Prostaglandin E1 as a regulator of lymphocyte function

CELLULAR

6, 457-465 (1973)

IMMUNOLOGY

SHORT COMMUNICATION Prostaglandin Selective

Action

E, as a Regulator

of Lymphocyte

on B Lymphocytes and Synergy with in Depression of Immune Responses 1

Function Procarbazine

FRANCO QUAGLIATA,’ VICTOR J. W. LAWRENCE,~ AND JULIA M. PHILLIPS-QUAGLIATA Rheumatic Diseases Study Group, Depavtmc+zt of Medicine rind Departmrut New York University School of Medicine, New York, New York

Received Awkst

of Pntholngy, 10016

21, 1972

The possibility that prostaglandin (PG) E1 can regulate lymphocyte function in z&o was studied. In conjunction with procarbazine hydrochloride, a powerful immunosuppressive agent whose main target is the thymus and thymus-derived (T) cells, PGE, had a synergistic effect in prolonging homograft survival, though alone it had no effect on this immune response. The main lymphocyte target of PGEl appeared to be bone marrow-derived (B) as determined from direct assessment of total, complement receptor and 6’positive lymphocytes in the spleen and by histological evaluation.

INTRODUCTION The activities of cells involved in inflammation and immune reactions are likely to be under the control of several regulators. Regulation may occur at the cellular level, affecting numbers and types of cells involved and at the subcell&r level, affecting the functions of these cells. Prostaglandins are fatty acids widely distributed in mammalian tissues and those of the E type are thought to be present in at least some kinds of inflammatory site ( 1, 2). They have multiple biological effects, some of which may be related to their ability to influence the intracellular levels of cyclic adenosine 3’,5’monophosphate (cAMP) through stimulation of adenyl cyclase activity (3-S). PGEr has been shown to have a proinflammatory effect, increasing vascular permeability and causing edema formation in viva and being slightly leukotactic in vitro (6). In contrast, the effects of PGEr on mast cells, inhibiting histamine release (7, zi), and on phagocytic cells, inhibiting lysosomal enzyme release (9) in vitro. suggest that it may also have an antiinflammatory effect. In support of this idea it was found that PGEr suppressed adjuvant disease and the hemagglutinin response to 1 Supported by U.S.P.H.S. Grants TOl-AM-05064, POl-AM-01431, and ROl-AI-09647. 2 P.D. fellow of the Arthritis Foundation. 3 Recipient of a student fellowship from the New York Chapter of the Arthritis Foundation.

Copyri ht All rig&

1973 by Academic Press, reproduction in any form

Inc. reserved

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sheep red blood cells (SRBC) without suppressing delayed hypersensitivity (DH) to PPD in rats (lo), thus suggesting that PGEr might even be immunosuppressive through an effect on B lymphocytes. To check this hypothesis, we contrasted the effects of PGEr with those of procarbazine hydrochloride, a powerful immunosuppressive agent ( 11) whose main site of action is on the thymus and T lymphocytes, though it also depresses B lymphocytes to some extent (12, 13). We studied these two agents alone and in combination, in their effect on different lymphocyte populations and on several parameters of the immune response in the mouse. The results indicate that the two agents synergize in prolonging homograft survival; PGEl probably acts by depleting a class of B lymphocytes, procarbazine mainly T cells, though it also affects B cells. MATERIALS

