Discrete Signaling Pathways Dendritic Cell-Like Characteristics Through

Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic Cell-Like Characteristics Through Discrete Signaling Pathways This information is current as of September 27, 2021. Gary K. Koski, Gretchen N. Schwartz, David E. Weng, Brian J. Czerniecki, Charles Carter, Ronald E. Gress and Peter A. Cohen J Immunol 1999; 163:82-92; ; http://www.jimmunol.org/content/163/1/82 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Calcium Mobilization in Human Myeloid Cells Results in Acquisition of Individual Dendritic Cell-Like Characteristics Through Discrete Signaling Pathways

Gary K. Koski,1* Gretchen N. Schwartz,* David E. Weng,2* Brian J. Czerniecki,† Charles Carter,‡ Ronald E. Gress,* and Peter A. Cohen3*

We have shown previously that calcium ionophore (CI) treatment of various myeloid origin cells results in rapid acquisition of properties associated with mature, activated dendritic cells. These properties include increased CD83 and costimulatory molecule expression, tendencies to form dendritic processes, loss of CD14 expression by monocytes, and typically an enhanced capacity to sensitize T lymphocytes to Ag. We here analyze the intracellular signaling pathways by which CI induces acquisition of such 2؉ properties. Thapsigargin, which raises intracellular Ca levels by antagonizing its sequestration, induced immunophenotypic and Downloaded from morphologic changes that paralleled CI treatment. CI-induced activation was broadly attenuated by the Ca2؉ chelating compound EGTA and by calmodulin antagonists triﬂuoperazine dimaleate and W-7. However, antagonists of signaling pathways downstream to calmodulin displayed more selective inhibitory effects. Calcineurin antagonists cyclosporin A and the FK-506 analogue, ascomycin, diminished costimulatory molecule and CD83 expression, as well as formation of dendritic processes in CI-treated myeloid cells, and strongly attenuated the T cell allosensitizing capacity of CI-treated HL-60 cells. These calcineurin antagonists displayed minimal effect on CI-induced CD14 down-regulation in monocytes. In contrast, the calmodulin-dependent protein kinase antag- http://www.jimmunol.org/ onists, K252a and KT5926, while displaying only modest effects on CI-induced costimulatory molecule and CD83 expression, -strongly blocked CD14 down-regulation. These results are consistent with a Ca2؉-dependent mechanism for CI-induced differ entiation of myeloid cells, and indicate that multiple discrete signaling pathways downstream to calcium mobilization and calmodulin activation may be essential in regulating this process. The Journal of Immunology, 1999, 163: 82–92.

n previous studies, we demonstrated that the addition of cal- The fact that a single class of pharmacologic agents induced the cium ionophore (CI)4 to human peripheral blood monocytes coordinate expression of a number of surface proteins associated or immature dendritic cells (DC) resulted in the acquisition of with an activated APC phenotype, additionally promoting remark- I by guest on September 27, 2021 many immunophenotypic, morphologic, and functional properties able functional and morphologic changes, suggested the existence characteristic of activated myeloid DC (1). These included the rap- of an important, previously uncharacterized, signaling pathway op- idly (within 20 h) up-regulated expression of CD80, CD86, CD83, erative in many myeloid cells. The elucidation of this pathway is CD40, and CD54 (ICAM-1), the loss of CD14 expression by critical for determining how myeloid-lineage cells at many stages monocytes, and the later (48–96 h) acquisition of dendritic pro- of ontogeny may rapidly respond to their environment by acquir- cesses, as well as a capacity to sensitize T lymphocytes to Ag at ing properties typically associated with activated DC and should efficiencies equivalent to that of purified, activated DC (1). We provide clues to the regulation, at the molecular level, of a wide have also shown that CI induced many similar immunophenotypic ϩ variety of genes associated with APC function. and morphologic effects in 6-day cultured human CD34 bone mar- In the studies presented here, we used well-characterized phar- row cells, the promyelocytic leukemia line, HL-60, and chronic my- macologic antagonists that block Ca2ϩ-mediated signaling path- elogenous leukemia cells freshly obtained from patients (2). ways at defined points to elucidate the mechanisms by which CI promotes the acquisition of particular DC-associated properties in *Medicine Branch, National Cancer Institute, Bethesda, MD 20892; †Department of a wide variety of human myeloid cells, including both transformed Surgery, University of Pennsylvania Medical Center, Philadelphia, PA 19104; and (leukemia) cells and nontransformed cells at various stages of on- ‡Department of Transfusion Medicine, Warren Grant Magnuson Clinical Center, Na- tional Institutes of Health, Bethesda, MD 20892 togeny (monocytes and cultured bone marrow progenitors). Strat- Received for publication August 24, 1998. Accepted for publication April 8, 1999. egies employing these agents have similarly been used by others to characterize Ca2ϩ-dependent signaling pathways in both T cell The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance and myeloid systems (3–5). The human acute myeloid leukemia with 18 U.S.C. Section 1734 solely to indicate this fact. line HL-60, previously shown to mimic many of the responses to 1 Address correspondence and reprint requests to Dr. Gary K. Koski, Medicine CI observed in normal monocytes, proved of particular value for Branch, National Cancer Institute, Building 10, Room 12N226, 9000 Rockville Pike, Bethesda, MD 20892. E-mail address: [email protected] or [email protected] its ability to withstand prolonged exposure to certain drug combi- 2 nations. We also performed, for purposes of confirmation, limited Current address: Hematology/Oncology Department, Cleveland Clinic Foundation, ϩ 9500 Euclid Avenue, Cleveland, OH 44195 studies on 6-day cultured CD34 bone marrow progenitor cells, 3 Current address: Center for Surgery Research, FF50, Cleveland Clinic Foundation, because a large subpopulation of these cells acquires a number of 9500 Euclid Avenue, Cleveland, OH 44195 DC characteristics upon exposure to CI (2). 4 Abbreviations used in this paper: CI, calcium ionophore; DC, dendritic cell; CsA, Among the acquired DC characteristics we examined in these cyclosporin A; CM, culture medium; TpD, trifluoperazine dimaleate; CaMK, calmodulin-dependent protein kinase(s), Chel Cl, chelythrine chloride; rh, recombinant studies are the enhanced surface expression of costimulatory mol- human. ecules B7.1 (CD80) and B7.2 (CD86), which exist on the surface

