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1,25-Dihydroxyvitamin D3 enhances the generation of nonspecific suppressor cells while inhibiting the induction of cytotoxic cells in a human MLR
CELLULAR
IMMUNOLOGY
l@doo-d@
(19%)
1,2!5Dihydroxyvitamin D3 Enhances the Generation of Nonspecific Suppressor Cells while Inhibiting the Induction of Cytotoxic Cells in a Human MLR MICHELLEA.MEEHAN, RONALDH. KERMAN, AND JACQUESM. LEMIRE’ Division of Pediatric Nephrology, Department of Pediatrics, and Division of Organ Transplantation, Department of Surgery, The University of Texas Medical School at Houston, Houston, Texas 77030 Received June 3, 1991; accepted December 7, 1991 The active vitamin D metabolite, 1,25-dihydroxyvitamin D, (1,25-Ds), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effectsof the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response.Suppressor-cellactivity of MLR-generated effector cells was enhanced by calcitriol ( IO-r0 to 10-sM). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppressa primary MLR with stimulators and/or respondersheterologous to the effector-generating MLR. Calcitriol ( 1O-9to lo-’ M) was also effective in preventing the generation of cytotoxic T cells when tested in a 5’Cr releaseassay.While no differenceswere observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D,-treated cells and control cells, a significant reduction of classII antigen expressionwas observedin the presenceof the hormone. The effectsof academic press, IDC. calcitriol on human MLR are similar to those observedwith cyclosporine. o 1992
INTRODUCTION
The steroid hormone 1,25-dihydroxyvitamin D3 ( 1,25-D3)2has been shown to exert immunosuppressive properties in vitro (l-4). Following antigenic or mitogenic challenge and upon exposure to the hormone, human lymphocyte DNA synthesis and immunoglobulin production are significantly inhibited ( l-4). The CD4+ subsetappears to be particularly sensitive to the action of the active metabolite. 1,25-D3 has been shown to suppressCD4+ T-cell proliferation and inhibit CD4+ effector function such asantibody production by B cells (5-8). The human mixed lymphocyte reaction (MLR) provides a system for analyzing the interactions between subpopulations of alloantigen specific helper-inducer, cytotoxic-suppressor cells. Since CD4+ cells constitute a major population of responding cells in an MLR, their activity is essential in inducing other lymphocyte subsets, or their precursors, to differentiate into effecters of cytotoxicity or suppression(9). We therefore investigated the potential role of 1,25-D3 in modulating the functional activity of cells generated in a human MLR. ’ To whom correspondence should be addressedat University of California, San Diego Department of Pediatrics, M-0009-G La Jolla, CA 920934009. 2 Abbreviation used: 1,25-Ds, 1,25-dihydroxyvitamin DS. 400 0008-8749/92 $3.00 Copyright 0 1992 by Academic Press,Inc. AU rights of reproduction in any fom reserved.
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MATERIALS AND METHODS Vitamin D3 Preparation Crystalline 1,25-D3 was generously provided by Dr. Milan Uskokovic (HoffmannLa Roche, Inc., Nutley, NJ). The vitamin D3 metabolite was dissolved in absolute ethanol and added to culture media in concentrations ranging from lo-*’ to 10m8M. Control cells were cultured in the hormone vehicle (0.025% ethanol). Lymphocyte Isolation Human peripheral blood mononuclear cells were obtained from normal adult volunteers and separatedby centrifugation of heparinized venous blood on Ficoll-Paque (Pharmacia LKB Biotechnology, Inc., Piscataway,NJ). The isolated fraction contained >95% mononuclear cells. The cells were suspended in complete media containing RPM1 1640 (Cellgro, Mediatech, Washington, DC) supplemented with 10% heatinactivated fetal calf serum, penicillin (50 p/ml), streptomycin (50 pg/ml), and Lglutamine (0.29 mg/ml). Mixed Lymphocyte Reaction for Generation of Modulators A mixed lymphocyte reaction was initiated by adding 1 X lo5 irradiated (3000 R) stimulator cells to 1 X lo5 responder cells (each in a volume of 0.05 ml) in a 96-well round-bottom microtiter plate (Corning Glass Works, Coming, NY). Cells were cultured in the presence of 1,25-D3 in concentrations ranging from lo-” to 10e8M. Control cells were incubated with 0.025% ethanol. After 7 days incubation, the cells were harvested, washed, and irradiated with 2500 rad to be used as effector cells. To further determine the antigen specificity of the response,four groups of effecters were tested in a primary MLR. In the first group, the effecters were autologous to the responders. In the second group, the effector cells were autologous to the responders of the primary MLR but with a different stimulator. In the third group, the effecters were heterologous to the respondersbut with identical stimulators. In the fourth group, the effecters, responders, and stimulators were completely heterologous. Suppressor-Cell Assay Responder cells were suspended in complete media at 1 X lo5 cells/well. Effector cells, 2 X lo5 cells/well, were added to a primary MLR, autologous and heterologous to the original MLR, and allowed to incubate for 4 days, at which time they were pulsed with 1.0 &i of [3H]thymidine (5 Ci/mmol, Amersham Int., Amersham, UK). Eighteen hours later, the cells were harvested with a multiple automated sample harvester unit onto glass fiber filter paper. The filter papers were dried and processed for liquid scintillation counting in a beta scintillation counter ( 10). Results are expressed both as absolute and Acpm and as mean percentagesuppressionor enhancement of the D3-treated cell cultures compared to the control MLR + standard error of the mean. Phenotypic Analysis of Modulators Effector cells generated in the presence or absence of 1,25-D3 were submitted to phenotypic analysis by flow cytometry techniques. Cells were suspended in complete media to a final concentration of 5 X 106/ml. FACS analysis was performed as pre-
402
MEEHAN, KERMAN,
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viously described (11). Briefly, 10 ~1 of fluoresceinated anti-human leukocyte monoclonal antibody was incubated with 100 ~1of cell suspension at 4°C for 30 min. The cells were washed extensively, fixed with 0.5 cc of 1%paraformaldehyde, and analyzed in a flow cytometer (FACScan, Becton-Dickinson, Mountain View, CA). The following monoclonal antibodies were used: Anti-Leu-Sb-CD2 (Becton-Dickinson) Anti-Leu-3a-CD4 (Becton-Dickinson) Anti-Leu-2a-CD8 (Becton-Dickinson) Anti-Leu- 12-CD 19 (Becton-Dickinson) Anti-TCR- 1-T-cell receptor (Becton-Dickinson) Anti-HLA-DR-DR (Becton-Dickinson) Anti-IL-2 receptor-CD25 (Becton-Dickinson) Anti-Ta- 1RD- 1-activated T cell (Coulter Immunology, Hialeah, FL).
