doi:10.1182/blood-2006-03-014126Prepublished online May 9, 2006;   

 Anna RubartelliSonia Carta, Sara Tassi, Claudia Semino, Gianluca Fossati, Paolo Mascagni, Charles A Dinarello and -containing secretory lysosomes: role of microtubules

βHistone deacetylase inhibitors prevent exocytosis of interleukin-1

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Histone deacetylase inhibitors prevent exocytosis of interleukin-1β-

containing secretory lysosomes: role of microtubules

Sonia Carta1, Sara Tassi1, Claudia Semino1, Gianluca Fossati2, Paolo Mascagni2,

Charles A. Dinarello3, and Anna Rubartelli1

1Laboratory of Experimental Oncology E, National Cancer Research Institute, Genova,

Italy; 2Center for Research, Italfarmaco, Cinisello Balsamo, Milano, Italy; 3Department of Medicine, University of Colorado at Denver and Health Sciences Center,

Denver, CO 80262, USA.

Short title: HDAC inhibitors block secretion of IL-1β

Supported in part by grants from Associazione Italiana per la Ricerca sul Cancro, Ministero della Salute, CIPE (02/07/2004, CBA project) and NIH Grants AI-15614 (CAD) and HL-68743 (CAD) G.F. and P.M. are employed by Italfarmaco SpA, Milano, Italy whose potential product ITF2357 was studied in the present work. Currently, Italfarmaco SpA has no commercial products related to the compounds discussed in the present article. S.C. and S.T. contributed equally to this study. A.R., S.C., S.T. and C.S. designed research. S.C., S.T. and C.S performed research. P.M. and G.F. contributed ITF2357 and ITF2375 and critically reviewed the paper. C.A.D. contributed to the design of the research, to critically review the experiments and the manuscript. A.R. supervised the experiments and wrote the paper. Address correspondence to: Dr. Anna Rubartelli Experimental Oncology E National Cancer Research Institute Largo Rosanna Benzi, 10 16132 Genova, Italy e-mail: anna.rubartelli@istge.it Phone: +39 010 5737379 Fax: +39 010 352855 Scientific category: Immunobiology Total text word count: 4960 Abstract word count: 200

Blood First Edition Paper, prepublished online May 9, 2006; DOI 10.1182/blood-2006-03-014126

Copyright © 2006 American Society of Hematology

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Abstract

A number of agents reducing IL-1β activity are being developed as novel

immunomodulatory and anti-inflammatory therapies. However, the elucidation of their

molecular mechanism of action is required in the context of medical management of

inflammatory diseases. Inhibitors of histone deacetylases (HDAC) are promising

anticancer agents with pleiotropic activities. Of these, suberoylanilide hydroxamic acid

has been reported to inhibit the production of several pro-inflammatory cytokines. In the

present study, we investigated the effects of two HDAC inhibitors on IL-1β secretion:

suberoylanilide hydroxamic acid and a newly developed hydroxamic acid-derived

compound ITF2357. These HDAC inhibitors do not affect the synthesis or intracellular

localization of IL-1β but both strongly reduce the levels of extracellular IL-1β by

preventing the exocytosis of IL-1β-containing secretory lysosomes. At nanomolar

concentrations, ITF2357 reduces the secretion of IL-1β following ATP activation of the

P2X7 receptor. Whereas the inhibition of HDAC results in hyperacetylation of tubulin,

acetylation of HSP90 was unaffected. The reduction in IL-1β secretion appears to be due

to disruption of microtubules impairing lysosome exocytosis. Together, these

observations indicate that a functional microtubule network is required for IL-1β secretion

and suggest that disruption of tubulin is the mechanism by which inhibitors of HDAC

reduce the secretion of IL-1β.

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Introduction

Interleukin-1 β (IL-1β) is a potent proinflammatory cytokine; subnanomolar

concentrations following intravenous injection into humans are sufficient to cause several

inflammatory responses.1 Thus it is not surprising that the production of active IL-1β is

tightly controlled, at more than one level.2 A first level is transcriptional: IL-1β is not

expressed in healthy individuals. A second is translational, as high levels of IL-1β mRNA

can be present in cells in the absence of translation.3 The third and perhaps the most

complex is the pathway by which the initial translational product, the inactive

IL-1β precursor, is cleaved into an active form and released extracellularly. The cleavage

of the inactive IL-1β precursor is accomplished by caspase-1 and both cleavage and

secretion appear to be linked. However, the activation of caspase-1 itself from an inactive

form is also tightly controlled by mechanisms that only recently have been partially

elucidated.4,5 Similar to other clinically relevant cytokines, IL-1β lacks a secretory signal

peptide and avoids the classical exocytotic route.6 Two major steps can be envisaged in

the IL-1ß secretion process in human monocytes, the primary sources of IL-1β. First, toll

like receptor ligands such as lypopolysaccharide (LPS), peptidoglycans or the

intracellular nucleotide oligomerization domain (NOD) agonists induce gene expression

and synthesis of the IL-1β precursor;2 however it accumulates in the cytosol, and only a

portion of the total synthesis enters a specialized subset of secretory lysosomes, where

inactive pro-caspase-1 is also present.7,4 Once stimulated, monocytic cells release

approximately 20% of the IL-1β slowly over 24-48 hours into the extracellular

compartment, unless a stimulus-triggered secretion event takes place. A powerful

stimulus of secretion is exogenous ATP that acting in an autocrine manner on P2X7

receptors expressed on the surface of monocytic cells8 facilitates IL-1β secretion. In vivo,

ATP accumulates at the site of inflammation, released by dying cells or actively secreted

by monocytes or other inflammatory cells, such as platelets, and thus accelerates the

secretion of the mature cytokine.8

Engagement of P2X7 receptors triggers a series of events, leading to IL-1β

processing and secretion, both of which have been dissected and partially clarified.