AND

METHODS

A/J, CBA/J, C3HeBFe/J, and AKR/J mice obtained from Jackson Laboratories (Bar Harbor, ME) and CFl mice obtained from Car-worth Laboratory Animals (New City, NY) were kept on a standard pellet diet and water ad libitum. PGEr was a gift from Dr. J. E. Pike of Upjohn Co., Kalamazoo, MI. It was dissolved to 1 mg and 2 mg/ml in 0.06 M sodium phosphate buffer and 100 pg for the first grafting experiment and 200 pg for all subsequent experiments were injected subcutaneously (SC) twice daily at approximately 12hr intervals, throughout the experiments. This schedule was adopted because of the very short halflife of the substance (14). Procarbazine hydrochloride (Natulan, Matulane), was a gift from Dr. W. E. Scott of Hoffman La Roche, Nutley, NJ. Subcutaneous injections of 360 or 300 mg/kg/day were given in 0.9% w/v NaCl solution (saline) for 4 days. The dose was reduced in the second grafting experiment because the mice became very susceptible to the action of Nembutal after procarbazine treatment as previously noted (Floersheim, G. L., personal communication). This lower dose was used in all subsequent experiments. Control mice received sc injections of saline. A/J strain tail skin, removed after washing with soap, was cut into pieces of approximately 2.5 mm2 and grafted according to the method described (15) on the right flank of CBA mice, anesthetized by ip injections of 0.01 ml/g body wt Nembutal (Abbott Laboratories, North Chicago, IL), diluted to 4.5 mg/ml in saline. Eight days later the plasters were removed and the number of surviving grafts per group was recorded daily throughout the experiments. Blue-stained Brucella abortw (ring test antigen) suspension was obtained from the U.S. Dept. of Agriculture, Agricultural Research Service, Animal Health Div., IA. For immunization it was diluted 1:5 and 0.1 ml (about 10 x lo6 killed organisms) were injected ip. For the determination of the anti-Brucella titers the mice were bled at weekly intervals from the time of immunization from the retroorbital venous plexus and the serum was stored frozen, diluted 1:5 in saline. One tenth-milliliter doubling dilutions in 0.1% bovine serum albumin (Pentex, Kankakee, IL) in phosphatebuffered saline, pH 7.2, were made in disposable “U” plates (Cooke Engineering Co., Alexandria, VA). Duplicate plates contained antibody treated with 0.1 M 2-mercaptoethanol (2-&E) for 30 min at 37°C before dilutions were made. One tenth-milliliter volumes of Bruce&z abortw diluted 1: 100 in the same diluent were added and agglutinations were read after standing overnight at room temperature.

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Complement receptor lymphocyte (CRL) determinations were performed on splenic lymphocytes suspended in RPMI-1640 (GIBCO, Grand Island, NY) as described (16) using SRBC (obtained from the City of New York Department of Health, Bureau of Laboratories) sensitized with a 1: 500 dilution of amboceptor (rabbit anti-SRBC hemolysin, BBL, Baltimore, MD). Fresh CFl mouse serum diluted 1 :20 in veronal-buffered saline containing calcium and magnesium (VBS) (17) was used as the source of complement. Tests were done on Day 11 after initiation of PGEl or saline treatment or after the last injection of procarbazine in the CBA strain and on Day 11 from the beginning of treatment with either agent or saline in the AKR strain. Anti-&AKR serum was prepared in C3HeBFe female mice. These received six weekly injections of 1.2 x lo7 AKR female thymocytes ip and were bled 1 week after the last injection. The serum was inactivated at 56°C for 30 min and absorbed at 4°C for 1 hr with an equal volume of a mixture of AKR kidney-lung homogenate and AKR erythrocytes washed with saline five and three times, respectively. After centrifugation to remove the absorbing material, the serum was filtered through a 0.20-pm membrane in a Nalgene Filter Unit (Nalge, Rochester. NY). For the cytotoxic assay, fresh normal guinea pig serum (complement), also absorbed with the AKR kidney-lung homogenate and erythrocytes mixture and filtered was diluted 1 : 10 in VBS. Cell suspensions from individual mouse spleens were labelled with 50 &i 51Cr (Amersham Searle, Chicago, IL) per 100 x 10” cells per 2.5 ml lor 40 min at 37°C. The cells were then washed three times in 50 ml RPM1 and resuspeuded to 5 x 106/ml in RPMI. One tenth-milliliter antiserum dilutions were made in VBS and 0.1 ml cell suspension was added followed by 0.1 ml complement. After incubation at 37°C for 30 min, 1 ml of cold (4°C) saline was added and the tubes were centrifuged at 15OOg for 15 min. A 0.65-ml aliquot of the supernatant from each tube was counted in a Nuclear Chicago Autogamma Counter. Maximal 51Cr release (high control) was taken as that in the supernatant of cells frozen and thawed three times. Spontaneous 51Cr release was measured in tubes containing complement but no antibody (low control). Percentage of Xr released from individual cell suspensions was calculated as: cpm in supernatants