of matured DC and certain other APC as counterreceptors for from these donors were studied under a variety of culture conditions. Pre- CD28 and CTLA-4 on T lymphocytes. We also focused on the liminary experiments established that maximum viability was achieved enhanced expression of CD83, a surface protein of undetermined during pharmacologic inhibitor studies by modifying our prior culture method (1) as follows: monocytes were plated in serum-free medium function whose expression is strongly associated with DC activa- (Macrophage-SFM; Life Technologies, Grand Island, NY) with 50 ng/ml tion/differentiation. Where technically practicable, we examined added rhGM-CSF (H. Nguyen et al., manuscript in preparation), aliquoting additional characteristics of DC including cellular morphology and cells at 3 ϫ 106 or 5 ϫ 106 per well in 24-well cluster plates (Costar). The APC function. The similar effects observed in each myeloid cell monocytes were exposed to optimal doses of the inhibitory agents over- 2ϩ night before CI coexposure. The optimal activating dose of CI A23187 was type is consistent with the presence of a Ca -triggerable activa- 150 ng/ml for monocytes plated at 3 ϫ 106 cells/well and 225 ng/ml for tion/differentiation pathway with common elements observed monocytes plated at 5 ϫ 106 cells/well. Finally, the monocytes were har- among the cell types tested. This pathway appears to use a pivotal vested within 20 h after addition of CI to provide maximum viability dur- calmodulin/calcineurin/calmodulin-dependent kinase axis for ing analysis. Qualitatively similar results, but typically with lower viabil- transmission of its activation signal through the cytoplasm, with ity, were observed in other media (e.g., RPMI 1640 with 5% human AB serum (1)), in the absence of rhGM-CSF, when CI treatment was initiated some CI-induced characteristics proving more sensitive to cal- sooner after addition of the inhibitory agents, or when CI treatment was cineurin antagonists such as cyclosporin A (CsA), and others more continued in the presence of the inhibitory agents beyond 20 h (not shown). sensitive to antagonists of calmodulin-dependent protein kinase(s) ϩ Culture and evaluation of human CD34 cells (CaMK). This bifurcating pathway thus appears to share some fea- ϩ tures with the putative Ca2ϩ-dependent arm of the cellular activa- Normal donor bone marrow cells enriched for CD34 cells (purity 91– tion pathway in T lymphocytes (3, 4) and also suggests the pos- 98%) were obtained from Poietic Technologies (Gaithersburg, MD) or from cadaveric vertebral bodies by positive immunomagnetic selection us- sibility that clinically important immunosuppressive calcineurin ing a CD34 isolation kit (Miltenyi Biotech, Auburn, CA). To promote Downloaded from antagonists such as CsA may interfere significantly in certain cir- enrichment and expansion of intermediate DC precursors (9, 10), CD34ϩ cumstances with the aquisition of activated DC characteristics cells were resuspended to 4 ϫ 104 cells/ml and cultured for 6 days in an in vivo. enriched IMDM (11) supplemented with 10% FCS (HyClone, Logan, UT), 20 ng/ml rh-c-kit ligand, 10 ng/ml rhGM-CSF, and 10 ng/ml rhTNF-␣; fresh medium and factors were added on day 3. After the 6-day expansion Materials and Methods and enrichment phase, cells were harvested, washed, and replated in 48- ϫ 5 Reagents and Ab well cluster plates (Costar) at 5 10 cells/well in fresh medium contain- http://www.jimmunol.org/ ing rhGM-CSF and rhTNF-␣ but not rh-c-kit ligand. Then, 45 min after CI A23187 was obtained from Sigma (St. Louis, MO). Thapsigargin, replating, CsA was variably added, and an optimal activating dose of CI EGTA, trifluoperazine dimaleate (TpD), W-7, chelythrine chloride (Chel (375 ng/ml) was variably added 45 min later. Wells were harvested 20 h Cl), okadaic acid, ascomycin, KT5926, KT5720, KT5823, and K252a were later for analysis. obtained from Calbiochem (La Jolla, CA). CsA was obtained from Sandoz (East Hanover, NJ). These agents were chosen for their established spec- Abs and FACS analysis ificity in open-cell systems and determined low toxicity in closed-cell systems. Exhaustive dose-response titrations were performed for each agent in The methods employed and the Ab reagents used against CD80, CD86, our closed-cell myeloid cultures to determine the dose range in which CD14, ICAM-1 (CD54), CD40, and CD1a were identical to previous pub- toxicity was low and phenotypic modulations were reproducibly observed. lished reports (1) except that PE-conjugated anti-CD83 Ab (Coulter) was substituted for indirect staining. Where available (see Results), drug analogues with identical toxicity pro- by guest on September 27, 2021 files but different enzymatic specificities were examined to enhance the Photomicroscopy interpretation of data. (3–8). All biochemical reagent stocks were stored at Ϫ70°C before use, and individual aliquots were thawed only once imme- Cells to be analyzed were resuspended in fresh medium and transferred to diately before culture addition. In some experiments, cultures were sup- Lab-Tek 8 glass chamber slides (Nunc, Naperville, IL) previously coated plemented with the following recombinant human (rh) cytokines: rh-c-kit with 1% polylysine (Sigma) and incubated for 30 min at 37°C, 5% CO2 (1). ligand (sp. act. 2.7 ϫ 105 U/mg), rhTNF-␣ (sp. act. 1.1 ϫ 108 IU/mg), and Slides were then examined and photographed with an Olympus IX-70 in- rhIL-4 (sp. act. 2.9 ϫ 107 IU/mg) (all R&D Systems, Minneapolis, MN), verted microscope using Nomarski differential interference contrast optics. and rhGM-CSF (sp. act. 5.6 ϫ 106 IU/mg (Amgen, Thousand Oaks, CA). Some preparations were fixed in absolute ethanol and Wright’s stained before photography. Culture and evaluation of HL-60 Allosensitization studies The human promyelocytic leukemia-derived cell line, HL-60 (CCL 240) was obtained from American Type Culture Collection (Manassas, VA) and Human T lymphocytes obtained from lymphocyte-rich elutriation fractions maintained as described previously (2) in RPMI 1640 with 10% FCS in were purified using T cell isolation columns (R&D Systems) (1). HL-60 ␮ exponential growth by serial passage in 162-cm2 tissue culture flasks cells preincubated with or without CsA (0.05 g/ml) were subjected to CI (Costar, Cambridge MA). To evaluate the effects of particular biochemical (180 ng/ml) for 72 h and harvested. To ensure that any observed inhibitory agents, HL-60 cells were washed and seeded in fresh culture medium (CM) effects of CsA resulted from direct inhibition of HL-60 cells, rather than into 24-well tissue culture plates (Costar) at a density of 0.5 ϫ 106 cells/ from CsA “carryover” into T cell coculture, positive control HL-60 cells well (2 ml total volume). The following day, calcium-mobilizing agents treated with CI for 72 h without CsA were briefly exposed at the time of ␮ A23187 (previously optimized dose range of 180–375 ng/ml) or thapsi- harvest to an equivalent dose of CsA (0.05 g/ml) and then immediately gargin were variably added to stimulate cultures; in experiments evaluating washed five times in CsA-free CM in parallel with all other treatment the effects of other agents on CI treatment, the agents were added to culture groups. This procedure normalized the amount of trace CsA carried over 45 min before calcium-mobilizing agents. All pharmacologic agents used into T cell coculture for both control and test groups. HL-60 cells were then ϩ ␥ to either induce or to inhibit Ca2 -dependent activation of myeloid cells -irradiated to 30 Gy and cocultured with T lymphocytes in 96-well flat- were exhaustively dose-titrated from ranges of high activity to extinction of bottom plates (Costar, Cambridge MA). The T cell number was held con- ϫ 5 activity. Doses reported in Results represent optimized concentrations that stant at 1 10 cells/well while the irradiated HL-60 cells were added to manifested the highest activities at minimal to absent toxicities during the each well in graded numbers. Cocultures were incubated for 96 h, then pulsed with 1 ␮Ci/well [3H]TdR and harvested and counted 18 h later. specified time in culture, bearing in mind the previously established IC50, and/or the concentrations previously used by others in closed-cell systems to obtain inhibiton of enzyme targets. Cells were maintained in the pres- Results ence of inhibitors for the entire culture period. Cells were harvested for Treatments that modulate biologic availability of intracellular FACS analysis at 20 h and later time points. Ca2ϩ or inhibit the regulatory protein calmodulin broadly affect Culture and evaluation of human peripheral blood monocytes HL-60 expression of costimulatory, activation, and adhesion molecules Human CD14ϩCD33ϩ peripheral blood monocytes from six healthy vol- unteers (typically 95% purity) were prepared by leukapheresis and elutria- We had previously demonstrated (2) that treatment of the acute tion and cryopreserved as described previously (1). Thawed monocytes promyelocytic leukemia line HL-60 with optimized doses of CI 84 CALCIUM SIGNALING PATHWAY ANTAGONISTS BLOCK MYELOID CELL ACTIVATION