Fundamental to the engagement of the purinergic P2X7 receptor is the efflux of K+ from

the cell, which occurs simultaneously with ATP activation. K+ efflux appears crucial for the

generation of active caspase-1 from its inactive precursor9-11 through activation of calcium-

independent phospholipase A2 (iPLA2).12 The exit of K+ is then followed by Ca2+ entry13

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and the sequential activation of phosphatidylcholine-specific phospholipase C (PC-PLC)

and calcium-dependent phospholipase A2 (cPLA2), which is required for lysosome

exocytosis and secretion of IL-1β.4 The small peptide LL37, released by activated

neutrophils and epithelial cells, triggers P2X7 receptors, and thus also can facilitate

secretion of IL-1β.14

An important cellular process that remains to be clarified is whether and how the

cytoskeleton contributes to IL-1β-containing vesicle movement and/or exocytosis. The

involvement of the cytoskeleton in vesicle and organelle transport is a longstanding

concept. However, the molecular constituents vary considerably depending on the cell

type or the cargo vesicles themselves, and the complete picture of the roles of the

cytoskeleton in vesicle transport and exocytosis remains unclear. For instance, the

combined action of actin-myosin and microtubule networks is required for intracellular

lysosome movement.15 In contrast, only the microtubule network seems involved for

secretory lysosomes and secretory granule exocytosis in different cells;16 in mastcells,

actin filaments have been shown to down-regulate degranulation.17

Despite the tight regulation of processing and release of IL-1β, several

inflammatory disorders are known, each of which result from a defective secretion of IL-

1β. Among these are the autoinflammatory syndromes such as Muckle-Wells syndrome,

familial cold-induced auto-inflammatory syndrome, familial mediterranean fever as well as

systemic juvenile rheumatoid arthritis, which affect growing children, and are particularly

devastating.5 Importantly, blocking the IL-1 receptors in each of these diseases with

treatment by the IL-1 receptor antagonist (IL-1Ra) returns these patients to

normalcy,18,19,20 thus confirming the exquisite dependency of excessive secretion of IL-1β

in the pathogenesis of these syndromes.

Although IL-1Ra therapy is safe and effective, it requires daily injections and its

efficacy is directly related to occupancy of IL-1 type I receptors, which are present on

nearly all cells.2 Furthermore, the relatively short in vivo half-life of the antagonist and

urinary excretion necessitates the use of large quantities of the recombinant protein. For

these reasons, blocking the secretion of IL-1β would offer a specific therapy for the

inflammation due to IL-1β. Although developed to increase pro-apoptotic genes for the

treatment of cancer,21 the inhibitor of histone dehacetylases (HDAC), suberoylanilide

hydroxamic acid (SAHA), reduces secretion of mature IL-1β from human peripheral blood

mononuclear cells in vitro and the level of circulating IL-1β in LPS-injected mice, without

affecting the steady-state levels of IL-1β mRNA or the intracellular levels of precursor IL-

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1β.22 However, SAHA does not target caspase-1, at least in vitro, and the mechanism

underlying its ability to reduce the secretion of IL-1β remains to be clarified.22 Most

HDACs are nuclear enzymes, involved in the epigenetic regulation of gene expression by

modulating the acetylation state of core histones.23 Accordingly, the major and most

studied substrates of HDAC are histones. Several transcription factors and nuclear import

factors have also been shown to be regulated by processes of acetylation and

deacetylation.24 In addition, two cytoplasmic targets of HDACs have been identified: α-

tubulin26 and heat shock protein 90 (HSP90).27 Deacetylation of both proteins is mediated

by HDAC6, which acts cytoplasmically.25,26 In addition, α-tubulin is a target of the NAD-

dependent histone deacetylase SIRT2.28 Based on this information we hypothesized that

the inhibition of HDAC activity could alter the microtubule organization leading to the

inhibition of IL-1β secretion. Here we investigated the molecular components of IL-1�

secretory pathway, which may be affected by two HDAC inhibitors, SAHA and ITF2357,

both hydroxamic acid-derived, orally active compounds with known with anticancer

activities29 but also anti-inflammatory properties.30

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Methods

Chemicals and antibodies

LPS, ATP, trichostatin A (TSA), HC-toxin, paclitaxel (Taxol), nocodazole, proteinase K,

Ponceau S, Tween-20 were purchased from Sigma-Aldrich (Milano, Italy). Arachidonyl

trifluoromethylketone (AACOCF3) and bromoenol lactone were obtained from Alexis

Biochemicals (Lausen, Switzerland). SAHA, ITF2357 and ITF-nil (ITF2375) were

synthesized by Italfarmaco SpA, Cinisello Balsamo, Italy.29,30 Tubacin31,32 was a kind gift of

Drs. Mazitschek and Schreiber (Harvard University, Cambridge, MA, USA). The following

antibodies were used: 3ZD anti-IL-1β monoclonal antibody (mAb) (IgG1, obtained from the

National Cancer Institute Biological Resources Branch, Frederick, MD); rabbit anti

caspase-1 antiserum R105 (kind gift of Dr. DK Miller, Merck Research Laboratories,

Rahway, NJ); anti-cathepsin D mAb (IgG2a, Calbiochem, Milano, Italy), anti-α-tubulin and

anti acetyl α-tubulin mAb (Sigma-Aldrich); rabbit anti-histone H4 and rabbit anti-acetylated

histone H4 (Upstate, Lake Placid, NY), anti-HSP90 mAb (Stressgen, Victoria, BC) and

anti-acetyl-lysine mAb (Cell Signaling Technology, Beverly, MA). Horseradish peroxidase-

conjugated goat anti-mouse or anti-rabbit IgG were from DAKO A/S, Denmark; fluorescein

isothiocyanate (FITC) or cyanine 3 (Cy3) labeled secondary reagents were from Jackson

ImmunoResearch, Soham, United Kingdom.