containing

anti-BAKR

-

cpm in low control

supernatants

100 x cpm in high control

supernatants

-

cpm in low control

supernatants

Plateau values for thymocytes (100% %r release) were found between 1: 10 and 1: 160 dilutions of the antiserum, those for spleens between 1: 10 and 1: 40. The percentage of 0 AKR-positive cells in the spleens of normal AKR mice was somewhat low iu comparison to values reported for 8 C3H-positive cells in spleens of other strains, but is comparable to that observed in AKR mice by others (18). These determinations were carried out on Day 14 from the beginning of treatment. Hematocrit values were determined in heparinized capillary tubes after centrifuging for 5 min in a microhematocrit centrifuge (Clay Adams, Inc., New York, NY) according to standard procedure. WBC counts were done on blood diluted in Unopette disposable blood-diluting pipettes No. 5855 (Becton, Dickinson and Co., Rutherford, NJ) using Neubauer improved counting chambers. WBC differential counts were done on smears stained with McNeil tetrachrome stain, counting 200 cells per slide. All these determinations were done on the Day 11

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after initiation of PGE, and saline treatment and after the last injection of procarbazine. For histology, thymi, spleens, and lymph nodes were fixed in formol-acetic acid for 4 hr, then transferred to 70% alcohol and the sections stained with hematoxylin and eosin and with methylgreen and pyronine. RESULTS The survival of A/J tail skin grafts on CBA/J mice was markedly prolonged by treatment with procarbazine for 4 days before application of the grafts. In two experiments shown in Fig. 1, an increase of 5 days in the median survival time was induced by either 360 (Fig. la) or 300 (Fig. lb) mg/kg/day procarbazine. Though daily treatment with PGE, alone from the time of grafting did not significantly prolong homograft survival, daily treatment with 100 (Fig. la) or 200 (Fig. lb) pg PGEl/day f or 20 days after procarbazine pretreatment resulted in longer graft survival than after procarbazine alone, the median survival time being 8 days longer than in controls. The effect of PGEl on graft survival appeared more pronounced in those mice in which prolongation had already been most effectively induced by procarbazine. This was deduced from the differing slopes of the survival/time curves. In the group treated with the two agents (Fig. lb) in combination, some of the grafts were still in place s* ‘y i ; :i I!__ CL,_., 9 : i ‘.., : L.1 ’ i :i., ,

90

70

-

CONTROlS

--------

PROC*RP.*Z,NE

112)

,.............,.

POE, (,3)

(6)

_ _ -. _. _. PROCARBAZ,NE+

PGE,(7)

50

-CONTROLS

:.....

-------; :..., j ._!_._.( : ‘I. $2. ..i ,I L-L ._.,

(IO)

PROCARBAi!lNC PROCARBAZlNC

(13)

PGE, (12) PGE, (12) .-._. -.- - PROCARBAZINE+

PGE, (111

*death

30

lo

lb

DAYS

;ORDM

GRA&ING

FIG. 1. Survival of A/J tail skin grafts on CBA/J mice. (la) 360 mg/kg/day procarbazine for 4 days prior to grafting and/or 100 pg/day PGEI for 20 days from the day of grafting. (lb) 300 mg/kg/day procarbazine and/or 200 fig/day PGEl as above. Controls for both experiments received saline for 20 clays. Figures in parentheses represent numbers of mice per group at the time of deplastering.