Cell-Mediated Lympholysis An MLR wasinitiated aspreviously describedwith 1,25-Dsin concentrations ranging from 10m9to 10m7M. A control MLR was generated with 0.025% ethanol. After 7 days incubation, cells were harvested and used as effector cells in varying effector: target ratios ranging from 50: 1 to 1.5:1. Target cells were prepared by suspending naive responder cells in complete media at a concentration of 2 X 106/ml in a 25-cm* tissue culture flask with phytohemagglutinin A at a 1/ 1000 dilution (DIFCO Laboratories, Detroit, MI). After 7 days, target cells were washed and counted. Chromium (“Cr) labeling of target cells was achieved with 100 PCi 5’Cr/2 X lo6 target cells (sp act 502.15 mCi/mg, ICN Biomedicals, Inc., Costa Mesa, CA). After 1 hr of incubation at 37°C cells were washed and resuspended in complete media and allowed to incubate for 30 min at 37°C. The target cells were again washed and resuspended in complete media at 1 X lo4 cells/well. Effector and target cells were incubated in round-bottom microtiter plates in a final volume of 0.20 TABLE 1 Inhibition of Primary MLR by 1,25-Da-Treated Effector Cells
Fresh MLR vs effectorgenerating MLR Autologous Heterologous Same stimulator, different responder Same responder, different stimulator
MLR + effector cells from untreated original MLR (cm4
MLR + effector cells from 10-s M 1,25-D4 original MLR @pm)
Acpm
% Suppression
34,832 41,853
11,251 23,731
23,581 18,122
68 43
38,823
28,612
10,211
26
37,587
23,304
14,283
38
Note. The first column compares the fresh MLR to the original MLR from which the effector cells were generated. Effector cells from 1,25-Dg-treated MLR suppressedthe primary MLR in all four groups tested, reported as maximum suppression observed ( lOm8M). Results are expressed as mean cpm, Acpm, and % suppression.
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403
MLR ACTIVITY
50 40 E
30 -
2E
20-
2
10 -
* p < 0.05
-70 -
*
-90 t -100 1
I
I
Control
lo-‘”
.^
I
^
10 -=
I 10”
M
1,25 - (OH), -D, FIG. 1. Antigen specific suppressionby 1,25-DJ-treated effector cells of autologous primary MLR. Primed lymphocytes from control and 1,25-D3(lo-” to lo-* M)-treated MLR cultures were used as effecters (2 X lo6 cells/ml, 2500 R) in a primary MLR (2 X lo6 responder cells/ml; 2 X lo6 stimulator cells/ml, 3000 R). Cells were cultured for 4 days and pulsed with t3H]thymidine for an additional 18 hr. Data represent the effect (% enhancement or % suppression) of effecters on the primary autologous MLR compared to control MLR in the absenceof effector cells.
ml. After 4 hr of incubation at 37”C, the plates were centrifuged at 800 rpm for 5 min, and 100 ~1of supernatant was removed and counted for releaseof 5’Cr. Maximum 5’Cr releasewas obtained by adding 0.1 cc of 10%Tween to labeled targets.Spontaneous “Cr release was obtained by adding 0.1 cc of complete media to 1 X lo4 targets. Percentagespecific “Cr releasewas calculated according to % specific 5’Cr release =
cpm experimental - cpm spontaneous x 100. cpm maximum - cpm spontaneous RESULTS
Enhancement of Suppressor-Cell Activity by Autologous Efector Cells Generated with 1,250, When Day 7 MLR-generated mononuclear effector cells were added to a primary MLR, a 32.9% inhibition of MLR proliferation was seen,thus identifying the presence
404
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of effecters with suppressor activity. When 1,25-D3 was added at the initiation of the effector MLR (in increasing concentrations of lo-” to lOPaM) the Day 7 effecters exerted enhanced suppressoractivity when added to a primary MLR, with maximum suppressor effect of 67.7 + 2.7% MLR inhibition observed at lo-* M 1,25-D3 (P < 0.05, Table 1 and Fig. 1). The same Day 7 effecters did not display suppressor activity in a primary MLR with autologous responders when stimulators were different than those used in the initiating MLR. However, generation of effecters in the presence of 1,25-Ds resulted in the Day 7 effector cells displaying significant suppressor cell activity despite stimulator disparity (P < 0.02, Table 1 and Fig. 2). Again, lo-* M 1,25-D3was the optimal concentration for maximal suppressor activity. Reduction of Allogenicity and Induction of Suppressor-Cell Activity with I,25D3 In a limited number of controlled experiments (n = 4), when effecters generated from an MLR were allogeneic to the respondersof a primary MLR, a mild proliferative response was observed despite identical stimulators. However, the addition of 1,25-
5oI .-
*
E
z8
30-
s 2 Lu s .5
20 -
5
p-zo.02
lo-
-50
I Control
I _-
I
10 -‘”
1,25-
*
I
10’
M
(OH):~D,
FIG. 2. Antigen nonspecific suppressionby 1,25-D&eated effector cells. Effector cells were prepared and culture processedas in Fig. 1. The effector cells were added to a primary MLR with autologous responders but allogenically different stimulators. Data are expressedas described in the legend to Fig. 1.