Cell cultures

Human monocytes isolated from buffy coats from healthy donors, enriched by adherence

in RPMI 1640 medium containing 10% fetal bovine serum (all from Sigma-Aldrich) were

activated with 1 µg/ml LPS for 4 hours or 18 hours at 37°C in RPMI 1640 medium

supplemented with 1% Nutridoma-HU (Roche Applied Science, Monza, Italy) as

described.7,4 In each experiment, supernatants were first collected and then replaced with

RPMI/1% Nutridoma in the presence or absence of 1 mM ATP or other drugs as indicated

and incubated for the indicated times. After the addition of ATP, supernatants were

collected, and cells were lysed in 1% Triton X-100 lysis buffer.7,4

Microglial N9 cells were obtained from Dr. C. Verderio and maintained as described.33 One

million cells were activated with 100 ng/mL of LPS for 4 hours followed by 1 mM ATP for

20 minutes as described.33

Subcellular Fractionation by Differential Ultracentrifugation

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Subcellular fractionation was performed as described.7,4 Briefly, monocytes were washed,

resuspended in homogenizing buffer (250 mM sucrose, 5 mM EGTA, 20 mM HEPES-

KOH, pH 7.2) at 5 x 107/ml and disrupted in a Dounce homogenizer. The postnuclear

supernatants (PNS) were diluted 10-fold and centrifuged at 50,000 g for 5 minutes,

resulting in a pellet enriched in endo-lysosomes, as confirmed by electron microscopy (not

shown); the pellet was treated with 0.1 mg/ml proteinase K in the presence or absence of

1% Triton X-100 for 30 minutes on ice. The 50,000 g generated supernatants were

subjected to 40 minutes at 100,000 g to obtain soluble cytosol.

ELISA analyses.

IL-1β, IL-8 and TNF-α content of supernatants from human monocytes cultures was

determined by ELISA (R&D Systems, Inc., Minneapolis, MN).

The IL-1ß content of supernatants of N9 cells was assayed by ELISA (Pierce Endogen,

Woburn, MA). Triton X-100 (2%) was added to supernatants to solubilize microvesicles.33

Western blot analysis

Cell lysates, pellets from subcellular fractionation, trichloroacetic acid-concentrated

supernatants or cytosolic fractions were boiled in reducing Laemmli sample buffer,

resolved on 12% SDS-PAGE and electrotransferred as described7,4. Filters were probed

with the different Abs followed by the relevant secondary Abs as indicated and developed

with ECL-plus (Amersham Pharmacia Biotech, Milan, Italy). Densitometric analyses were

performed by analyzing at least three different exposures of the same blot.

Flow Cytometry

Cells were processed and stained with anti α-tubulin or anti acetyl-tubulin followed by the

relevant secondary reagents as described.34 Data acquisition was performed using a

FACSort cytometer (Becton Dickinson, Milano, Italy). The percentage of positive cells and

mean values of fluorescence intensity were evaluated using CellQuest.

Determination of phospholipase activity

PC-PLC and PLA2 activity was measured using the Amplex Red PC-PLC assay kit

provided by Molecular Probes Europe (Leiden, The Netherlands) and the cPLA2 Assay Kit

by Cayman Chemical (Ann Arbor, MI) according to the manufacturer’s instructions. Briefly,

50 µg of cell lysates were used for each sample. PLC activity (mU/µg) was determined by

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comparison with the positive controls.4 PLA2 activity (nmol/µg/minute) was calculated as

indicated in the kit instructions.4

Calcium mobilization assay

Monocytes adherent on a glass coverslip, activated with 1 µg/ml LPS for 4 hours at 37°C

in the presence or absence of HDAC inhibitors as indicated, were loaded with the

acetoxymethyl-ester of Fura-2 (Fura-2-AM, 1 M) for 1 hour, placed under an AXIOVERT

10 microscope (Zeiss) maintained at 37°C, and stimulated with 1 mM ATP. Fura-2-AM was

excited at 340 and 380 nm, and emitted light was filtered at 510 nm. The fluorescence

ratio at 340/380 was evaluated by a charged-coupled device camera (Atto Instruments,

Rockville, MD). Results are presented as the mean of fluorescence of at least 50 cells in

each experiment monitored for 15 minutes. [Ca2+]i increase was calculated as described.4

Measurement of K+ efflux

Two million monocytes, which had been incubated for 4 hours with 1 �g/ml of LPS in the

presence or absence of HDAC inhibitors were exposed to 1 mM ATP for 30 minutes. The

reaction was stopped by removal of the medium followed by lysis of the cells in 10% nitric

acid. Cell extracts were then spun at 100,000 g for 1 hour and the K+ content was assayed

in an atomic absorption spectrophotometer.9,12

Cytolytic assay

NK cell cytolytic activity against K562 was tested in a 51Cr-release assay as described.34

Briefly, K562 cells were loaded with 51Cr and co-cultured for 4 hours with NK cells (pre-

treated for 4 hours with the different drugs) used as effector cells, at a different effector-

target (E/T) ratio. Results are expressed as percentage of cytotoxicity as described.34

Mixed lymphocyte reaction

Allospecific T cells were obtained by co-culturing purified T cells with allogenic irradiated

peripheral blood mononuclear cells as described.35 After one week of co-culture, 105

allospecific T cells pre-treated for 4 hours with the inhibitors were co-cultured with 104

allogeneic dendritic cells (DCs) in 96-well plates for 3 days. Cells were pulsed with 1 µCi

of [3H]thymidine (Amersham Pharmacia Biotech, City, Country) per well for the last 18

hours of culture, harvested, and counted in a ß-counter (Packard). Tests were conducted

in triplicate, and results are expressed as mean cpm ± SD.

