CLINICAL IMMUNOLOGY AND IMMUNOPATHOLOGY 61,410-420 (1991)

In Vitro IgE Production by Interleukin 4-Stimulated Human

Peripheral Blood Mononuclear Cells Is Suppressed by Rapamycin 1 HONGYU

Luo,*

H U I F A N G C H E N , ~ PIERRE D A L O Z E , ~ JOSEPH C H A N G , ~ G I L L E S S T - L O U I S , * AND J I A N G P I N G W U *'2

*Laboratory of Nephrology and Transplantation Immunology and tLaboratory of Surgical Research, Notre-Dame Hospital Research Center, University of Montreal, Montreal, Quebec, Canada H2L 4311; and tWyeth-Ayerst Research, Princeton, New Jersey 08543-8000 Rapamycin (RAPA) is a new immunosuppressant which is 50-fold to 100-fold more potent than cyclosporin A (CyA) in inhibiting cellular immune responses and allograft rejection in animal models of organ transplantation. The drug's effect on in vitro IgE synthesis by interleukin (IL)4-stimulated human peripheral blood mononuclear cells was examined and compared with CyA's effect in this study. RAPA was found to be about 1000-fold more potent than CyA in inhibiting lgE synthesis. Its inhibitory effect on IgE production was significant if it was added to the culture before Day 6 of a 14-day culture. The suppression was accompanied by the inhibitory effect on cell proliferation and on IgE-binding factor (IgE-BF) production. IL2 was able to partially reverse CyA- but not RAPA-induced inhibition of IgE production. Commercial B cell growth factor (cBCGF) was not able to reverse either RAPA- or CyA-induced suppression of lgE synthesis. The strong inhibitory effect of RAPA in IgE synthesis may be useful in certain clinical applications where overproduction of pathogenic lgE is a key issue. RAPA can also be used as a tool to dissect the regulation of IgE production. 9 1991AcademicPress. Inc.

INTRODUCTION Rapamycin (RAPA) is a natural product similar in structure to FK506 (1). It was discovered in 1975 as an anti-fungal drug (2) and has attracted great attention recently because it is found to be several times more potent than cyclosporin A (CyA) in preventing allograft rejection (3, 4). Recent studies have strongly suggested that RAPA acts through a different mechanism from CyA and FK506 in that, unlike CyA or FK506, it does not inhibit the accumulation of the mRNA of early phase T cell activation genes such as interleukin (IL) 2, interferon (IFN)-/, and c-myc (5). Yet at the molecular level, RAPA and FK506 bind with similar affinity to a cis- and trans-rotamase termed FK binding protein (FKBP), and both are potent inhibitors of the rotamase (6). This paradox suggests that different mechanisms, in addition to inhibition of rotamase activity, are essential in explaining their immunosuppressive effects. For the humoral immune response, RAPA's inhibitory effect on IgE-like Ab production in rats and its inhibitory effect t This work was supported by a grant from the Cancer Research Society, Canada, to J. Wu. J. Wu is a scholar of Fonds de la Recherche en Sante du Quebec. 2 To whom correspondence and reprint requests should be addressed at Laboratory of Transplantation Immunology, Room 1-3159, Pavilion Deschamps, Notre-Dame Hospital, 1560 Sherbrooke St. East, Montreal, Quebec, Canada H2L 4MI. 410 0090-1229/91 $1.50 Copyright9 1991by AcademicPress. Inc. All rightsof reproductionin any formreserved.

RAPA INHIBITS lgE PRODUCTION

411

on mouse B cell activation have been documented (7, 8). We have also found recently that RAPA strongly suppresses hz vitro IgG, IgM, and IgA production by human lymphoeytes (9). In the present study, RAPA's inhibitory effect on in vitro-IL4-stimulated IgE production by human peripheral blood mononuclear cells (PBMC) was investigated and the underlying mechanisms discussed. MATERIALS AND METHODS

Reagents RAPA was a generous gift from Wyeth-Ayerst Research and CyA was from Sandoz. Human recombinant IL4 and IL2 were purchased from Genzyme Co. (Boston, MA) and commercial B cell growth factor (cBCGF) was from Cellular Products Inc. (Buffalo, NY). HB101 culture medium was purchased from Hana Biol. Inc (Berkeley, CA); RPMI 1640, fetal calf serum (FCS), penicillin, and streptomycin were from Flow Laboratories (McLean, VA); and Lymphoprep was from NYCOMED (Oslo, Norway). Affinity-purified goat antibodies to human ~/, Ix, or t~ chains were purchased from Sigma (St. Louis, MO) and peroxidaseconjugated affinity-purified goat anti-human ~/, Ix, or et chain-specific Ab were from TAGO (Burlingame, CA). All other reagents used were purchased from regular commercial suppliers and represented the best grade available.

Mononuelear Cell Preparation and Cuhure Conditions Peripheral blood mononuclear cells were isolated from heparinized blood of healthy volunteers by Lymphoprep gradients according to the manufacturer's instructions. Cells were cultured at 1.5 x 106 cells/ml/well in 48-well plates in HB101 medium supplemented with 5 mM L-glutamine, 5% FCS, and antibiotics. IL4 was used at 10 ng/ml, which was found to be optimal for IgE production in our system. After 12-14 days of culture, the IgE in the supernatants was measured.

