hypothesis nonadherent PBM were cultured with PGE2 (3000 nA4) and/or HC (lop6 M) from Day 0 to Day 2 or from Day 0 to Day 7 (Fig. 5). When the compounds are present during the whole culture period (full bars) the already-described effects are observed. When they are present during the first 48 Percent
oontrole A-
of -I-
200
I
100 1---------_ 50
i
T
_------
HC
F
ilIP-
FIG. 5. HC does not interact with the stimulatory efkct of PGE2. Nonadherent PBM were cultured in the presence of 3 x 10e6 M indomethacin. The cultures received no addition (control group) or PGEz (3000 nkf) and/or HC (10m6 M) on Day 0. In one experimental group (full bars) the cultures were untouched until Day 7. In the other group (empty bars) the cultures were collected on Day-2, washed, and then cultured without addition until Day 7. Results as means (k SE) of the percentage of corresponding controls in four experiments.
B-CELL-RESPONSE
SUPPRESSION
411
BY HYDROCORTISONE
hr (empty bars) both PGE2 and HC are stimulatory and they do not seem to interact. We then examined whether the suppression by PGEz (added on Day 2) is potentiated by HC (added on Day 0). To approximate the situation where prostagladins are produced endogeneously (see below), we used 300 nM PGE2 and lo-’ M HC. In five experiments HC, which potentiated the response (189 + 64% of controls), increased the suppression by PGE2 (from 55 + 14 to 15 + 6% of controls; P < 0.05). Possible Interactions between HC and Endogeneous Prostaglandins We examined this point by two indirect approaches. The first was to determine whether indomethacin could prevent the effect of HC on the response of nonadherent cells cocultured with 5 x IO5 adherent cells (Table 2). Indomethacin inhibits the suppression by IO-’ M HC but does not reproducibly affect that exerted by 1O-6 M HC. The second approach was to determine to what extent HC inhibits the endogeneous production of PGE;! in our conditions. Cultures of 5 x lo6 unfractionated PBM were set up and after 48 hr, the supernatants were collected and PGE2 concentrations measured. In control cultures 393 ti PGE* was produced, and this was suppressed below 5 nM by indomethacin. When HC was present in the culture medium 10e6 M profoundly inhibited PGE2 production (41 n&Q but IO-’ M only partially suppressed it (105 nM) and 1O-8 M had no effect (372 nM). HC Administered in Vivo Potentiates the Suppressive Effect of PGE2 Six normal volunteers received 400 mg HC hemisuccinate intravenously. Nonadherent PBM were isolated from blood drawn immediately before (control PBM) or 4 hr after (treated PBM) the injection. They were cultured with indomethacin and the effects of 30 and 300 ti PGE;! on the response were determined (Table 3). After HC injection the anti-TNP response is decreased. More importantly, the effect of PGE2 is clearly different in control and in treated PBM. Whereas exogeneous PGE;! tends to stimulate the response of the former cells, the response of treated PBM is suppressed by PGE2 even at the 30 I&! concentration. DISCUSSION We have shown that the effect of HC on the in vitro specific human B-cell response depends on the nature of the cell preparations used in culture. The antiTABLE
2
INDOMETHACIN PARTIALLY PREVENTS THE EFFECT OF HC” Indomethacin (3 x 10 -6 M) HC 0 lo-‘M 1O-6 M
-
t
316 k 66b
435 2 24 354 t 596 98 k 28
73 f 24b 49 k 32
u Response as PFC/106 (mean k SE in four to five experiments). b P < 0.01.
412
GALANAUD
ET AL.
TABLE HC in Vivo
POTENTIATES
THE in Vitro
Anti-TNP PGE2 in cultures 0
30 nM 3OOlLw
3 SUPPRESSION
BY EXOGENEOUS
PGEz
response of nonadherent PBM collected”
Before HC
After HC
1279 t 475 1547 -c 486 1681 Z!Y610
348 T 87h 139 2 54tJ 110 k 5hb
u Means f SE in six experiments involving different subjects. b P < 0.05 by the paired t test.