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Results

SAHA and ITF2357 inhibit the secretion of mature IL-1β but do not affect synthesis

or intracellular distribution of the IL-1β precursor.

Monocytes were stimulated for 4 hours with LPS in the presence or absence of increasing

concentrations of SAHA or ITF2357, and then exposed for an additional 15 minutes to

ATP. Secreted IL-1β was determined by western blotting. As shown in Figure 1, secretion

of IL-1β during the 4 hours of LPS stimulation is low (lane 6); in contrast, when LPS-

stimulated monocytes are exposed to ATP, a marked increase in the secretion of IL-1β is

observed (lane 7). The two inhibitors do not affect the amount of precursor IL-1β

synthesized by LPS-stimulation (compare lanes 4 and 5 with lane 2) but impressively

reduced the portion of secreted IL-1β (lane 9 and 10). ATP-induced IL-1β secretion is

paralleled by secretion of caspase-136,4 and of the lysosomal enzyme cathepsin D.4

Treatment with SAHA or ITF2357 prevents the ATP-induced secretion of the three proteins

(Figure 1A and B), suggesting that the two compounds exert their inhibitory effects on IL-

1ß secretion at the level of lysosomal exocytosis. As shown in Figure 1B, SAHA is active

at 0.1 µM (70% of inhibition), but loses efficacy at lower concentrations. In contrast,

ITF2357 maintains its inhibitory effect at low concentrations; for example at 20 nM

ITF2357 inhibits IL-1β secretion by 60%.

To further investigate the mechanism of action of the two compounds, we studied

the effects of SAHA and ITF2357 on the intracellular localization of pro-IL-1β, pro-

caspase-1 and cathepsin D. Monocytes stimulated with LPS in the presence or absence of

the inhibitors were subcellularly fractionated and the presence of IL-1β in the lysosomal-

enriched fraction was analyzed. Figure 1C shows that the relative amount of pro-IL-1β

detected in the lysosomes of untreated cells (about 10% of the total cellular content) was

equivalent to that found in the same fraction of cells exposed to SAHA or ITF. Similarly,

the pro-caspase-1 content in the same lysosomal fraction was unaffected by treatment

with the two compounds. As a control, the endolysosomal marker cathepsin D, which co-

fractionates with pro-IL-1ß and pro-caspase-1 in comparable amount (about 40% of the

total content) is present in both untreated and treated cells. Panel D shows a

representative experiment, with similar amount of pro-IL-1β (upper panel), pro-caspase-1

(middle panel) and cathepsin D (lower panel) in lysosomal fractions (Ly) and post nuclear

supernatants (PNS) of untreated or ITF2357-treated monocytes.

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These data indicate that the two compounds do not interfere with the intracellular targeting

of pro-IL-1β and caspase-1.

Both SAHA and ITF prevent PLA2 but not PLC activation

We have previously shown that IL-1β secretion by monocytes is driven by a series of

sequential events involving PC-PLC and cPLA2 activation.4 Therefore, we investigated

whether SAHA or ITF2357 interfere with phospholipase functions. As shown in Figure 2

whereas PLC activation is unaffected by ITF2357 or SAHA, both compounds inhibit PLA2

activation by 50%.

Kinetics of SAHA and ITF inhibitory effects

To investigate the effect of SAHA or ITF2357 on cells actively synthesizing pro-IL-1β,

monocytes were stimulated with LPS and treated with the two compounds for the entire

time of culture (4 hours) or exposed to the inhibitors for the last 2 or 1 hours. At the end of

the incubation time, cells were triggered for 10 minutes with ATP and secretion of IL-1β,

caspase-1 and cathepsin D was evaluated. As shown in Figure 3, 4 hours of treatment

with SAHA or ITF2357 lead to a complete inhibitory effect on IL-1β secretion. Only about

25% of inhibition is obtained when monocytes cultured for 2 hours with LPS were exposed

to the compounds for additional 2 hours before triggering with ATP, and no inhibition is

observed when treatment is restricted to the last hour before addition of ATP.

HDAC inhibitors with different specificity block IL-1β secretion

SAHA possesses a well known HDAC inhibitory activities;22,37 ITF2357 has recently been

shown to cause H3 and H4 histone hyperacetylation in an hepatoma cell line and in

PBMC, respectively.29,30 We reasoned that the inhibition of lysosomal exocytosis observed

with SAHA and ITF2357 could be due to their inhibitory effects on HDAC, and that the time

elapsed between exposure to the inhibitors and block of IL-1β secretion (Figure 3) may be

required for the hyperacetylation of a substrate, in turn responsible for blocking the

exocytosis of the secretory lysosomes. We therefore investigated whether other HDAC

inhibitors37 affect IL-1β secretion. Among the compounds tested, TSA, like SAHA, results

in nonselective inhibition of all HDACs. In addition, the fungal metabolite, HC-toxin, is a

trapoxin-related inhibitor, selective for class I HDACs, whereas tubacin specifically binds to

the catalytic domain of HDAC6,31,32 which is responsible for the deacetylation of α-

tubulin25,26,32 and HSP90.27 As shown in Figure 4A, exposures of monocytes to each of the

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different HDAC inhibitors resulted in a block of secretion of IL-1β without any effect on its

intracellular accumulation. Similar inhibition was observed on caspase-1 and cathepsin D

secretion (data not shown). HDAC inhibitors did not affect ATP induced K+ efflux and Ca2+

entry, ruling out that their inhibitory effects on IL-1β secretion is due to interference with

the proximal signaling responses to P2X7 activation (Figure 4B and C). In keeping, the

different compounds decrease the amount of IL-1β secreted by monocytes during 18

hours of culture with LPS, without additional ATP stimulation, from 50 to 80% (Figure 4D).