Radioimmunoassays for IgE The assays were performed as described previously (10). Briefly, 96 polyvinyl microtiter plates (Dynatech, Alexandria, VA) were coated with mouse mAb specific to human IgE (clone 89). After blocking of nonspecific binding sites with 10% FCS in Hanks' solution, 75-Ixl samples were added to the wells and incubated overnight at room temperature. ,After washing, the wells were reacted overnight with a n/SI-labeled IgE-specific mAb (clone 4.15 from Dr. A. Saxon, UCLA). The results were expressed as the net IgE synthesis by subtracting the IgE levels measured in cycloheximide-treated parallel cultures from that measured in the testing samples.

ELISA for IgG, IgM, and IgA ELISA for IgG, IgM, and IgA were established according to a protocol described by Lipsky (11). Briefly, polystyrene microtiter plates (Maxisorb, Nunc) were coated with I Ixg/ml, 100 Ixl/well affinity-purified goat antibodies specific for ~/, Ix, or a chains in carbonate buffer (0.01 M, pH 9.5). After overnight incubation at room temperature, plates were blocked with 0.5% bovine serum albumin in

412

L U O ET A L .

carbonate buffer for 1.5 hr. After washing, the plates were incubated with samples (100 ~tl/well) for 1.5 hr at 37~ The plates were again washed, and peroxidaseconjugated affinity-purified Ab to % I~, or c~ chains (I:10,000 dilution in phosphate-buffered saline, I00 rd/well) were added. After a 2-hr incubation at 37~ the plates were washed, and 100 ixl/well of enzyme substrate (O-phenylenediamine, 0.4 mg/ml in phosphate-citrate buffer containing 0.4%, v/v, H2Oz) was added. After 20 min, the reaction was terminated with 50 p.l 2.5 M HCI, and samples were read at 492 nM with an ELISA reader. The sensitive ranges of the ELISA were 2 to 125 ng/ml for IgG and IgA, and 4 to 250 ng/ml for IgM. The specificity of the assays was verified in that up to 500 ng/ml of irrelevent isotypes of Ab had only less than 2% cross-reactivity with the isotype tested. RIA for lge-BF Ige-BF was measured by RIA as described previously (I0). The sensitivity of the assay was 2 U/ml. One unit was the equivalent of 150 pg protein. Assays for [3H]thymidine uptake [3H]Thymidine uptake was used as an indicator for cell proliferation. Total PBMC were cultured at 2 • 105 cells/well in 96-well plates. Cells were pulsed with [3H]thymidine (0.5 i.tCi/well) for 6 hr before harvest, and radiolabeled thymidine uptake was measured in a scintillation counter. RESU LTS

RAPA Inhibits IL4-sthnulated IgE Production by PBMC Inasmuch as RAPA showed a strong inhibitory effect on both T and B cell activities (8, 9, 12), we investigated its effect on IL4-stimulated IgE production, which is a T cell-dependent process (13). Both RAPA and CyA inhibited IgE production by PBMC in a dose-dependent fashion (Fig. 1A). In a typical 14-day culture, 1 nM RAPA suppressed 95% of the IgE production and I i.tM CyA suppressed 97%. RAPA was about 1000 times more potent than CyA in causing 50% inhibition of maximal IgE production in this model. RAPA also dose dependently suppressed other isotypes of Ig in this system in the presence of IL4 (Fig. 1B). At 1 nM, RAPA suppressed 75, 90, and 58% of IgG, IgM, and IgA production, respectively. We also examined RAPA's effect on the production of the IgE-binding factor, which has been reported to be involved in the regulation of IgE synthesis (14). IgE-BF synthesis was enhanced 10-fold by IL4, from 84 to 982 U/ml in a 14-day culture of PBMC. RAPA suppressed the IL4-enhanced IgE-BF dose dependently, and at 1 riM, the suppression was 45% (Fig. 2). Kinetics of RAPA 's Inhibitory Effect on IgE Production To examine the kinetics of RAPA and CyA's effect on IgE production, 1 nM RAPA or I FtM CyA was added to the culture at Days 0, 2, 4, 6, 8, and 10. Supernatants were collected at Day 14 and IgE was measured. RAPA had its strongest inhibitory effect When added at Day 0. This effect was, however, gradually lost when the addition of the drug was delayed until Days 8--10 (Fig. 3). CyA

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FIG. 1. Dose-dependent suppression exerted by RAPA or CyA on IgE, IgG, IgM, and IgA synthesis by IL4-stimulated PBMC. PBMC (1.5 x 106/ml) were cultured for 14 days in the presence of IL4 (10 ng/ml). Various concentrations of RAPA or CyA were added at the beginning of the culture. Supernatants were harvested on Day 14 for the quantitation of IgE (A) and for IgG, IgM, and IgA (B). In (A), the cultures without and with IL4 produced

In vitro IgE production by interleukin 4-stimulated human peripheral blood mononuclear cells is suppressed by rapamycin.

Rapamycin (RAPA) is a new immunosuppressant which is 50-fold to 100-fold more potent than cyclosporin A (CyA) in inhibiting cellular immune responses ...
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