FIGURE 1. Thapsigargin treatment elevates costimulatory, adhesion, and activation molecule surface expression in HL-60 cells. HL-60 cells were plated at 0.5 ϫ 106 per well in 2.0 ml CM in 24-well tissue culture plates and cultured overnight before the addition of A23187 (180 ng/ml) or thapsigargin (250 nM). Cells were harvested 20 h later, stained with PE-conjugated mouse control (light line), anti- human CD80, CD86, CD54 (ICAM-1), or CD83 Ab (heavy line), and subjected to FACS analysis. Data is representative of three experiments.

A23187 (188–375ng/ml) led to the rapid up-regulation of surface To clarify the role of calcium mobilization in CI-induced dif- CD80, CD86, CD54, and CD83 within 20 h (see Figs. 1 and 2). ferentiation, we began the present studies by examining the effects During this culture period, CI-induced increased expression of nu- of various calcium-dependent signaling pathway agonists and an- Downloaded from clear-localized RelB was also observed (L. Lyakh et al., manu- tagonists. When, instead of CI treatment, HL-60 cells were ex- script in preparation). At later time points (40–96 h), additional posed to an optimized dose of thapsigargin (250 nM), a compound CI-induced modulations occurred, including the enhanced expres- which raises intracellular Ca2ϩ concentrations by impeding its se- sion of CD40, CD1a, the formation of dendritic processes, and an questration (5, 12), the cells also up-regulated their expression of enhanced capacity to allosensitize T cells (2) (see Figs. 3, 4, and 5). CD80, CD86, CD54, and CD83 (Fig. 1) and furthermore acquired http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 2. Pharmacologic inhibition of short-term A23187-induced surface expression of CD80, CD86, and CD83. HL-60 cells were plated at 0.5 ϫ 106 per well in 2.0 ml CM in 24-well tissue culture plates and cultured overnight before the addition of various inhibitors. EGTA (1 mM), TpD (15 ␮M), W-7 (40 ␮M), K252a (0.25 ␮M), okadaic acid (10 nM), ascomycin (0.005 ␮g/ml), and CsA (0.5 ␮g/ml) were added 45 min before the addition of A23187. Cells were maintained in culture for 20 h, harvested, stained with PE-conjugated mouse control (light line), anti-human CD80, CD86, or CD83 (heavy line), and subjected to FACS analysis. Data is representative of a minimum of three experiments for each agent. The Journal of Immunology 85