To test the specificity of the effect of HDAC inhibitors on cytokine release, we

compared the secretion of IL-1β with that of IL-8 and TNF-α. IL-8 is released by default via

the ER-Golgi pathway.38 On the other hand, TNF-α is trafficked from the Golgi to the

recycling endosomes, from which it is transported to the cell surface.39 As shown in Figure

4D, 18 hour secretion of IL-8 was almost unaffected by HDACs, the highest inhibition

being 25 and 30% upon treatment with TSA and HC-toxin, respectively. Secretion of TNF-

α was decreased by the various HDAC inhibitors, although considerably less than that of

IL-1β.

We then compared the effects of HDAC inhibitors on the acetylation state of nuclear

(histones) and cytoplasmic (α-tubulin and HSP-90) targets of HDAC in activated

monocytes. Treatment with each of the HDAC inhibitors except tubacin resulted in marked

hyperacetylation of histone H4 (Figure 5A). In contrast, none of the compounds tested

induced detectable acetylation of HSP90 (Figure 5B). Exposure to HDAC inhibitors

resulted in different hyperacetylation of tubulin when evaluated both by cytofluorimetry

(panel C) and western blotting (panel D). However, HC-toxin did not result in detectable

change in the acetylation state of tubulin. Of note, ITF2357 induced H4 and tubulin

acetylation, even if to a lesser extent than the other inhibitors, and at a concentration

greater than that required for inhibition of IL-1β secretion (100nM versus 50nM).

Cytoskeletal inhibitors interfere with lysosome exocytosis

To further explore the role of cytoskeleton on lysosomal exocytosis, we studied the effects

of different cytoskeleton-perturbing agents on the secretion of IL-1β. As shown in Figure 6,

treatment with the actin depolymerizing fungal metabolite cytochalasin D17 increased IL-

1β secretion in the absence of ATP triggering (panel A, lane 3), confirming our previous

data.40 We next examined the effects of two different substances, nocodazole and taxol,

which act as microtubule disrupting and stabilizing agent, respectively, the net effect being

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the impairment of microtubular function41. The results show that distinct from cytochalasin

D, both drugs influence only slightly basal secretion (lane 2 and 4), but substantially

impaired ATP-induced secretion (lanes 6 and 8). However, at high concentrations of taxol

(20 and 40 µM), a paradoxical effect was often observed, with increased lysosomal

exocytosis and secretion (lanes 9 and 10). As shown, taxol-induced hyperacetylation of

tubulin was consistently dose-dependent (Figure 6B). Together, these results indicate that

microtubules participate in lysosomal exocytosis and that the levels of tubulin acetylation

must be tightly balanced to allow a correct vesicle transport.

In the attempt to understand the molecular basis of the stimulatory effect of

cytochalasin D on IL-1β processing and secretion, we exposed LPS-treated monocytes to

cytochalasin D together with compounds that block processing and/or secretion of IL-1β at

different levels. Figure 6C shows that Ca2+-depletion, which prevents ATP triggered IL-

1β secretion,7,13 also inhibits the secretion induced by cytochalasin D (lane 7 versus lane

1). Likewise, AACOCF3, which blocks cPLA2, essential for IL-1β externalization,4 strongly

inhibits secretion (lane 5). Interestingly, cytochalasin D-induced secretion is also prevented

by pre-treatment of monocytes with HDAC inhibitors (lanes 2 to 4). However, treatment

with BEL, an inhibitor of iPLA2, that prevents processing of the precursor form of IL-1β,4,12

results in increased secretion of unprocessed pro-IL-1β (lane 6).

Effects of deacetylase inhibitors on T lymphocyte and NK cell activity

Secretory lysosomes and tubulin rearrangement are involved in a number of

immunological related responses, including NK cell mediated cytotoxicity and antigen

presentation16,42. To assess whether HDAC inhibitors have any effects on these

responses, we tested the different compounds in two in vitro assays, NK killing and a

secondary MLR. As shown in Figure 7A, neither SAHA nor ITF2357 have significant

effects on NK cell lysis of the tumor cell line K562. In contrast, a small reduction in the

killing was observed after exposure to TSA, HC-toxin or tubacin. Of note, a clear

correlation with induction of tubulin acetylation in NK cells is absent (Figure 7B). It remains

unclear why ITF2357-treated NK cells, unlike monocytes, do not display detectable

acetylation of tubulin even at higher concentrations (100 nM). When the different

compound were tested on alloreactive T cells toward allogenic DCs, ITF2357 and HC-toxin

did not reduce or enhance proliferation; a low levels of inhibition was observed with SAHA,

TSA and tubacin (Figure 7C).

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Discussion

The development of novel therapeutic strategies for inhibition of IL-1β activity is

central for the treatment of many systemic inflammatory diseases caused by this cytokine.

Understanding the mechanisms that control IL-1β processing and release is therefore of

considerable importance not only as a matter of basic mechanisms but also for the

development of innovative anti-inflammatory therapies. We have previously identified a

subset of secretory lysosomes as vehicles for the extracellular delivery of IL-1β, and

outlined the key role of the sequential activation of phospholipases C and A2 in regulating

exocytosis.4,7

Here we extend the understanding of the IL-1β secretion pathway by defining the

involvement of cytoskeleton in the process of exocytosis. We report that microtubule

targeting agents, including taxol and nocodazole, impair IL-1β secretion, indicating that a

functional microtubule network is required for the release of this cytokine. Microtubules

play important roles in the immune-inflammatory response. Tubulin rearrangement in

dendritic cells is critical not only for the organization of the immune synapse at the

interface with T cells42 but also for the polarized release of cytokine-containing secretory

lysosomes at the synaptic cleft.34,34 This is in agreement with the function of microtubules

as tracks along which organelles and vesicles are transported through the cell and support

previous observations that microtubular cytoskeleton is essential for exocytosis of

secretory lysosomes in hemopoietic cells.16

A major finding of this study is that HDAC inhibitors are highly effective in reducing the

secretion of IL-1β. Remarkably, all the HDAC inhibitors assessed in this study, apart from

HC-toxin, prevent IL-1β secretion and induce tubulin acetylation. Tubulin undergoes

acetylation by the cytoplasmic HDAC625,26 or by the recently described type III HDAC

SIRT2.28 Since the HDAC inhibitors we have explored are directed to class I or II enzymes,

with no activity on class III, as it is for ITF2357 (not shown), their target is likely to be

HDAC6 rather than HDAC SIRT2.