TNP response can be induced in cultures of PBM isolated by centrifugation over Ficoll-Hypaque (unfractionated PBM) (15) or in preparations of cells additionally filtered on nylon colunms (nonadherent PBM) (18). The B-cell response of nonadherent PBM is usually higher, in agreement with the original report of Luzzatti et al. (17). This response, like that of unfractionated PBM (24), is specific, T dependent, and monocyte dependent (22). In this respect the 3-5% monocytes present in nonadherent PBM are sufficient to support the response. Unlike the response of unfractionated PBM, which is 80% inhibited by low (IO-‘-BY7 h4) concentrations of HC (7), that of nonadherent PBM is resistant to HC. As HC may increase nonspecific human B-cell response (3, 4), this lack of suppression could reflect an enhancement of the anti-TNP background by HC. Our results exclude this hypothesis: the low or null anti-TNP background response observed in the absence of TNP-PAA is not increased by HC, and the total nonspecific Bcell response (measured by a reverse hemolytic plaque assay) is not affected. We thus looked for an adherent cell able to render nonadherent PBM response sensitive to HC. The addition of 5 or 10% plastic-adherent cells to cultures of nonadherent PBM allows 10e6 and IO-’ M HC to completely inhibit the antiTNP response. Thus, HC suppresses the anti-TNP response only in the presence of a threshold proportion of monocytes. The active cell can be identified as the monocyte based on the following arguments: the preparations of adherent cells contain more than 85% peroxydase-positive cells; the effect is radio resistant; and it can be obtained with adherent cells isolated from E- cells. Although we cannot exclude the possibility that HC interacts with other cells (T or B cells) lost on the nylon column, these results show that monocytes potentiate the suppressive effect of HC in our conditions. The most likely interpretation of our results is that HC positively interacts with the suppressive function of monocytes to inhibit the in vitro B-cell response. Two lines of evidence suggest that this suppression results from a real interaction and not from the addition of two suboptimal suppressions. In individual experiments the association of HC and monocytes could inhibit the response although both were stimulatory when introduced separately into cultures of nonadherent PBM (results not shown). More importantly, the dose-effect curves are suggestive of such an interaction. Two broad mechanisms can be envisaged for this phenomenon: (i) HC can modify the functional activity of monocytes, increasing their suppressive effect; and (ii)
B-CELL-RESPONSE
SUPPRESSION
BY HYDROCORTISONE
413
HC can render the responding lymphocytes more sensitive to monocyte-mediated suppression. No argument could be found in favor of the former mechanism: neither monocytes preincubated with dexamethasone nor their dialyzed supernatants suppress the response (results not shown). In contrast, the latter mechanism is suggested by our demonstration that activation on nonadherent PBM for 48 hr in the presence of HC renders them sensitive to the inhibition by subsequent addition of adherent cells. A well-defined monocyte product (25) able to modulate the anti-TNP response of human PBM is PGEz (23). Moreover, prostaglandins can have a synergistic effect with corticosteroids in some models of lymphocyte activation (26, 27). We show that HC does not interfere with the stimulatory effect of PGE2 but potentiates its suppressive effect. This delineation provides an approach to define the target(s) of the synergistic effects of HC and PGE2. In addition, we show that, after a high intravenous dose of HC, PBM are more susceptible to the in vitro suppression by PGE2. This phenomenon could play a role in the influence of the stress on the prostaglandin sensitivity of lymphocyte response (28). Our in vitro results are obtained with concentrations of PGE,? (as low as 30 nM) which are present in the inflammatory sites (29) and concentrations of HC (1O-7-1O-6 A4) o bt ained with therapeutic regimens. The precise role of this interaction in the suppression of the B-cell response by HC and monocytes is more difficult to figure out. The following indirect elements suggest that the synergy between HC and PGE2 can be implicated in the suppressive effect of low concentrations (lo-‘M) of HC. (i) This concentration of HC only partially suppresses PGE2 production. (ii) Indomethacin counteracts the suppressive effect of IO-’ M HC. Although indomethacin may have several effects, the inhibition of cyclooxygenase is probably involved, as exogeneous PGE2 can render HC suppressive in the presence of indomethacin. However, other mechanisms should be operative and would be predominant when higher concentrations of HC-which inhibit PGE2 production-are used. HC could render the responding cells more susceptible to non-prostaglandin-mediated suppression by monocytes (30). Our results outline the different effects of corticosteroids on human and murine B-cell responses. In the latter case activated monocytes or their products protect from the inhibition by HC (6, 31). This suggests that HC acts essentially by inhibiting the production of interleukins, a mechanism apparently not involved in the human system. The fact that corticosteroids and monocytic cells can interact in two opposite ways may have an in vivo relevance. As outlined by Bradley and Mishell (31), the nonspecific stimulation of macrophages in infections could protect B cells from suppression by endogeneous corticosteroids. In contrast, an exaggeration of monocyte-mediated suppression may be present in situations such as systemic lupus erythematosus ((32) and our unpublished results) or aging (33). This could potentiate the immunosuppressive effect of endogeneous or therapeutically administered corticosteroids. ACKNOWLEDGMENTS This work was supported by grants from CNRS and from CRAMIE
414
GALANAUD
ET AL.