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when the mice were killed 1 month after grafting. Some deaths, probably not attributable to the anesthesia, occurred after deplastering in the groups receiving the combined treatment. As no autopsies were performed, the cause of death is not known. All the survivors appeared healthy. These results suggested that PGEl might bc depressive either by deletion or inactivation of some cell population involved late in graft rejection. Such a population might include cells making cytotoxic antibody and cells capable of causing cytolysis in the presence of antibody. The activities of such cells might become apparent only after deletion (by procarbazine) of specifically immune T cells initially responsible for rejection. To investigate directly which cell populations are affected by PGE, and procarbazine we determined the percentages of CRL in the spleens of mice treated with PGE1, procarbazine or saline. As shown in Table 1, the total numbers of spleen cells were reduced by either PGE, or procarbazine treatment. However, in two experiments in two strains of mice (CBA and AKR) the percentages of CRL in the spleen were significantly increased by procarbazine treatment and significantly decreased hy PGE, treatment. Calculating the numbers of CRL per spleen from the data in Table 1 it appears that procarbazine and PGE, reduced them by the same amount (to about 7 X lo6 from 19.95 x lo6 in the controls). An opposite effect was obtained from the assay for &positive cells in the spleens of AKR mice receiving PGEI, procarbazine or saline (Table 2). Anti-8 AKR serum released a higher percentage of radioactivity from YZr-labeled spleen cells of PGEI-treated mice and a lower percentage from those of procarbazine-treated mice than from those of controls. This indicated that the percentages of &positive TABLE

1

PIJKCENT COMPLEMENT RECEPTOR LYMPHOCYTES IN SPLEEN AFTER VARIOUS TREATMENTS” AKR

CBA

Treatment

(2)

None Procarbazinc PGE, Proc. + PGEl

22.7 40.1 f 12.6 f 22.2

u Mean values f

SD ; figures in parenthescs

3.80 (3) 3.38 (5) (1) indicate

TABLE PEKCENT VK

Total spleen cells X lo6

18.6 zk 2.89 (5) 27.3 f 1.14 (5) 11.4 i 1.86 (5) ND

103 f 27.4 26 f 11.6 65 f 23.2 ND

number of mice per grollp.

2

RELEASED FKOM AKR MOUSE CELLS UPON T~cuuxr~oN AKR SERUM AND GUINEA PIG, C

Treatment

Cell source

None Procarbazine PGEI None

Spleen Spleen Spleen Thymus

a Mean f

Percent CRL

Percent CRL

SD (SE).

No/group (5) (5) (4) (1)

WITH C3H ANTI-0

Values at plateau10.15 f 5.80 f 20.20 f

4.1 (1.9) 4.6 (2.0) 6.4 (3.2) 100

.-

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TABLE EFFECT

3 IN AKR

OF PROCARBAZINE AND PGEl ON BLOOD PICTURE 10 DAYS AFTER BEGINNING OF TREATMENT

Treatment

Hematocrit

None Procarbazine PGE,

40.6 f 34.6 f 43.5 f

WBC

0.37 1.80 0.45

MICE”

X lo3 b

6.2 f 1.93 2.6 f 1.02 2.4 zk 1.36

a Mean f SD, five mice per group. b Differential WBC counts showed no gross differences.

(T) cells were increased in the spleen of PGEr- and decreased in those of procarbazine-treated mice. Calculating the numbers of o-positive cells, using the whole spleen counts from Table 1, it appears that they were not reduced after PGE1, but were, markedly, after procarbazine (13 X lo6 and 1.6 X 106, respectively) as compared with the controls (10 X 106). These results indicated that the effect of procarbazine was greater on T than on B cells, whereas that of PGE, was mainly confined to B cells. The peripheral white counts were reduced by treatments both with PGEr and procarbazine but, as shown in Table 3, the hematocrits, which were reduced by procarbazine, were increased in the group treated with PGE1. This might have been due to hemoconcentration caused by loss of fluid to the tissues, which would account for their edematous appearance on histological examination (Quagliata, F., unpublished observations). Anti-Brucella titers were measured in the CBA mice of the second grafting experiment shown in Fig. lb. They were immunized on Day 8 after grafting. The results are shown in Fig. 2. PGE, treatment alone did not significantly depress .4 ,’,J

TOTAL 12

2-ME

RESISTANT

,;’,’,,’,,,‘,* ,\ ,: ..sl,1,’

10

0 SALINE .

PROCARSAZINE

-

PGE,

d

+ PROC. BOOST 8

15

21

29

s

15

21

29

DAYS

FIG. 2. Anti-Brucelkc

ubortus antibody

response in five mice per group.