A role for tubulin acetylation has been proposed in cell motility25 but the true

significance of this post-translational modification as well as its role in other microtubule

functions is still debated.43 It is conceivable that tubulin acetylation affects the activity of

microtubule-associated proteins or motors, for this would result in an impaired vesicle

transport. Interestingly, the microtubule stabilizing agent taxol, which induces a

concentration-dependent acetylation of tubulin, is less effective in blocking secretion of

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IL-1β at concentrations resulting in greater acetylation. This is reminiscent of the

paradoxical effect reported for SAHA on IL-1β secretion: at high concentrations (that

results in stronger tubulin acetylation, not shown) the inhibitory effects on IL-1β secretion

decreased or even reversed.22 These observations suggest that the degree of tubulin

acetylation must be under a tight balance to allow correct progression of exocytosis. For

example, an excessive acetylation may result in a loss of contact with regulatory proteins

associated to microtubules. Alternatively, high doses of taxol or HDAC inhibitors may

deregulate other post-translational modifications of tubulin43 resulting in enhanced rather

than reduced exocytosis.

The lack of detection of intracellular mature IL-1β and caspase-1 upon treatment with

HDAC inhibitors suggest that these drugs also attenuate the activity of either caspase-1 or

other inflammasome regulatory proteins.5 As iPLA2, that is implicated in caspase-1

activation,7,11,12 is also involved in various cytoskeleton-mediated processes of membrane

trafficking,44 it is tempting to speculate that tubulin hyperacetylation inhibits iPLA2 activity.

The discrepancy between the effects of HC-toxin on lysosomal exocytosis that is

blocked and tubulin acetylation, which apparently is unaffected (Figure 5 and Haggarty et

al),32 suggests that either a cellular target different from tubulin is responsible for the

inhibitory effects of this compound on IL-1β secretion, or that low levels of tubulin

acetylation, undetectable in our assays (cytofluorimetry and western blot) are sufficient to

prevent lysosomal exocytosis. Consistent with the latter possibility, high concentrations of

ITF2357 induced tubulin acetylation, but low concentrations effectively block IL-1β

secretion without detectable modifications on the acetylation state of tubulin. In any case,

the kinetics of inhibition of IL-1β secretion by HDAC inhibitors (Figure 3 and not shown)

reveals that 4 hours of exposure to the drugs are required to achieve the inhibitory effect,

suggesting that one or more hyperacetylated substrates participate in reducing lysosomal

exocytosis. The possibility that HDAC inhibitors, acting on nuclear substrates, promote the

synthesis of unknown proteins able to contrast lysosome exocytosis is unlikely, as blocking

protein synthesis in LPS-activated monocytes does not prevent the secretion of previously

synthesized IL-1β.6 Our data do not, however, rule out the possibility that other HDAC6-

sensitive molecules may also (or alternatively) participate in the secretion inhibitory

process.

Treatment of monocytes with HDAC inhibitors does not affect the proximal signaling

responses to P2X7 activation, e.g. K+ efflux and Ca2+ influx,8 but inhibits the activity of

cPLA2 (Figure 2), which is required for IL-1β-containing lysosome exocytosis.4 Functional

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association between microtubular cytoskeleton and phospholipases has been

demonstrated in several models.45-48 In particular, cPLA2 is implicated in EGF- or FGF-

induced cytoskeletal reorganization and has been found to interact with microtubules.47-48

Based on these observations, it is possible that in monocytes the activation of cPLA2, as

proposed above for iPLA2, requires a microtubule rearrangement: induction of tubulin

acetylation even if weak, such as in the case of ITF2357, could prevent this activation.

In contrast to microtubule poisons, cytochalasin D induces IL-1β secretion, confirming

our previous results.40 This enhanced secretion is likely to be mediated by Ca2+ entry and

cPLA2 activation, since monocytes exposed to cytochalasin D in the presence of Ca2+

chelators or PLA2 inhibitors do not exhibit increased secretion. These findings are in

agreement with previous reports indicating that microfilament disruption leads to increased

activity of cPLA217 and that cytochalasin D elicits Ca2+ mobilization and activates cPLA2 in

polymorphonuclear leukocytes.49

Studies on macrophage or microglia cell lines have suggested that low efficiency

ATP-triggered IL-1β secretion is mediated by microvesicles shed from the plasma

membrane.33,53 When HDAC inhibitors were added to the N9 microglia cell line, the

release of IL-1β was unaffected (data not shown) suggesting that tubulin and cPLA2 are

not implicated in this pathway.

The HDAC inhibitors explored in this paper display a relevant specificity for IL-1 β

release, either induced by LPS only or by LPS plus exogenous ATP. Indeed, the same

HDAC inhibitors only slightly affect the release of the chemokine IL-8, a pro-inflammatory

protein secreted through the classical exocytotic pathway.38 In contrast, the amount of

TNF-α39 secreted by HDAC-treated monocytes is decreased, although to a lesser extent

than IL-1 β. In the case of IL-1 β, HDAC inhibitors prevent only the secretion but not the

synthesis of the precursor (Figure 1A and 4A). In the case of TNF-α, inhibition occurs at

the level of gene expression (not shown), as reported for SAHA22 and ITF2357.30

HDAC inhibitors have recently emerged as novel agents with potential therapeutic

value for the treatment of cancer and are currently in development or early phase clinical

investigation.51 Our present data suggest that these drugs can also be suitable for the

treatment of inflammation. In particular, ITF2357 given its very high activity and specificity

is a good candidate to be developed as an anti-inflammatory drug that could have wide

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utility in numerous IL-1β-mediated inflammatory conditions.