FIGURE 3. Inhibition by CsA of long-term A23187-induced surface expression of CD80, CD86, CD54, CD40, and CD1a. HL-60 cells were plated at 0.5 ϫ 106 per well in 2.0 ml CM in 24-well tissue culture plates and cultured overnight. CsA (0.5 ␮g/ml) was added to some HL-60 groups before the addition of A23187 (260ng/ml). Cells were maintained in culture for an additional 96 h, harvested, and stained with PE-conjugated mouse control (light line), anti-human CD80, CD86, CD54, CD40, or CD1a (heavy line) Abs and subjected to FACS analysis. Data is representative of three experiments. Downloaded from dendritic processes (Fig. 4). In related studies, HL-60 cells prein- Antagonists of protein phosphatase 2B (calcineurin) strongly cubated with the Ca2ϩ chelating compound, EGTA (1 mM), inhibit immunophenotypic, morphologic, and functional proved largely refractory to the CD80, CD86, and CD83 up-reg- activation of HL-60 induced by calcium-mobilizing agents ulating effects of A23187 (Fig. 2), as were HL-60 cells treated An enzyme target of the regulatory protein calmodulin is the 2ϩ http://www.jimmunol.org/ with A23187 in the presence of Ca -free medium (not shown). serine-threonine protein phosphatase, calcineurin (PP2B) (15). These data were consistent with the hypothesis that elevated 2ϩ Two compounds which inhibit the activity of calcineurin are the intracellular Ca was an essential initiating event in CI-induced well-characterized immunosuppressive agents, cyclosporin A and myeloid activation/differentiation. Because increases in intracellu- the FK-506 analogue, ascomycin (3, 5, 16); inhibition is achieved lar Ca2ϩ levels in turn positively affect the activity of the regula- via complexes these agents form with cellular proteins (cyclophilin tory protein calmodulin (13), we next used several compounds and FK506-binding protein, respectively). Therefore, we tested the commonly used to antagonize calmodulin activity in both open and ability of these drugs to interfere with the CI-induced expression of closed-cell systems (3, 6, 7, 14) to look for evidence of calmod- DC-associated surface protein in HL-60 cells and compared these ulin’s role in the CI-induced expression of DC-associated surface agents to okadaic acid, a compound which has little activity against by guest on September 27, 2021 proteins in HL-60 cells. Preincubation of HL-60 cells with either calcineurin, but instead inhibits protein phosphatases 1 (IC ϭ an optimized dose of TpD (15 ␮M; IC ϭ 13 ␮M) or W-7 (40 50 50 10–20 nM) and 2A (IC ϭ 0.1 nM). Preincubation with 10 nM ␮M; IC ϭ 28 ␮M), proved to strongly attenuate the expression 50 50 okadaic acid had virtually no effect on the CI-induced expression of CD80, CD86, and CD83 resulting from 20-h treatment with CI of CD80, CD86, or CD83 in HL-60 cells after 20 h CI treatment (Fig. 2). (Fig. 2). On the other hand, ascomycin caused an almost total Longer exposure of HL-60 cells to calcium-mobilizing agents ablation of CI-induced expression of CD80, as well as strong in- plus EGTA, TpD, or W-7 proved excessively toxic, preventing the hibition of CD86 and CD83 expression at 20 h (Fig. 2). The second meaningful study of these agents in morphologic and functional calcineurin antagonist, CsA, had virtually identical effects at 20 h assays requiring analyses at time points beyond 20 h of culture (not (not shown). Because, in contrast to EGTA and calmodulin inhib- shown). itors, optimized doses of calcineurin antagonists were not notably toxic to HL-60 during longer culture periods, we next tested the Antagonists of CaMK modestly inhibit CI-induced expression of capacity of CsA to block CI-induced alterations that are not ob- costimulatory molecules and CD83 served until several days posttreatment, as well as CsA’s ability to Calmodulin is known to regulate the activity of members of a sustain inhibition of otherwise early appearing immunophenotypic family of calmodulin-dependent serine-threonine protein kinases characteristics. HL-60 cells pretreated for 45 min with CsA before (CaMK I, II, and IV/Gr). We looked for evidence that CaMK(s) a 96-h exposure to A23187 not only remained inhibited in their participated in CI-induced up-regulation of surface expression of expression of CD80, CD86, and CD54, but also failed to express costimulatory molecules and CD83 on HL-60 cells using the de novo CD40 and CD1a, which normally appear after 72–96 h ϭ CaMK inhibitor K252a (IC50 2.8 nM) (8). K252a had a very exposure to CI (Fig. 3) (2). modest yet consistent capacity to inhibit CI-induced up-regulation We also investigated the effect of CsA on acquisition of mor- of CD80, CD86, and CD83 (Fig. 2). A second CaMK inhibitor, phologic features characteristic of DC induced by calcium-mobi- ϭ KT5926 (IC50 5.9 nM) (4, 8), had similar modest effects on the lizing agents. As reported previously, untreated HL-60 cells cul- expression of these molecules (not shown). In contrast to EGTA tured for 72 h and transferred onto polylysine-coated glass slides and calmodulin inhibitors, optimized doses of CaMK inhibitors displayed no dendritic processes (Fig. 4A); the addition of CsA were not notably toxic to HL-60 during longer culture periods; alone (0.5 ␮g/ml) throughout the culture period had no apparent nonetheless, they did not block the CI-induced acquisition of den- effect (Fig. 4B). In contrast, as reported previously, cells treated dritic processes apparent at the end of such cultures (not shown). with an optimal dose of A23187 (180 ng/ml) displayed pro- These results suggested that calmodulin-dependent kinase(s) may nounced morphologic alterations, with numerous cellular pro- play only a subsidiary role in the particular modulations observed cesses typical of activated DC (Fig. 4C). However, when HL-60 when HL-60 is treated with calcium-mobilizing agents. cells were preincubated with CsA for 45 min before the addition of 86 CALCIUM SIGNALING PATHWAY ANTAGONISTS BLOCK MYELOID CELL ACTIVATION

FIGURE 4. CsA strongly inhibits acquisition of dendritic processes by HL-60 cells treated with calcium- mobilizing agents. HL-60 cells were plated in 2.0 ml

CM in 24-well tissue culture plates. Untreated cells (A), Downloaded from cells treated with 0.5 ␮g/ml CsA only (B), cells treated with an optimized dose of A23187 (180 ng/ml) (C), or cells treated with 0.5 ␮g/ml CsA for 45 min before the addition of A23187 (D) were cultured an additional 72 h. Then, cells were harvested, resuspended in fresh CM, and transferred onto polylysine-coated glass chamber slides. After 30 min incubation at 37°C, cells were http://www.jimmunol.org/ examined by Nomarsky differential interference contrast microscopy and photographed. HL-60 cells left untreated (E), treated with 250 nM thapsigargin (F), or treated with thapsigargin in the presence of 0.5 ␮g/ml CsA (G) were similarly processed, with the exception that these cells were ethanol-ﬁxed and Wright’s stained before photography. Data is representative of three experiments for CsA and two experiments for