CALCITRIOL
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HUMAN
405
MLR ACTIVITY
50 40 -
* p < 0.07
E g8
30-
s 2
20 -
.-O-
2 cn 8
-20 -30 * -40 -50
I Control
I _^
10 -'"
I 10 -=
I lo*
M
1,25 - (OH), -D, FIG. 3. 1,25-D,-treated effector cells suppressthe primary MLR in a MHC nonrestricted fashion. Effector cells were prepared as in Fig. 1 and added to a primary MLR. Effector cells were heterologous to responders of the primary MLR while stimulators were identical.
D3 to the effector MLR not only reduced the MLR-enhancing effectbut also contributed to effecters with up to 26.3% suppressive effect at lo-* M 1,25-D3 (P < 0.07, Table 1 and Fig. 3). Finally, with the use of effectersfrom an MLR where the respondersand stimulators are heterologous to the primary MLR, an MLR-enhancing responseof 52.0 f 25.0% was observed (Fig. 4). However, incubation of the effector MLR with 10-l’ to lo-* A4 1,25-D3 generated effectorswith lessenhancing activity, reducing the MLR response by 43.3% at lo-* M (P < 0.02, Table 1 and Fig. 4).
1,25Dihydroxyvitamin D3 Inhibition of the Generationof Cytotoxic Cells The human MLR results in the generation of a cytotoxic response against 51Crlabeled stimulator targets at 7 days from the initiation of the culture. As shown in Fig. 5, a significant reduction in the degreeof cytotoxicity was observed in cells previously incubated with both 1O-* and 1OP9Mconcentrations of 1,25-D3 with maximum effect observed at lo-* A4 (P < 0.03). The difference observed between the controls and 1,25-D,-treated cells was present at all effector to target ratios studied, ranging from 1.5:1 to 5O:l.
406
MEEHAN, KERMAN, AND LEMIRE 100 90 E ifi s 5
*
60-
p
7060
-
5
*
5 % 'n 5 KJ 9 0
-10
-
-20
-
-30
-
-40
-
-50
I Control
I 10 -'O
I 10 -g
I loa
1,25 - (OH), -D, FIG. 4. 1,25-D3-treatedeffecters suppressheterologous primary MLR. Effector cells were prepared as in Fig. 1 and added to a primary MLR, heterologous for both respondersand stimulators. Data are expressed as percentage enhancement of MLR.
Effect of 1,25-Dihydroxyvitamin D3 on PhenotypicAnalysis of MLR Modulators To determine if the enhanced suppressor cell function of 1,25-D3 MLR-generated effecters was due to the selection of CDV lymphocyte subsets,effecters incubated in the presence or absence of 1,25-D3 were submitted for FACS analysis. As shown in Table 2, 1,25-D3 did not allow for the selection of a particular cell subpopulation. A significant reduction of HLA-DR antigens (21.2% vs 13.3%, P < 0.03) was observed following 1,25-D3treatment, whereas arithmetic but insignificant reductions were observedfor the IL-2 and Ta 1 receptors.The expression of ClassI antigens was unaffected by 1,25-D3. DISCUSSION The active vitamin D metabolite, 1,25-D3, has been shown to have significant in vitro immunomodulatory properties including inhibition of DNA synthesis and antibody production in activated human peripheral blood lymphocytes (l-4). Further studies have shown the CD4+ subset to be the preferential target for the action of the hormone (5-8). CD4+ cell proliferation and CD4+ cell-driven B-cell immunoglobulin
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* **
407
pc0.03 p
1,25-Ds[M] 60 L!is 010’ 50 % .= 0 ‘E 0 5 40 E s
30
20
10
0 50
25
12.5
Effector to Target Cell Ratio FIG. 5. Inhibition of generation of cytotoxic cells by 1,25-D3. After a 7-day MLR culture in the presence or absence of 1,25-D3 ( 10m9to lo-’ M), the lymphocytes were used as effector cells against “0-labeled PHA-stimulated lymphocytes. Data represent the percentage cytotoxicity at effector to target ratios ranging from 5O:l to 12.5:1.
production are particularly sensitive to the action of the hormone. Theseresults suggest an inhibitory action of the metabolite on T-helper activity. To the contrary, CD8+ cells appear to be insensitive to the action of the hormone. Moreover, suppressor activity of CD8+ cells on immunoglobulin production was found to be increased in the presence of 1,25-D3 when exogenous recombinant human IL-2 was added to cultures ( 12). Effector MLR cultures generatemodulators with suppressorcell activity when added to a primary autologous MLR. However, when the effecters were cultured in the presence of increasing concentrations of 1,25-D3, they exerted enhanced suppressor activity with an additional 34.8% MLR inhibitory effect at 10m8A4 1,25-D3. This suppressoractivity was not antigen specific; the sameautologous effecters also exerted enhanced suppressor activity despite different stimulators. Heterologous effecters, as predicted, when added to a primary MLR exerted an enhancing effect resulting in increased proliferation. This enhanced MLR responsewas reduced when the effecters were generatedwith 1,25-D3; absolute MLR inhibition wasalso observedwhen identical stimulators were used (Fig. 3). Thus, 1,25-D3 results in the generation of nonspecific suppressor cells.