Acknowledgments

We thank Drs S.L. Schreiber and R. Mazitschek (Cambridge, Ma, USA) and HHMI and

ICG (initiative for Chemical Genetics–National Cancer Institute) for kindly providing us with

tubacin, the National Cancer Institute Biological Resources Branch, Frederick, MD for

providing 3ZD anti-IL-1β mAb, Dr. D.K. Miller (Merck Research Laboratories, Rahway, NJ)

for the kind gift of the rabbit anti caspase-1 antiserum R105, Dr. A. Poggi and Mr. G. Rossi

(Genova, Italy) for the help in Ca2+ and K+ measurement respectively and the Blood

Centers of Ospedale Galliera and S. Martino (Genova, Italy) for buffy coats.

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Legends to Figures

Figure 1. Both SAHA and ITF2357 inhibit IL-1 β secretion but do not affect IL-1 β

intracellular accumulation and targeting. (A) Monocytes were activated 4 hours with

LPS in the absence (lanes 1, 2, 6 and 7) or presence of 50 nM ITF2357 (lane 4 and 9) or

50 nM of the negative compound ITF2375 (ITF-nil, lane 3 and 8) or 0.5 µM SAHA (lane 5

and 10), and then exposed to 1 mM ATP for 15 minutes (lanes 2 to 5 and 7 to 10).

Supernatants and cell lysates were analysed by western blotting for the presence of IL-1ß

(upper panel), caspase-1 (middle panel), and cathepsin D (Cath-D, lower panel). One

representative experiment out of 10 performed is shown.

(B) Densitometric analyses of western blots of mature IL-1 β, caspase-1 and cathepsin D

secreted following exposure to different amounts of the three drugs as indicated. Results

are expressed as percent of inhibition of secretion ±SE of a single experiment performed

in triplicate. One representative experiment out of at least 5 performed is shown. Data

were validated by ELISA (not shown). In 13 different donors, IL-1 β secreted in 15 minutes

of ATP stimulation by 0.5x106 cells ranged between 5 and 15 ng (not shown).

(C,D) Monocytes were cultured 4h with LPS, in the absence or presence of 50 nM ITF2357

or 0.5 µM SAHA and a post-nuclear supernatant (PNS) and a endolysosome enriched

fraction were obtained as described in material and methods.

(C) Densitometric analyses of western blots showing the percent of pro-IL-1 β, pro-

caspase-1 and cathepsin-D in the lysosomal fractions of monocytes activated with LPS in

the absence (-) or presence of SAHA or ITF2357. (D) Western blot of a representative

experiment showing endolysosomal-enriched fractions (Ly, lanes 1 and 2) and aliquots

(1/10) of post nuclear supernatants (PNS, lanes 3 and 4) from monocytes exposed 4h to

LPS (lanes 1 and 3) or to LPS and ITF2357 (lanes 2 and 4). Filters were hybridized with:

anti-IL-1 β (upper panel); anti caspase-1 (middle panel), anti cathepsin D (lower panel).

For each panel, one representative experiment out of at least 3 performed is shown.

Figure 2. Both SAHA and 2357 prevent PLA2 activation. Monocytes stimulated for 4

hours with LPS in the absence or presence of 50 nM ITF2357 or 0.5 µM SAHA. The cells

were then incubated for 2 minutes with 2 mM ATP and the presence of active PC-PLC or

PLA2 in cell lysates was determined. Data are expressed as percent of PL activity of

control (LPS-ATP stimulated) cells. PLC activity (milliunits/µg) and PLA2 activity

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(nmol/µg/min) in a representative experiment were, respectively, 1.15±0.002 and

0.44±0.01 after LPS-ATP treatment and 1.21±0.004 and 0.22±0.018 after LPS-ATP

treatment in the presence of ITF2357 or SAHA, respectively.

Figure 3. Four hour treatment with SAHA and ITF2357 is required to prevent IL-1ß

secretion. Monocytes were stimulated with LPS and treated without or with 50 nM

ITF2357 or 0.5 µM SAHA for the entire time of culture (4h) or left untreated the first 2

hours and treated the last 2 hours (2h), or left untreated 3 hours and treated the last 1 hour

(1h). At the end of the incubation time, cells were triggered for 10 minutes by ATP and

secretion of IL-1 β, caspase-1 and cathepsin D was evaluated by western blot following by

densitometric analyses. Data are expressed as percent of inhibition of secretion of IL-1 β,

caspase-1 and cathepsin D. One representative experiment out of at 4 performed is

shown.

Figure 4. Different inhibitory effects of HDAC inhibitors on IL-1 β, IL-8 and TNF-α

secretion (A) Monocytes were stimulated for 3 hours with LPS in the absence (lanes 1

and 2) or presence of TSA (1 µM, lane 3), HC-toxin (HC-T, 1 µM, lane 4), SAHA (0.5 µM,

lane 5) or tubacin (tub, 20 µM, lane 6) and then exposed to 1 mM ATP for 15 minutes

(lanes 2 to 6). Supernatants and cell lysates were analysed by western blotting for the

presence of IL-1 β. (B) HDAC inhibitors do not affect ATP-induced K+ efflux. Monocytes as

in A, untreated or exposed to 1 mM ATP for 20 minutes were analysed for the K+

intracellular content. K+ depletion is expressed as percent of intracellular K+ in LPS-

stimulated monocytes (control cells). (C) HDAC inhibitors do not affect ATP-induced [Ca2+]i

rise. Monocytes as in A were loaded with Fura-2-AM and exposed to 1 mM ATP. Results

are the mean of fluorescence of at least 50 cells in each experiment monitored for 15

minutes, and are expressed as increase in [Ca2+]i above non ATP stimulated cells

(∆[Ca2+]i)

(D) Monocytes were cultured for 18 hours with LPS and supernatants were assayed for

the presence of IL-1 β (open bars), IL-8 (striped bars) or TNF-α (closed bars) by ELISA.