thapsigargin. by guest on September 27, 2021

CI, such striking morphologic changes were virtually absent (Fig. for 72 h in the presence or absence of 0.05 ␮g/ml CsA, the lowest 4D). Similar CsA-blockable effects were seen when dendritic mor- dose of this drug in dose-titration studies found to give maximal phology was induced in HL-60 with thapsigargin (Fig. 4, E–G). sustained inhibition of HL-60 cells (not shown). After rigorous Our previous studies demonstrated that CI treatment of HL-60 washing and irradiation, treated HL-60 cells were tested for their increased its allosensitizing capacity by up to 25-fold, although on capacity to induce normal donor T lymphocyte alloproliferation. a per cell added basis CI-treated HL-60 remained less allostimu- As demonstrated previously (2), untreated HL-60 displayed a neg- latory than CI-treated normal monocytes, consistent with the ab- ligible capacity to allosensitize T lymphocytes (Fig. 5). In contrast, errant MHC regulation often observed in this type of leukemia (2, HL-60 cells treated with CI demonstrated markedly enhanced al- 17–19). Such dysregulation could be corrected partially by com- losensitizing capacity. However, coexposure to CsA during CI bining CI treatment with adjunct rhGM-CSF and rhIFN-␥, further treatment virtually abolished the enhanced allosensitizing capacity tripling HL-60’s allosensitizing capacity (2). In the present studies, conferred by CI treatment. The observed inhibition was not attrib- we examined the functional impact of CsA on HL-60 cells treated utable to carryover of CsA from the antecedent HL-60 culture into with CI alone. HL-60 cells were either untreated or treated with CI the T cell coculture (see Materials and Methods). The Journal of Immunology 87

determined to cause optimal overnight activation of day 6 cultured CD34ϩ cells (2). CD34ϩ cells harvested after the initial 6-day culture were het- erogeneous with respect to the expression of both costimulatory molecules and CD83, with only a small minority of cells displaying high expression of CD80, CD83, or dendritic processes (2). This phenotypic distribution was unchanged after replating and overnight culture in the absence of CI, whether or not CsA was included, indicating that CsA treatment per se did not reverse pre- ceding differentiation events (Figs. 6 and 7). However, as reported previously, overnight CI treatment (in the absence of CsA) caused cells to develop near uniform high expression of CD80 and CD86, with a majority of cells also acquiring strong CD83 expression and dendritic processes (Figs. 6 and 7). The addition of CsA markedly FIGURE 5. CsA attenuates A23187-induced enhancement of HL-60 al- inhibited CI-enhanced surface expression of CD80 and CD86, with losensitizing capacity. HL-60 cells were either left untreated (negative con- an apparent, though more modest, inhibition of CD83 up-regula- trol), cultured in the presence of an optimized activating dose of 180 ng/ml tion and dendritic process acquisition (Figs. 6 and 7). A23187 (positive control), or cultured in the presence of 180 ng/ml A23187 plus 0.05 ␮g/ml CsA and harvested 72 h later. This minimum Downloaded from effective dose of CsA was used (rather than the usual 0.5 ␮g/ml) to min- Calcium pathway antagonists downstream to calmodulin have imize carryover of CsA into T cell coculture. To normalize for the potential discrete effects on costimulatory molecule and CD83 vs CD14 effects of such CsA carryover, positive control HL-60 cells were spiked at regulation in peripheral blood monocytes the time of harvest with 0.05 ␮g/ml CsA, and all treatment groups immediately washed five times in 10 ml CM. Following irradiation (30 Gy), As previously reported, treatment of normal peripheral blood graded numbers of HL-60 cells were cocultured in 96-well flat-bottom monocytes with CI induces many phenotypic modulations typical ϫ 5 of activated myeloid DC (1). Therefore, we tested the effects of plates with a fixed number (1 10 ) of MHC nonmatched purified human http://www.jimmunol.org/ CD4ϩ T cells. Cells were cocultured for 96 h before the addition of 1 individual pharmacologic agents on CI-treated normal human pe- ␮Ci/well [3H]TdR, harvested 18 h later, and analyzed by liquid scintilla- ripheral blood monocytes to determine whether their response tion spectroscopy. Error bars represent SEM from triplicate wells. Data is would parallel those observed in HL-60 cells. Because, in contrast representative of three experiments. to HL-60 and bone marrow cells, monocytes constituitively and uniformly express high levels of CD14, down-regulation of CD14 expression induced by CI treatment could also be analyzed in this Effects of calcineurin antagonist CsA on normal cultured ϩ population (1). CD34 cells parallel those observed in HL-60 As for HL-60, CI activation of monocytes proved to be calcium-

To determine whether the marked effects of calcineurin antagonists dependent. Up-regulation of costimulatory molecule expression by guest on September 27, 2021 on CI-treated HL-60 cells extended to normal myeloid progenitors, and CD14 down-regulation were markedly impaired when CI we investigated CsA’s effects on cultured CD34ϩ cells, which also treatment of monocytes was conducted in Ca2ϩ-free medium (not experience rapid immunophenotypic and morphologic activation shown). As similarly observed in HL-60 (Fig. 1), thapsigargin during CI exposure (2). Following initial harvest from healthy vol- treatment, which raises intracellular Ca2ϩ levels by antagonizing unteer bone marrow, puriﬁed CD34ϩ cells were cultured for 6 its sequestration, resulted in increased monocyte expression of days in rhGM-CSF, rhTNF-␣, and rh-c-kit ligand, then replated in CD80, CD86, CD54, and CD83, as well as down-regulated CD14 fresh medium identical except for the absence of c-kit ligand. At expression, similar to modulations induced by CI treatment (not the time of replating, individual groups were either untreated or shown). Such studies conﬁrmed that calcium mobilization can treated with a dose of CsA determined to be optimal in HL-60 serve as a profound differentiating stimulus for monocytes. How- studies; 45 min later, individual groups were either untreated or ever, concomitant exposure of monocytes both to CI and to cal- treated for 20 h with a dose of CI A23187 (375 ng/ml) previously cium signaling pathway antagonists proved to be more toxic to