408
MEEHAN, KERMAN, AND LEMIRE TABLE 2 Phenotypic Analysis of 1,25-Ds-Treated Effector Cells Monoclonal antibody
Control
1,25-D3
Leu-5b (CD2) Leu-3a (CD4) Leu-2a (CD8) Leu-12 (CD19) TCR HLA-DR IL-2 Ta-IRD-1 Class I
90.5 62.4 26.1 6.1 84.1 21.2 21.7 17.2 100
87.5 59.9 27.5 9.1 83.0 13.3* 14.2 11.7 100
Note. Effector cells generated from a 7day MLR in the presence ( lOm8M) or absence of 1,25-D, were submitted for phenotypic analysis by cell sorting techniques (FACS). The data are expressedas percentage of cells positive for the respective antigens.
* P < 0.03.
The enhanced nonspecific suppressor-cell activity generated with 1,25-D3 was not the result of a selection of CD8+ lymphocytes since flow cytometric analysis of controls and 1,25-D3-treated effecters revealed no significant differences. However, 1,25-D3 reduced expression of activation markers on the effecters, including a significant reduction in MHC class II, while not affecting class I antigen expression. The active D3 metabolite has previously been shown to affect DR expression ( 13, 14). A decreasein the expression of class II antigens may contribute to a reduction of the allogenicity observed. The effect of the reduction of DR expression on suppressor cell activity is unclear. A predominant class I expression may allow for suppressor determinant exposure. Since an absolute increasein the number of suppressoreffecterscannot account for the marked inhibition of the MLR, a potential effect of 1,25-D3 on an “intermediate” cell in the suppressor cascadecannot be excluded. Despite its apparent preferential effect for CD4+ cells, 1,25-D, could exert a selective inhibition of certain CD4+ cells while allowing full expression of others such as the CD4+ helper-inducer of suppression or nonspecific macrophage suppressors. The MLC generation of T-cell cytotoxic activity was also reduced after incubation with 1,25-D3. The hormone was effective in preventing the development of effecters of cytotoxicity. No inhibition of CD8+ cells was observed in the presence of 1,25-Dj and it is possible that the CD4+ helper-inducer of cytotoxicity could have been targeted by the active metabolite. However, an earlier effect of 1,25-D3 on precursors of cytotoxic cells cannot be excluded. The mixed lymphocyte reaction represents the in vitro model of allograft response. Regulation of the MLR by 1,25-D3 suggestsa potential immunosuppressive role in alloimmune regulation. Indeed, use of the potent anti-rejection drug, cyclosporine, results in MLR responsessimilar to those described for the active vitamin D metabolite. Cyclosporine facilitates the induction of suppressor T cells by alloantigens ( 15). The suppression observed was found to be both antigen specific and antigen nonspecific. Moreover, the induction of cytotoxic T lymphocytes is completely inhibited by cyclosporine (15). The primary target of cyclosporine is the CD4+ cell. However, Hess et al. (16) have shown that in the presence of cyclosporine, the T-helper-inducer of
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suppression could still be activated, suggesting a relative resistance of the suppressor T-cell pathway to cyclosporine. Thus, these results suggest a potential role for 1,25-D3 as an immunoregulatory agent for use in transplant and autoimmune patients (17). ACKNOWLEDGMENT This work was partially supported by National Institutes of Health Grant R29-DK-39024 (J.M.L.).
REFERENCES 1. 2. 3. 4. 5.
Tsoukas, C. D., Provvedini, D. M., and Manolagas, S. C., Science 224, 1438, 1984. Lemire, J. M., Adams, J. S., Sakai, R., and Jordan, S. C., J. Cfin. Invest. 74, 657, 1984. Rigby, W. F. C., Stacy, T., and Fanger, M. W., J. Clin. Invest. 74, 1451, 1984. Bhalla, A. K., Amento, E. P., Scrog, B., and Glimcher, L. G., J. Zrnmunol. 133, 1748, 1984. Lemire, J. M., Adams, J. S., Kermani-Arab, V., Bakke, A. C., Sakai, R., and Jordan, S. C., J. Immunol. 134,3032, 1985. 6. Sumiko, I., Takahashi, T., Kura, F., Sugiyama, H., and Hoshino, T., J. Immunol. 136,4427, 1986. 7. Provvedini, D. M., and Manalagas, S. C., J. Clin. Endocrinol. Metab. 68(4), 1989. 8. Vanham, G., Ceuppens, J. L., and Bonillon, R., Cell. Immunol. 124, 320, 1989. 9. Thomas, F. T., Lee, H. M., Lower, R. R., and Thomas, J. M., Surg. Clin. North Am. 59,253, 1979. 10. Kerman, R. H., Flechner, S. M., Van Buren, C. T., Lo&r, M. I., and Kahan, B. D., Transplantation 43, 205, 1987. 11. Kennan, R. H., Kimball, P. M., Van Buren, C. T., Lewis, R. M., and Kahan, B. D., Trunspluntation 51, 338, 1991. 12. Jordan, S. C., Lemire, J. M., Sakai, R. S., Toyada, M., and Adams, J. S., Mol. Immunol. 27(l), 95, 1990. 13. Rigby, W. F. C., Waugh, M., and Grozrano, R. F., Blood 76( 1), 189, 1990. 14. Carrington, M. N., Tharp-Hiltbold, B., Knoth, J., and Ward, F. E., J. Immunol. 140, 4013, 1988. 15. Hess, A. D., and Tutschka, P. J., J. Zmmunol. 124, 2601, 1980. 16. Hess, A. D., Donnenberg, A. D., Tutschka, P. J., and Santos, G. W., J. Immunol. 130,7 17, 1983. 17. Lemire, J. M., and Archer, D. C., J. Clin. Invest. 87, 1103, 1991.