(Mean

log* titers * SE)

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463

the response, whereas procarbazine either alone or in association with PGEl significantly depressed the initial phase of the response particularly affecting the production of Z-ME-resistant antibody ; procarbazine alone somewhat prolonged Z-ME-sensitive antibody production, a phenomenon similar to that observed with 6-mercaptopurine treatment (19). A slight, but not statistically significant, early increase in the production of Z-ME-resistant antibody in the PGEr-treated group is under further investigation. The histological appearance of the lymphoid tissues after treatment with the two agents is shown in Fig. 3. The thymi of procarbazine-treated mice were

FIG.

3. Histology of thymus

of treatment. eosin stain.

1 = saline-treated

(T) and spleen (S) of AKR mice 10 days after the beginning ; 2 = procarbazine-treated ; 3 = PGEI-treated. Hematoxylin and

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markedly shrunk in toto with the margin between cortex and medulla well preserved. Those of PGE,-treated mice were only moderately reduced in size, but the margin between cortex and medulla is not clearly defined probably due to depletion of cortical thymocytes. In the spleen, after procarbazine treatment, the perifollicular areas were depleted, while after PGE, treatment there was reduction of the red pulp so that the follicles appear closer to one another. The lymph nodes were also examined: while procarbazine depleted the paracortical areas as previously observed (12, 13), PGEl had no apparent effect at the dose and schedule used. DISCUSSION Our results show that, although PGE, treatment alone had no effect on homograft survival, when mice were treated with procarbazine in such a way that their homograft reactivity was depressed, an additional impairment of this function could be produced by treatment with PGE1. The PGE1-induced effect became obvious only late after grafting, suggesting that the cells involved were those which only became important in the rejection mechanism late after sensitization. The histological changes in the thymi and spleens and the differential reductions in the percentages of splenic CRL and &positive cells caused by the two agents seem to imply that they have different mechanisms of action. That PGEl has a directly depressing effect on B lymphocytes was shown by the reduction in splenic CRL and relative increase in the number of splenic &positive cells. The reduction in total lymphocyte counts both in the spleen and peripheral blood supports the idea that PGEl treatment actually causes depletion of B lymphocytes and not merely masking or removal of the complement receptor from their surface. B cells, which might be postulated to participate late in graft rejection, could be producers of cytolytic antibody or could be cells capable of participating nonspecifically in target cell destruction in cooperation with specific antibody. Such cytolytic B lymphocytes have been described (20, 21). We did not find a suppressive effect of PGEl alone on anti-Brucella antibody production ; this argues against the idea that suppression of antibody production is responsible for the prolongation of graft survival. Therefore, at present we favor the idea that the lymphocytes chiefly inactivated by PGEl treatment in Z&JOare cytolytic, but probably not specific antibody-producing B cells. However, suppression of the cytolytic activity of immune lymphoid cells in vitro after incubation with PGEl or even other agents which increase intracellular concentrations of CAMP has been observed in a system in which the cytolytic lymphocytes are considered to be T cells (5). It is, therefore, possible that PGEl acts in viva on cytolytic T cells without killing them (&positive cell percentages are increased), though it might then be expected to affect normal graft rejection and delayed hypersensitivity reactions. PGEl treatment does deplete the thymus of small lymphocytes. This could be due to an effect on B-derived thymocyte precursors in the circulation, as the thymic cortex was strongly affected, but a direct effect of PGEl on cells already in the thymus cannot be excluded. A third possibility is that the depletion might he caused by a PGE1-mediated increase in adrenal steroid activity (22-24). That PGE, can directly affect T cells at least in vitro seems likely from the observation that it can inhibit blast cell transformation by phytohemagglutinin (25).