FIGURE 6. CsA inhibits enhanced expression of CD83 and costimulatory molecules in A23187-treated CD34ϩ bone marrow cells. CD34ϩ bone marrow cells expanded in culture for 6 days in the presence of cytokines were harvested and recultured in 24-well tissue culture plates (see Materials and Methods). Cells were treated or untreated for with 0.5 ␮g/ml CsA for 45 min before the addition of 375 ng/ml CI A23187. Cells were cultured for an additional 20 h, harvested, stained with PE-conjugated mouse control (light line) or anti-human CD80, CD86, or CD83 Abs (heavy line), and analyzed by FACS. Data is representative of three experiments. 88 CALCIUM SIGNALING PATHWAY ANTAGONISTS BLOCK MYELOID CELL ACTIVATION

FIGURE 7. CsA modestly inhibits A23187-induced tendency to acquire dendritic processes in normal myeloid progenitors. Day 6 cultured CD34ϩ bone marrow cells (see Methods and Materials) were plated in 1.0 ml CM in 48-well tissue culture plates. Untreated cells (A), cells treated with 0.5 ␮g/ml CsA only (B), cells treated with an optimized dose of A23187 (375 ng/ml) (C), or cells treated with 0.5 ␮g/ml CsA for 45 min before the addition of A23187 (D) were cultured an additional 20 h. Then, cells were harvested, resuspended in fresh CM, and transferred onto polylysine-coated glass chamber slides. After 30 min incubation at 37°C, cells were examined by Nomarski differential interference contrast microscopy and photographed. Data is representative of two experiments. Downloaded from

monocytes than to the HL-60 cell line. Therefore, it was necessary CD83 expression (Figs. 8 and 9). Strikingly, however, CI-induced

to modify our existing monocyte culture system to improve via- CD14 down-regulation was inhibited by the calmodulin antago- http://www.jimmunol.org/ bility during multiagent exposure. nist, W-7, but minimally affected by the calcineuin antagonist This proved possible by substituting serum-free medium for hu- CsA. In contast, the CaMK antagonist KT5926, like W-7, mark- man serum-containing medium (1). In either culture condition, CI edly inhibited the CD14 down-regulation induced by CI (Fig. 8). treatment induced monocytes to up-regulate CD80, CD86, and Unlike W-7, KT5926 had little or no effect on CI-induced expres- CD83, as well as markedly down-regulate CD14, within 20 h of CI sion of CD80, CD86, or CD83 (Fig. 8). Structurally related com- exposure. However, several differences were observed: 1) a lower pounds with reduced activity against CaMK (the protein kinase G concentration of CI proved optimal for activation in serum-free antagonist KT5823 and the protein kinase A antagonist KT5720) medium (90–180 ng/ml vs 375–750 ng/ml with 10% human serum as well as the protein kinase C antagonist Chel Cl displayed little present); 2) whereas early (0–8 h) exposure to CI was necessary in suppression of A23187-induced CD14 down-regulation (Fig. 8). by guest on September 27, 2021 serum-containing medium to achieve maximal activation, CI ex- We also examined the inhibitory effects of a second CaMK antag- posure could be delayed for 24 h or longer in serum-free medium onist, K252a (Fig. 9). This compound, in comparison to CsA, be- without impeding subsequent acquisition of DC characteristics (H. haved identically to KT5926 in its selective block of CD14 down- Nguyen et al., manuscript in preparation); 3) serum-free medium regulation. Not only were costimulatory molecule and CD83 enabled preservation of high viability when monocytes were ex- expression not appreciably inhibited by K252a, but in roughly half posed to combinations of CI and inhibitors, particularly when the the donors tested, CD80 and CD86 expression was modestly en- monocytes were pretreated overnight with the inhibitors in the hanced, even in the face of profoundly blocked CD14 down-reg- presence of rhGM-CSF, followed by a limited (20–30 h) exposure ulation (Fig. 9). to CI. This enabled meaningful study of those immunophenotypic modulations that occurred within 20 h of CI exposure. Similar results were also obtained when pharmacologic inhibitors were Discussion added synchronously with CI treatment, when rGM-CSF was not The present studies employed a variety of pharmacologic agents, included, and when CI treatment was extended to 40 h, but with predominantly enzymatic inhibitors, to gain insight into the mech- less acceptable viability. anism(s) by which CI induce a variety of myeloid-origin cells to When cultured in serum-free medium alone for 20 h, normal acquire many DC characteristics. Although the data derived by human monocytes typically maintained high CD14 expression, studying such inhibitors in whole-cell systems are by nature indi- failed to express detectable levels of CD80, displayed low/absent rect, the judicious comparison of structurally unrelated agents with CD83, and down-regulated expression of CD86 (Fig. 8). When similar specificity, as well as structural analogues with alternative rhGM-CSF alone was also added during the culture, a modest but specificities, markedly enhances the meaningful interpretation of variable degree of CD14 down-regulation typically occurred results (3, 4, 20–22). Furthermore, the use of very conservative whereas significant CD80 or CD83 expression did not occur, and concentrations of each inhibitor increases the likelihood that ob- little alteration was observed in CD86 expression (Figs. 8 and 9). servations in whole-cell studies will reflect the specific activity of In contrast, monocytes treated with CI for 20 h down-regulated these agents in open-cell studies. CD14 expression nearly to control levels, expressed de novo We originally hypothesized that the immunophenotypic, mor- CD80, and up-regulated CD86 and CD83 expression, similar to phologic, and functional changes in human myeloid cells induced effects previously demonstrated in serum-containing medium (1). by A23187 were linked to this compound’s well-characterized Paralleling prior observations in HL-60 cells (Fig. 2), exposure of properties as an intracellular calcium-mobilizing agent. This was monocyte cultures to the calmodulin inhibitor W7 or the cal- by no means a foregone conclusion, because secondary pharma- cineurin inhibitor CsA markedly inhibited CI-induced up-regula- cologic effects could be responsible for these observed changes. tion of CD80, CD86, and to a lesser and more variable extent Therefore, we conducted experiments to determine the ability of The Journal of Immunology 89 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. Effect of pharmacologic inhibitors on A23187-induced surface molecule expression in human peripheral blood monocytes. Previously cryopreserved human peripheral blood monocytes from a healthy volunteer were thawed and resuspended in Life Technologies Macrophage-SFM at 3 ϫ 106 per well in 24-well cluster plates as described in Materials and Methods. Then, 45 min after culture initiation, the following inhibitors were each added at the designated final concentration, which previously had been found to maximize inhibition while minimizing toxicity: W-7 (10 ␮M), CsA (0.5 ␮g/ml), KT5926, KT5823, and KT5720 (each at 1 ␮M), and Chel Cl (10 ␮M) 45 min after inhibitor addition, rhGM-CSF (final concentration 50 ng/ml) was added to selected groups, and 20 h later A23187 (final concentration 150 ng/ml) was added to selected groups. After 40 h total culture period, cells were harvested and analyzed by FACS as described above. Before culture, monocytes had an identical profile to the “No Treatment” group after 40 h culture, except for higher initial expression of CD86 (not shown). Data is representative of three to eight experiments for each tested reagent.