IMMUNOLOGY
l@doo-d@
(19%)
1,2!5Dihydroxyvitamin D3 Enhances the Generation of Nonspecific Suppressor Cells while Inhibiting the Induction of Cytotoxic Cells in a Human MLR MICHELLEA.MEEHAN, RONALDH. KERMAN, AND JACQUESM. LEMIRE’ Division of Pediatric Nephrology, Department of Pediatrics, and Division of Organ Transplantation, Department of Surgery, The University of Texas Medical School at Houston, Houston, Texas 77030 Received June 3, 1991; accepted December 7, 1991 The active vitamin D metabolite, 1,25-dihydroxyvitamin D, (1,25-Ds), has been shown in both in vitro and in vivo experiments to be immunoregulatory. We analyzed the effectsof the hormone on the human mixed lymphocyte reaction (MLR), the in vitro model of allograft response.Suppressor-cellactivity of MLR-generated effector cells was enhanced by calcitriol ( IO-r0 to 10-sM). This suppressor activity was nonspecific since calcitriol-generated effector cells could suppressa primary MLR with stimulators and/or respondersheterologous to the effector-generating MLR. Calcitriol ( 1O-9to lo-’ M) was also effective in preventing the generation of cytotoxic T cells when tested in a 5’Cr releaseassay.While no differenceswere observed in the phenotypic analyses of the MLR-generated effector cells between 1,25-D,-treated cells and control cells, a significant reduction of classII antigen expressionwas observedin the presenceof the hormone. The effectsof academic press, IDC. calcitriol on human MLR are similar to those observedwith cyclosporine. o 1992
INTRODUCTION
The steroid hormone 1,25-dihydroxyvitamin D3 ( 1,25-D3)2has been shown to exert immunosuppressive properties in vitro (l-4). Following antigenic or mitogenic challenge and upon exposure to the hormone, human lymphocyte DNA synthesis and immunoglobulin production are significantly inhibited ( l-4). The CD4+ subsetappears to be particularly sensitive to the action of the active metabolite. 1,25-D3 has been shown to suppressCD4+ T-cell proliferation and inhibit CD4+ effector function such asantibody production by B cells (5-8). The human mixed lymphocyte reaction (MLR) provides a system for analyzing the interactions between subpopulations of alloantigen specific helper-inducer, cytotoxic-suppressor cells. Since CD4+ cells constitute a major population of responding cells in an MLR, their activity is essential in inducing other lymphocyte subsets, or their precursors, to differentiate into effecters of cytotoxicity or suppression(9). We therefore investigated the potential role of 1,25-D3 in modulating the functional activity of cells generated in a human MLR. ’ To whom correspondence should be addressedat University of California, San Diego Department of Pediatrics, M-0009-G La Jolla, CA 920934009. 2 Abbreviation used: 1,25-Ds, 1,25-dihydroxyvitamin DS. 400 0008-8749/92 $3.00 Copyright 0 1992 by Academic Press,Inc. AU rights of reproduction in any fom reserved.
CALCITRIOL
MODULATES
HUMAN
MLR
ACTIVITY
401
MATERIALS AND METHODS Vitamin D3 Preparation Crystalline 1,25-D3 was generously provided by Dr. Milan Uskokovic (HoffmannLa Roche, Inc., Nutley, NJ). The vitamin D3 metabolite was dissolved in absolute ethanol and added to culture media in concentrations ranging from lo-*’ to 10m8M. Control cells were cultured in the hormone vehicle (0.025% ethanol). Lymphocyte Isolation Human peripheral blood mononuclear cells were obtained from normal adult volunteers and separatedby centrifugation of heparinized venous blood on Ficoll-Paque (Pharmacia LKB Biotechnology, Inc., Piscataway,NJ). The isolated fraction contained >95% mononuclear cells. The cells were suspended in complete media containing RPM1 1640 (Cellgro, Mediatech, Washington, DC) supplemented with 10% heatinactivated fetal calf serum, penicillin (50 p/ml), streptomycin (50 pg/ml), and Lglutamine (0.29 mg/ml). Mixed Lymphocyte Reaction for Generation of Modulators A mixed lymphocyte reaction was initiated by adding 1 X lo5 irradiated (3000 R) stimulator cells to 1 X lo5 responder cells (each in a volume of 0.05 ml) in a 96-well round-bottom microtiter plate (Corning Glass Works, Coming, NY). Cells were cultured in the presence of 1,25-D3 in concentrations ranging from lo-” to 10e8M. Control cells were incubated with 0.025% ethanol. After 7 days incubation, the cells were harvested, washed, and irradiated with 2500 rad to be used as effector cells. To further determine the antigen specificity of the response,four groups of effecters were tested in a primary MLR. In the first group, the effecters were autologous to the responders. In the second group, the effector cells were autologous to the responders of the primary MLR but with a different stimulator. In the third group, the effecters were heterologous to the respondersbut with identical stimulators. In the fourth group, the effecters, responders, and stimulators were completely heterologous. Suppressor-Cell Assay Responder cells were suspended in complete media at 1 X lo5 cells/well. Effector cells, 2 X lo5 cells/well, were added to a primary MLR, autologous and heterologous to the original MLR, and allowed to incubate for 4 days, at which time they were pulsed with 1.0 &i of [3H]thymidine (5 Ci/mmol, Amersham Int., Amersham, UK). Eighteen hours later, the cells were harvested with a multiple automated sample harvester unit onto glass fiber filter paper. The filter papers were dried and processed for liquid scintillation counting in a beta scintillation counter ( 10). Results are expressed both as absolute and Acpm and as mean percentagesuppressionor enhancement of the D3-treated cell cultures compared to the control MLR + standard error of the mean. Phenotypic Analysis of Modulators Effector cells generated in the presence or absence of 1,25-D3 were submitted to phenotypic analysis by flow cytometry techniques. Cells were suspended in complete media to a final concentration of 5 X 106/ml. FACS analysis was performed as pre-
402
MEEHAN, KERMAN,
AND LEMIRE
viously described (11). Briefly, 10 ~1 of fluoresceinated anti-human leukocyte monoclonal antibody was incubated with 100 ~1of cell suspension at 4°C for 30 min. The cells were washed extensively, fixed with 0.5 cc of 1%paraformaldehyde, and analyzed in a flow cytometer (FACScan, Becton-Dickinson, Mountain View, CA). The following monoclonal antibodies were used: Anti-Leu-Sb-CD2 (Becton-Dickinson) Anti-Leu-3a-CD4 (Becton-Dickinson) Anti-Leu-2a-CD8 (Becton-Dickinson) Anti-Leu- 12-CD 19 (Becton-Dickinson) Anti-TCR- 1-T-cell receptor (Becton-Dickinson) Anti-HLA-DR-DR (Becton-Dickinson) Anti-IL-2 receptor-CD25 (Becton-Dickinson) Anti-Ta- 1RD- 1-activated T cell (Coulter Immunology, Hialeah, FL).