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465

Despite the quite marked reductions in splenic CRL and total white cell counts both in spleen and periphery after treatment with PGEl alone, no reduction in anti-Brucella antibody titers was obtained. In subsequent experiments in ungrafted mice, even longer pretreatment with PGEr neither reduced nor enhanced antibody production to either Brucella or burro erythrocytes (Melton, J. W. III, Phillips-Quagliata, J. M., and Quagliata, F., unpublished observations). This suggests that a subpopulation of CRL, not directly involved in antibody production, is the main target of PGEr. The depressive effect previously noted in the antiSRBC response in adjuvant arthritic rats might be due to depletion of a “processing” macrophage-like cell (26). Finally, it should be noted that though the suppressive effect of PGEl on graft rejection was not very impressive, it was achieved with very low doses given systemically ; similarly low doses were effective in suppressing adjuvant disease (10) ; this is consistent with the idea that locally produced PGEr might be one of a series of physiological regulators of the function of lymphocytes participating in inflammation. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.

Willis, A. L., J. Pltarm. PiEarmacol. 21, 126, 1969. S$ndergaard, J., and Greaves, M. W., Fed. Proc. 29,419, 1970. Butcher, R. W., and Baird, C. E., J. Biol. Chem. 243, 1713, 1968. Marsh, J., Ann. N. Y. Acad. Sci. 185, 416, 1971. Henney, C. S., Bourne, H. R., and Lichtenstein, L. M., J. Immunol. 108, 1526, 1972. Kaley, G., and Weiner, R., Ann. N. Y. Acad. Sci. 185, 338, 1971. Mannaioni, P, F., Biocltem. Plinrmncol. 19, 1159, 1970. Bourne, H. R., Lichtenstein, L. M., and Melmon, K. L., J. Inzmzmol. 108, 695, 1972. Weissman, G., Dukor, I’., and Zurier, R. B., Nature (London) New Biol. 231, 131, 1971. Zurier, R. B., and Quagliata, F., Natrrrc (London) 234, 304, 1971. Floersheim, G. L., Scieltce 156, 951, 1967. Floersheim, G. L., Taub, R. N., Phillips-Quagliata, J. M., and Levey, R. H., Agents and Actions 1, 115, 1970. Quagliata, F., Phillips-Quagliata, J. M., and Floersheim, G. L., Cell. Innmc~zol. 3, 198 1972. Hamberg, M., and Samuelsson, B., J. Dial. Chem. 246, 6713, 1971. Billingham, R. E., and Medawar, P. B., /. Exp. Biol. 28, 385, 1951. Bianco, C., Patrick, R., and Nussenzweig, V., J. Exp. Med. 132, 702, 1970. Kabat, E. A., and Mayer, M. M., “Experimental Immunochemistry,” 2nd ed., Thomas, Springfield, IL, 1961. Bianco, C., and Nussenzweig, V., Science 173, 154, 1971. Sahiar, K., and Schwartz, R. S., Scieme 145, 395, 1964. MacLennan, I. C. M., and Harding, B., Nature (Londotz) 227, 246, 1970. VanBoxel, J. A., Stobo, J. D., Paul, W. E., and Green, I., Science 175, 194, 1972. Flack, J. D., Jessup, R., and Ramwell, P. W., Scictice 163, 691, 1969. DeWied, D., Witter, A., Versteeg, D. H. G., and Mulder, A. H., EndocvirzologJr 85, 561, 1969. Saruta, T., and Kaplan, N. M., Cl&z. Res. 19, 69, 1971. Smith, J. W., A. L., and Parker, C. W., J. Clin. Invest. 50, 422, 1971. Argyris, B. F., J. &p. Med. 129, 495, 1968.