Ca2ϩ per se to trigger differentiation in myeloid cells. Thapsigar- blood monocytes and HL-60 cells (Figs. 1 and 4). Furthermore, gin is a compound that raises cytoplasmic Ca2ϩ levels not through CI-induced acquisition of DC-associated characteristics was sen- the permeabilization of cell membranes to Ca2ϩ (as do CI agents sitive to the chelation of free Ca2ϩ by EGTA (Fig. 2) or its re- such as A23187), but rather through the pharmacologic inhibition moval from CM by dialysis (data not shown). of the smooth endoplasmic reticulum-associated ATP-dependent Such results render Ca2ϩ mobilization the probable explanation Ca2ϩ pump (12), thereby preventing endoplasmic reticulum se- for the observed effects of CI on myeloid cells. Furthermore, the questration of Ca2ϩ and causing higher cytoplasmic levels of Ca2ϩ signaling pathway antagonists employed in the present stud- Ca2ϩ. Consistent with the hypothesis that CI produces the ob- ies often resulted in impressive, if sometimes incomplete, inhibi- served effects in myeloid cells through Ca2ϩ mobilization, thap- tion of many DC-associated characteristics induced by calcium- sigargin induced qualitatively similar changes in human peripheral mobilizing agents. Because, in closed-cell studies, complete 90 CALCIUM SIGNALING PATHWAY ANTAGONISTS BLOCK MYELOID CELL ACTIVATION Downloaded from

FIGURE 9. Comparison of effect of CsA and K252a on A23187-induced surface molecule expression in human peripheral blood monocytes. Previously http://www.jimmunol.org/ cryopreserved human peripheral blood monocytes from a healthy volunteer were thawed and resuspended in Life Technologies Macrophage-SFM at 3 ϫ 106 per well in 24-well cluster plates as described in Materials and Methods. Then, 45 min after culture initiation, the following inhibitors were each added at the designated final concentration which had previously been found to maximize inhibition while minimizing toxicity: CsA (0.5 ␮g/ml), K252a (0.25 ␮M). 45 min after inhibitor addition, rhGM-CSF (final concentration 50 ng/ml) was added to selected groups, and 20 h later A23187 (final concentration 150 ng/ml) was added to selected groups. After 40 h total culture period, cells were harvested and analyzed by FACS as described. Data is representative of three experiments.

enzymatic inhibition cannot always be achieved with maintained CI-induced myeloid differentiation. An analogous approach, using by guest on September 27, 2021 high cell viability, we cannot rule out the formal possibility of pharmacologic antagonists of protein kinase C-dependent signal- ϩ some Ca2 -independent differentiating effects resulting from CI ing pathways, was recently employed successfully by others (20) treatment. Nonetheless, the bulk of evidence points to a leading to infer protein kinase CЈs role in phorbol ester-induced acquisition ϩ role for Ca2 mobilization in this process. of DC characteristics by in vitro cultivated human bone marrow There are several recognized mechanisms whereby increased progenitors. 2ϩ levels of cytoplasmic Ca may play a role in regulating gene In the present studies, our ability to monitor effects of the cal- expression. One of the best-characterized systems is the transcrip- cium chelator EGTA and the calmodulin antagonists W-7 and TpD tional activation of the IL-2 gene in T lymphocytes induced were limited to 24 h treatments, due to associated toxicities during 2ϩ through TCR stimulation (23, 24). Increased intracellular Ca longer cultures. Furthermore, only for HL-60 did it prove possible levels activate the regulatory protein calmodulin, which can bind to maintain viability during longer cultures that incorporated ex- to and positively modulate the activity of multiple enzymatic tar- posure to calcineurin antagonists or CaMK antagonists. Within gets. Among such targets are calmodulin-dependent serine/threo- these timing constraints, we were nonetheless able to perform key nine protein kinases (CaMKs). In particular, CaMK IV has been studies of costimulatory molecule and CD83 regulation not only in shown in T lymphocytes to induce or contribute to induction of the relatively robust HL-60 cell line, but also in normal bone mar- both c-jun (25) as well as p21ras-independent c-fos transcriptional row-derived myeloid progenitors and peripheral blood monocytes. activation (26). Both c-fos and c-jun are critical components of the Our combined observations among these myeloid cells, coupled transcriptional activator protein, AP-1, which participates in the with our studies of CD14 regulation in monocytes, are consistent regulation of a variety of genes. Calmodulin also positively mod- ulates the activity of the serine-threonine protein phosphatase 2B, with the presence of a calcium-dependent signaling pathway, calcineurin (15). Unlike many protein phosphatases, calcineurin which can lead to the acquisition of DC-like characteristics in hu- appears to have a very narrow substrate speciﬁcity (27). One such man myeloid cells. However, our studies employing antagonists of substrate, NFAT (28), is fundamental for subsequent IL-2 gene downstream regulatory targets of calmodulin suggest that this transcriptional activation in T lymphocytes. Dephosphorylation of pathway is at least bifurcatory in nature, with the enhanced ex- NFAT by activated calcineurin appears to unmask a nuclear lo- pression of costimulatory molecules and CD83 proving more sen- calization signal, leading to the translocation of NFAT across the sitive to calcineurin antagonists, and the down-regulation of CD14 nuclear membrane and into the nucleus. There, NFAT may form expression in monocytes conversely more sensitive to antagonists critical associations with AP-1 and interact with appropriate bind- of CaMKs. ing sites on the IL-2 promoter region. Therefore, we used a number We were also able to investigate the effects of prolonged expo- of well-characterized Ca2ϩ signaling pathway antagonists to look sure to CI plus calcineurin or CaMK inhibitors in the HL-60 cell for evidence of a calmodulin-calcineurin-CaMK signaling axis in line, due to the latter’s ability to maintain high viability in the face The Journal of Immunology 91 of such treatment. Notably, calcineurin antagonists inhibited CI- Other studies have suggested that phorbol esters, LPS, and TNF-␣ induced acquisition of dendritic processes in HL-60 and also pre- regulate ICAM-1 expression through NF-␬B as well (35, 36). vented CI enhancement of HL-60’s T cell-sensitizing capacity. Whereas there is presently no evidence that cAMP or treatment Such studies suggest that activation of the calcineurin pathway with LPS or phorbol esters achieved CD80 or ICAM-1 up-regu- can, at least in HL-60, lead not only to the expression of individual lation through elevations in intracellular Ca2ϩ levels, Ca2ϩ-mobi- molecules associated with enhanced Ag presentation, but also to lizing agents have been observed to contribute to the activation of the initiation of a multifaceted differentiation program. Consistent NF-␬B (37). Additionally, some studies have shown that the acti- with this hypothesis, we have recently determined that the induced vation of NF-␬B can also be inhibited by CsA, at least in part expression of nuclear-localized RelB in both HL-60 and mono- through modulating the degradation of the NF-␬B inhibitory sub- cytes, demonstrable within 20 h of CI treatment, is also signiﬁ- unit I␬B (38). Other studies have implicated RelB, a member of cantly blocked by cotreatment with calcineurin antagonists (L. the NF-␬B family, as playing a crucial role in DC development and Lyakh et al., manuscript in preparation). Such data reinforce the activation (39–42). Indeed, we have observed enhanced levels of possibility that calcineurin activation is critical to the full acqui- RelB induced by CI in both HL-60 and cultured peripheral blood sition of DC characteristics in myeloid cells following calcium monocytes (L. Lyakh et al., manuscript in preparation). We are mobilization stimuli. currently evaluating the role of NFAT, NF-␬B, and other nuclear Clinically important antagonists of calcineurin-dependent sig- factors in CI-induced myeloid differentiation. naling pathways, such as CsA and FK-506 are already used rou- Finally, it is of obvious interest to determine whether the calci- tinely to attenuate organ transplant rejection and to treat autoim- um-dependent signaling pathway we have described here is dis- mune disease. Although it is believed that these agents act in vivo tinct from, or overlaps with, activation pathways induced by the Downloaded from principally by interfering with the previously described calcium/ several cytokine/ligand treatments previously shown to induce DC calcineurin-dependent T cell activation pathway, other investiga- characteristics in myeloid cells (43–56). Because cytokine/ligand tors have provided evidence that calcineurin antagonists can also treatments typically require multiday culture to induce DC char- affect the maturation/activation of APC. For example, topically acteristics, it is challenging to incorporate studies with inhibitory applied FK-506 was observed to inhibit the expression of costimu- agents whose toxicity becomes more pronounced with prolonged