Cell-Mediated Lympholysis An MLR wasinitiated aspreviously describedwith 1,25-Dsin concentrations ranging from 10m9to 10m7M. A control MLR was generated with 0.025% ethanol. After 7 days incubation, cells were harvested and used as effector cells in varying effector: target ratios ranging from 50: 1 to 1.5:1. Target cells were prepared by suspending naive responder cells in complete media at a concentration of 2 X 106/ml in a 25-cm* tissue culture flask with phytohemagglutinin A at a 1/ 1000 dilution (DIFCO Laboratories, Detroit, MI). After 7 days, target cells were washed and counted. Chromium (“Cr) labeling of target cells was achieved with 100 PCi 5’Cr/2 X lo6 target cells (sp act 502.15 mCi/mg, ICN Biomedicals, Inc., Costa Mesa, CA). After 1 hr of incubation at 37°C cells were washed and resuspended in complete media and allowed to incubate for 30 min at 37°C. The target cells were again washed and resuspended in complete media at 1 X lo4 cells/well. Effector and target cells were incubated in round-bottom microtiter plates in a final volume of 0.20 TABLE 1 Inhibition of Primary MLR by 1,25-Da-Treated Effector Cells
Fresh MLR vs effectorgenerating MLR Autologous Heterologous Same stimulator, different responder Same responder, different stimulator
MLR + effector cells from untreated original MLR (cm4
MLR + effector cells from 10-s M 1,25-D4 original MLR @pm)
Acpm
% Suppression
34,832 41,853
11,251 23,731
23,581 18,122
68 43
38,823
28,612
10,211
26
37,587
23,304
14,283
38
Note. The first column compares the fresh MLR to the original MLR from which the effector cells were generated. Effector cells from 1,25-Dg-treated MLR suppressedthe primary MLR in all four groups tested, reported as maximum suppression observed ( lOm8M). Results are expressed as mean cpm, Acpm, and % suppression.
CALCITRIOL
MODULATES
HUMAN
403
MLR ACTIVITY
50 40 E
30 -
2E
20-
2
10 -
* p < 0.05
-70 -
*
-90 t -100 1
I
I
Control
lo-‘”
.^
I
^
10 -=
I 10”
M
1,25 - (OH), -D, FIG. 1. Antigen specific suppressionby 1,25-DJ-treated effector cells of autologous primary MLR. Primed lymphocytes from control and 1,25-D3(lo-” to lo-* M)-treated MLR cultures were used as effecters (2 X lo6 cells/ml, 2500 R) in a primary MLR (2 X lo6 responder cells/ml; 2 X lo6 stimulator cells/ml, 3000 R). Cells were cultured for 4 days and pulsed with t3H]thymidine for an additional 18 hr. Data represent the effect (% enhancement or % suppression) of effecters on the primary autologous MLR compared to control MLR in the absenceof effector cells.
ml. After 4 hr of incubation at 37”C, the plates were centrifuged at 800 rpm for 5 min, and 100 ~1of supernatant was removed and counted for releaseof 5’Cr. Maximum 5’Cr releasewas obtained by adding 0.1 cc of 10%Tween to labeled targets.Spontaneous “Cr release was obtained by adding 0.1 cc of complete media to 1 X lo4 targets. Percentagespecific “Cr releasewas calculated according to % specific 5’Cr release =
cpm experimental - cpm spontaneous x 100. cpm maximum - cpm spontaneous RESULTS
Enhancement of Suppressor-Cell Activity by Autologous Efector Cells Generated with 1,250, When Day 7 MLR-generated mononuclear effector cells were added to a primary MLR, a 32.9% inhibition of MLR proliferation was seen,thus identifying the presence
404
MEEHAN,
KERMAN,
AND
LEMIRE
of effecters with suppressor activity. When 1,25-D3 was added at the initiation of the effector MLR (in increasing concentrations of lo-” to lOPaM) the Day 7 effecters exerted enhanced suppressoractivity when added to a primary MLR, with maximum suppressor effect of 67.7 + 2.7% MLR inhibition observed at lo-* M 1,25-D3 (P < 0.05, Table 1 and Fig. 1). The same Day 7 effecters did not display suppressor activity in a primary MLR with autologous responders when stimulators were different than those used in the initiating MLR. However, generation of effecters in the presence of 1,25-Ds resulted in the Day 7 effector cells displaying significant suppressor cell activity despite stimulator disparity (P < 0.02, Table 1 and Fig. 2). Again, lo-* M 1,25-D3was the optimal concentration for maximal suppressor activity. Reduction of Allogenicity and Induction of Suppressor-Cell Activity with I,25D3 In a limited number of controlled experiments (n = 4), when effecters generated from an MLR were allogeneic to the respondersof a primary MLR, a mild proliferative response was observed despite identical stimulators. However, the addition of 1,25-
5oI .-
*
E
z8
30-
s 2 Lu s .5
20 -
5
p-zo.02
lo-
-50
I Control
I _-
I
10 -‘”
1,25-
*
I
10’
M
(OH):~D,
FIG. 2. Antigen nonspecific suppressionby 1,25-D&eated effector cells. Effector cells were prepared and culture processedas in Fig. 1. The effector cells were added to a primary MLR with autologous responders but allogenically different stimulators. Data are expressedas described in the legend to Fig. 1.