REFERENCES 1. Mantzouranis, E., and Borel, Y., Cell. Immunol. 43, 202, 1979. 2. Blomgren, H., J. Immunol. 3, 655, 1974. 3. Waldmann, T. A., Broder, S., Krakauer, R., Macdermott, R. P., Durm. M., Goldman, C., and Meade, B., Fed. Proc. 35, 2067, 1976. 4. Fauci, A. S., Pratt, R. K., and Whalen, G., J. Immunol. 119, 598, 1977. 5. Ilfeld, D. N., Krakauer, R. S., and Blaese, R. M., J. Immunol. 119, 428, 1977. 6. Mishell, R. I., Lucas, A., and Mishell, B. B., J. Zmmunol. 119, 118, 1977. 7. Galanaud, I?, Crevon, M. C., Hillion, D., and Delfraissy, J. E, Clin. Immunol. Immunopath. 18, 68, 1981. 8. Claman, H. N., N. Engl. J. Med 787, 388, 1972. 9. Fauci, A. S., and Dale, D. C., Blood, 46, 235, 1975. 10. Waldmann, T. A., Blaese, R. M., Broder, S., and Krakauer, R. S., Ann. Intern. Med. 88, 226. 1978. 11. Haynes, B. F., and Fauci, A. S., Cell. Immunol. 44, 157, 1979. 12. Gillis, S., Crabtree, G. R., and Smith, K. A., J. Zmmunol. 123, 1624, 1979. 13. Larsson, E. L., J. Immunol. 124, 2828, 1980. 14. Snyder, D. S., and Unanue, E. R., J. Immunol. 129, 1803, 1982. 15. Delfraissy, J. F., Galanaud, P., Dormont, J., and Wallon, C., J. Immunol. 118, 630, 1977. 16. Eby, W. C., Chong, C. A., Dray, S., and Molinaro, G. A., J. Immunol. 115, 1700, 1975. 17. Luzzatti, A. L., Heinzen, I., Hengartner, H., and Schreier, M. H., Clin. Exp. Zmmunot. 35, 405. 1979. 18. Galanaud, P., Delfraissy, J. F., Hillion, D., Segond, P., and Dormont, J., In “Human B-Lymphocyte Function. Activation and Immunoregulation” (A. S. Fauci and R. D. Ballieux, Eds.), pp. 169, Raven Press, New York, 1982. 19. Reinherz, E. L., Kung, P. C., Goldstein, G., and Schlossman, S. G., Proc. Natl. Acad. Sci. USA 76, 4061, 1979. 20. Salomon, J. A., and Flower, R. J., In “Hormones in Blood” (C. H. Gray and V. H. T. James, Eds.), p. 237, Academic Press, New York, 1979. 21. Dray, E, Charbonnel, B., and Maclousf, J., Eur. J. Clin. Invest. 5, 311, 1975. 22. Vazquez, A., Charron, D., and Galanaud, P., Cell. Immunol. 71, 148, 1982. 23. Emilie, D., Crevon, M. C., and Galanaud, P., C/in. Immunol. immunopathol. 29, 415-423. 1983. 24. Galanaud, P., Immunol. Rev. 45, 141, 1979. 25. Goodwin, J. S., Bankhurst, A. D., and Messner, R. P., J. Exp. Med. 146, 1719, 1977. 26. Mendelsohn, J., Multer, M. M., and Boone, R. E. J. Clin. Invest. 52, 2129, 1973. 27. Berenbaum, M. C., Cope, W. A., and Bundick, R. V., Glin. Exp. Immunol. 26, 534, 1976. 28. Goodwin, J. S., Bromberg, S., Staszak, C., Kaszubowski, P. A., Messner, R. E., and Neal, J. F., J. Immunol. 127, 518, 1981. 30. Gmeling-Meyling, R , and Waldmann, T. A., J. Immunol. 126, 529, 1981. 31. Bradley, L. M., and Mishell, R. I., Proc. Natl. Acad. Sci. USA 78, 3155, 1981. 32. Markenson, J. A., Lockshin, M. D.. Fuzesi, L., Warburg, M., Joachim, C., and Morgan, J. W.. J. Lab. Clin. Med. 95, 40, 1980. 33. Delfraissy, J. F., Galanaud, I?, Wallon, C., Balavoine, J. E, and Dormont, J.. Clin. Zmmunol. Immunopathol.
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Received December 28, 1982; accepted with revision March 23, 1983.