latory molecules on epidermal APC in a murine contact-sensitivity treatment. Logical candidates for cytokine/ligand treatments em- http://www.jimmunol.org/ model (29), and Panhans et al. (30) have described FK-506 sup- ploying calcium mobilization pathways are those whose action dis- pression of CD80 and CD86 expression by in vitro cultivated hu- plays similar rapidity to CI or thapsigargin treatment. Interestingly, man Langerhans cells. The data presented in such studies as well a recent study focusing on the transendothelial migration of human as in our own are consistent with the hypothesis that a wide variety monocytes demonstrated that monocytes can acquire pronounced of activated myeloid cell characteristics, including the expression DC characteristics within 2 days of exposure to collagen matrix of costimulatory molecules, the development of dendritic pro- and endothelial cells (57). This rapid time frame of conversion cesses, and perhaps the enhanced capacity to sensitize T cells can kinetically resembles CI-induced differentation more than the be regulated through a pathway that is sensitive to calcineurin lengthy differentiation process associated with typical cytokine/ antagonists. In addition, the apparent delayed toxicity observed in ligand treatments. We are continuing our efforts to define culture by guest on September 27, 2021 vitro when monocytes are calcium-mobilized in the presence of conditions to determine those myeloid differentiating treatments CsA raises the possibility that CsA may act therapeutically not that are sensitive to calcium-pathway antagonists. Furthermore, only by blocking calcium-activated DC differentiation, but also by refinements in our understanding of calcium-dependent activation killing calcium-activated APC. Thus, some of the clinically ob- pathways, especially if additional signaling branchpoints are found served immunosuppressive effects of CsA and FK-506 may be to exist, may allow specific pharmacologic agents to be used as exerted through suppression of myeloid APCs as well as T modulatory adjuncts to prepare APC with highly defined sensitiz- lymphocytes. ing, tolerizing, or apoptosing characteristics. Previous studies by others have suggested that the expression of CD80 (31) and CD54 (32, 33) in a variety of cell types may be Acknowledgments regulated, at least in part, at the transcriptional level. Because the data presented here suggest that elements of the Ca2ϩ-dependent We thank Drs. Nirbhay Kumar, Frances Hakim, Ludmila Lyakh, and Nancy Rice for helpful suggestions and encouragement, as well as Drs. signaling pathways found in myeloid cells bear similarities to Susan Leitman, E. J. Read, and Harvey Klein of the National Institutes of those found in T lymphocytes (3, 4), it seems possible or even Health Department of Transfusion Medicine. likely that some of the same or similar transcriptional factors used by T lymphocytes to regulate genes such as IL-2 might also tran- References scriptionally regulate CD80, CD54 and additional surface proteins in myeloid cells. The striking inhibition by CsA and an FK506 1. Czerniecki, B. J., C. Carter, L. Rivoltini, G. K. Koski, H. I. Kim, D. E. Weng, J. G. Rorors, Y. M. Hijazi, X. Shuwen, S. A. Rosenberg, and P. A. Cohen. 1997. analogue of CI-induced alterations in surface molecule expression Calcium ionophore treated peripheral blood monocytes and dendritic cells rapidly by myeloid cells raises the possibility that a likely candidate tran- display characteristics of activated dendritic cells. J. Immunol. 159:3823. scriptional factor may be NFAT or an NFAT-like protein. It should 2. Koski, G. K., G. N. Gchwartz, D. E. 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