Results are expressed as percent of secretion by LPS-stimulated monocytes (control

cells). One experiment out of three performed is shown. In this experiment, 18 hours

secretion by 0.7x106 LPS-stimulated monocytes was: 9.5±0.1 ng/ml for IL-1ß; 305±2 ng/ml

for IL-8; 6.1±0.3 ng/ml for TNF-α

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Figure 5. HDAC inhibitors induce acetylation of different intracellular targets. (A)

Monocytes stimulated for 3 hours with LPS in the absence or presence of ITF2357 (20 nM

or 100 nM), TSA (1 µM), HC-toxin (HC-T, 1 µM), SAHA (0.5 µM) or tubacin (20 µM) were

fractionated to obtain nuclei, that were subjected to SDS-PAGE and western blotting.

Filters were hybridized with anti histone H4 (lower panel) or anti acetyl-H4 (upper panel).

(B) Monocytes stimulated for 3 hours with LPS in the absence or presence of SAHA (0.5

µM), ITF2357 (0.1 µM), HC-toxin (HC-T, 1 µM), or tubacin (tuba, 20 µM) were lysed and

cell lysates were analysed by western blotting with anti HSP90 (upper panel) or anti-

acetyl-lysine (lower panel). The arrow in the upper panel indicates HSP90; the arrow in the

lower panel indicates a 50 K band, probably corresponding to acetylated tubulin-α. (C)

Monocytes treated as in A were fixed, permeabilized and stained with anti-tubulin-α (not

shown) or anti acetyl-tubulin-α and analyzed by cytofluorimetry. Monocytes treated with

the different inhibitors stained equally with anti tubulin-α (not shown) but differently with

anti acetyl-tubulin-α. Data are expressed as mean fluorescence intensity (mean FI) of cells

stained with anti acetyl-tubulin-α. (D) Monocytes treated as in B were lysed and analysed

by western blotting with anti tubulin-α (upper panel) or anti-acetyl-tubulin-α (lower panel).

Arrows indicate tubulin-α and acetyl-tubulin-α, respectively. For each panel, one

representative experiment of at least 4 performed is shown.

Figure 6. Microtubules are involved in IL-1ß secretion. (A) Monocytes were stimulated

for 4 hours with LPS in the absence (lanes 1 and 5) or presence of 1 µM cytochalasin D

(cytD, lane 3), 1 and 10 µM nocodazole (noc, lane 2, 6 and 7) or 10, 20 and 40 µM taxol

(lanes 4, 8-10). At the end of the 4 hours incubation, supernatants were analysed as such

(lanes 1 to 4) or 1mM ATP was added for 15 minutes (+ATP, lanes 5 to 10) before western

blot analysis for the presence of IL-1β. (B) Monocytes treated as in A were fixed,

permeabilized and stained with anti-tubulin-α (black columns) or anti acetyl-tubulin-α

(white column) and analyzed by cytofluorimetry. Data are expressed as mean

fluorescence intensity. For each panel, one representative experiment of at least 5

performed is shown.

C) Monocytes were activated 4 hours with LPS and 1 µM cytochalasin D in the absence (-,

lane 1) or presence of HC-toxin (1 µM, lane 2), ITF2357 (50 nM, lane 3), SAHA (0.5 µM,

lane 4), AACOCF3 (AA, 40 µM, lane 5), BEL (100 µM, lane 6), EGTA (5 mM, lane 7). At

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the end of the 4 hours of incubation, supernatants were analysed by western blot for the

presence of IL-1β.

Figure 7. HDAC inhibitors interfere barely with T lymphocyte or NK cell activity.

(A) NK cells were pre-treated for 4 hours with ITF2357 (50 nM), SAHA (0.5 µM), TSA (1

µM), HC-toxin (1 µM), or tubacin (10 µM) and co-incubated with 51Cr labeled K562 cells for

4 hours at a different effector-target (e/t) ratio, in the presence of the same HDAC

inhibitors. Results are expressed as percentage of cytotoxicity as described. (B) NK cells

treated for 4 hours with ITF2357 (50 nM), SAHA (0.5 µM), TSA (1 µM), HC-toxin (1 µM), or

tubacin (10 µM) were fixed, permeabilized and stained with anti-tubulin-α (not shown) or

anti acetyl-tubulin-α and analyzed by cytofluorimetry. The different cultures stained equally

with anti tubulin-α (not shown). Data are expressed as mean fluorescence intensity (mean

FI) of cells stained for acetyl-tubulin-α. (C) Allospecific T cells from 7-day MLR (105) were

pre-treated for 4 hours with ITF2357 (50 nM), SAHA (0.5 µM), TSA (1 µM), HC-toxin (1

µM), or tubacin (10 µM) and co-incubated with 104 allogeneic DCs for 18 hours in the

presence of the same HDAC inhibitors and of 1 µCi of [3H]Thymidine per well. Cells were

harvested and counted in a beta counter. Tests were conducted in triplicate, and results

are expressed as mean cpm ± SD. For each panel, one representative experiment out of

at least 3 performed is shown.

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Figure 1

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Figure 2

Figure 3

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Figure 4

Figure 5

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Figure 6

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Figure 7

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