CALCITRIOL
MODULATES
HUMAN
405
MLR ACTIVITY
50 40 -
* p < 0.07
E g8
30-
s 2
20 -
.-O-
2 cn 8
-20 -30 * -40 -50
I Control
I _^
10 -'"
I 10 -=
I lo*
M
1,25 - (OH), -D, FIG. 3. 1,25-D,-treated effector cells suppressthe primary MLR in a MHC nonrestricted fashion. Effector cells were prepared as in Fig. 1 and added to a primary MLR. Effector cells were heterologous to responders of the primary MLR while stimulators were identical.
D3 to the effector MLR not only reduced the MLR-enhancing effectbut also contributed to effecters with up to 26.3% suppressive effect at lo-* M 1,25-D3 (P < 0.07, Table 1 and Fig. 3). Finally, with the use of effectersfrom an MLR where the respondersand stimulators are heterologous to the primary MLR, an MLR-enhancing responseof 52.0 f 25.0% was observed (Fig. 4). However, incubation of the effector MLR with 10-l’ to lo-* A4 1,25-D3 generated effectorswith lessenhancing activity, reducing the MLR response by 43.3% at lo-* M (P < 0.02, Table 1 and Fig. 4).
1,25Dihydroxyvitamin D3 Inhibition of the Generationof Cytotoxic Cells The human MLR results in the generation of a cytotoxic response against 51Crlabeled stimulator targets at 7 days from the initiation of the culture. As shown in Fig. 5, a significant reduction in the degreeof cytotoxicity was observed in cells previously incubated with both 1O-* and 1OP9Mconcentrations of 1,25-D3 with maximum effect observed at lo-* A4 (P < 0.03). The difference observed between the controls and 1,25-D,-treated cells was present at all effector to target ratios studied, ranging from 1.5:1 to 5O:l.
406
MEEHAN, KERMAN, AND LEMIRE 100 90 E ifi s 5
*
60-
p
7060
-
5
*
5 % 'n 5 KJ 9 0
-10
-
-20
-
-30
-
-40
-
-50
I Control
I 10 -'O
I 10 -g
I loa
1,25 - (OH), -D, FIG. 4. 1,25-D3-treatedeffecters suppressheterologous primary MLR. Effector cells were prepared as in Fig. 1 and added to a primary MLR, heterologous for both respondersand stimulators. Data are expressed as percentage enhancement of MLR.
Effect of 1,25-Dihydroxyvitamin D3 on PhenotypicAnalysis of MLR Modulators To determine if the enhanced suppressor cell function of 1,25-D3 MLR-generated effecters was due to the selection of CDV lymphocyte subsets,effecters incubated in the presence or absence of 1,25-D3 were submitted for FACS analysis. As shown in Table 2, 1,25-D3 did not allow for the selection of a particular cell subpopulation. A significant reduction of HLA-DR antigens (21.2% vs 13.3%, P < 0.03) was observed following 1,25-D3treatment, whereas arithmetic but insignificant reductions were observedfor the IL-2 and Ta 1 receptors.The expression of ClassI antigens was unaffected by 1,25-D3. DISCUSSION The active vitamin D metabolite, 1,25-D3, has been shown to have significant in vitro immunomodulatory properties including inhibition of DNA synthesis and antibody production in activated human peripheral blood lymphocytes (l-4). Further studies have shown the CD4+ subset to be the preferential target for the action of the hormone (5-8). CD4+ cell proliferation and CD4+ cell-driven B-cell immunoglobulin
CALCITRIOL
MODULATES
HUMAN
MLR ACTIVITY
* **
407
pc0.03 p
1,25-Ds[M] 60 L!is 010’ 50 % .= 0 ‘E 0 5 40 E s
30
20
10
0 50
25
12.5
Effector to Target Cell Ratio FIG. 5. Inhibition of generation of cytotoxic cells by 1,25-D3. After a 7-day MLR culture in the presence or absence of 1,25-D3 ( 10m9to lo-’ M), the lymphocytes were used as effector cells against “0-labeled PHA-stimulated lymphocytes. Data represent the percentage cytotoxicity at effector to target ratios ranging from 5O:l to 12.5:1.
production are particularly sensitive to the action of the hormone. Theseresults suggest an inhibitory action of the metabolite on T-helper activity. To the contrary, CD8+ cells appear to be insensitive to the action of the hormone. Moreover, suppressor activity of CD8+ cells on immunoglobulin production was found to be increased in the presence of 1,25-D3 when exogenous recombinant human IL-2 was added to cultures ( 12). Effector MLR cultures generatemodulators with suppressorcell activity when added to a primary autologous MLR. However, when the effecters were cultured in the presence of increasing concentrations of 1,25-D3, they exerted enhanced suppressor activity with an additional 34.8% MLR inhibitory effect at 10m8A4 1,25-D3. This suppressoractivity was not antigen specific; the sameautologous effecters also exerted enhanced suppressor activity despite different stimulators. Heterologous effecters, as predicted, when added to a primary MLR exerted an enhancing effect resulting in increased proliferation. This enhanced MLR responsewas reduced when the effecters were generatedwith 1,25-D3; absolute MLR inhibition wasalso observedwhen identical stimulators were used (Fig. 3). Thus, 1,25-D3 results in the generation of nonspecific suppressor cells.
408
MEEHAN, KERMAN, AND LEMIRE TABLE 2 Phenotypic Analysis of 1,25-Ds-Treated Effector Cells Monoclonal antibody
Control
1,25-D3
Leu-5b (CD2) Leu-3a (CD4) Leu-2a (CD8) Leu-12 (CD19) TCR HLA-DR IL-2 Ta-IRD-1 Class I
90.5 62.4 26.1 6.1 84.1 21.2 21.7 17.2 100
87.5 59.9 27.5 9.1 83.0 13.3* 14.2 11.7 100
Note. Effector cells generated from a 7day MLR in the presence ( lOm8M) or absence of 1,25-D, were submitted for phenotypic analysis by cell sorting techniques (FACS). The data are expressedas percentage of cells positive for the respective antigens.
* P < 0.03.
The enhanced nonspecific suppressor-cell activity generated with 1,25-D3 was not the result of a selection of CD8+ lymphocytes since flow cytometric analysis of controls and 1,25-D3-treated effecters revealed no significant differences. However, 1,25-D3 reduced expression of activation markers on the effecters, including a significant reduction in MHC class II, while not affecting class I antigen expression. The active D3 metabolite has previously been shown to affect DR expression ( 13, 14). A decreasein the expression of class II antigens may contribute to a reduction of the allogenicity observed. The effect of the reduction of DR expression on suppressor cell activity is unclear. A predominant class I expression may allow for suppressor determinant exposure. Since an absolute increasein the number of suppressoreffecterscannot account for the marked inhibition of the MLR, a potential effect of 1,25-D3 on an “intermediate” cell in the suppressor cascadecannot be excluded. Despite its apparent preferential effect for CD4+ cells, 1,25-D, could exert a selective inhibition of certain CD4+ cells while allowing full expression of others such as the CD4+ helper-inducer of suppression or nonspecific macrophage suppressors. The MLC generation of T-cell cytotoxic activity was also reduced after incubation with 1,25-D3. The hormone was effective in preventing the development of effecters of cytotoxicity. No inhibition of CD8+ cells was observed in the presence of 1,25-Dj and it is possible that the CD4+ helper-inducer of cytotoxicity could have been targeted by the active metabolite. However, an earlier effect of 1,25-D3 on precursors of cytotoxic cells cannot be excluded. The mixed lymphocyte reaction represents the in vitro model of allograft response. Regulation of the MLR by 1,25-D3 suggestsa potential immunosuppressive role in alloimmune regulation. Indeed, use of the potent anti-rejection drug, cyclosporine, results in MLR responsessimilar to those described for the active vitamin D metabolite. Cyclosporine facilitates the induction of suppressor T cells by alloantigens ( 15). The suppression observed was found to be both antigen specific and antigen nonspecific. Moreover, the induction of cytotoxic T lymphocytes is completely inhibited by cyclosporine (15). The primary target of cyclosporine is the CD4+ cell. However, Hess et al. (16) have shown that in the presence of cyclosporine, the T-helper-inducer of
CALCITRIOL
MODULATES
HUMAN
MLR ACTIVITY
409
suppression could still be activated, suggesting a relative resistance of the suppressor T-cell pathway to cyclosporine. Thus, these results suggest a potential role for 1,25-D3 as an immunoregulatory agent for use in transplant and autoimmune patients (17). ACKNOWLEDGMENT This work was partially supported by National Institutes of Health Grant R29-DK-39024 (J.M.L.).
REFERENCES 1. 2. 3. 4. 5.
Tsoukas, C. D., Provvedini, D. M., and Manolagas, S. C., Science 224, 1438, 1984. Lemire, J. M., Adams, J. S., Sakai, R., and Jordan, S. C., J. Cfin. Invest. 74, 657, 1984. Rigby, W. F. C., Stacy, T., and Fanger, M. W., J. Clin. Invest. 74, 1451, 1984. Bhalla, A. K., Amento, E. P., Scrog, B., and Glimcher, L. G., J. Zrnmunol. 133, 1748, 1984. Lemire, J. M., Adams, J. S., Kermani-Arab, V., Bakke, A. C., Sakai, R., and Jordan, S. C., J. Immunol. 134,3032, 1985. 6. Sumiko, I., Takahashi, T., Kura, F., Sugiyama, H., and Hoshino, T., J. Immunol. 136,4427, 1986. 7. Provvedini, D. M., and Manalagas, S. C., J. Clin. Endocrinol. Metab. 68(4), 1989. 8. Vanham, G., Ceuppens, J. L., and Bonillon, R., Cell. Immunol. 124, 320, 1989. 9. Thomas, F. T., Lee, H. M., Lower, R. R., and Thomas, J. M., Surg. Clin. North Am. 59,253, 1979. 10. Kerman, R. H., Flechner, S. M., Van Buren, C. T., Lo&r, M. I., and Kahan, B. D., Transplantation 43, 205, 1987. 11. Kennan, R. H., Kimball, P. M., Van Buren, C. T., Lewis, R. M., and Kahan, B. D., Trunspluntation 51, 338, 1991. 12. Jordan, S. C., Lemire, J. M., Sakai, R. S., Toyada, M., and Adams, J. S., Mol. Immunol. 27(l), 95, 1990. 13. Rigby, W. F. C., Waugh, M., and Grozrano, R. F., Blood 76( 1), 189, 1990. 14. Carrington, M. N., Tharp-Hiltbold, B., Knoth, J., and Ward, F. E., J. Immunol. 140, 4013, 1988. 15. Hess, A. D., and Tutschka, P. J., J. Zmmunol. 124, 2601, 1980. 16. Hess, A. D., Donnenberg, A. D., Tutschka, P. J., and Santos, G. W., J. Immunol. 130,7 17, 1983. 17. Lemire, J. M., and Archer, D. C., J. Clin. Invest. 87, 1103, 1991.