Rapamycin Induces a Caspase-Independent Cell Death in Human Monocytes

American Journal of Transplantation 2006; 6: 1331–1341Blackwell Munksgaard

Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2006.01332.x

Rapamycin Induces a Caspase-Independent Cell Deathin Human Monocytes

A. Mercallia, V. Sordia, M. Ponzonib, P. Maffic,F. De Taddeoc, G. Gattid, P. Servidae,M. Bernardie, L. Bellioe, F. Bertuzzic, A. Secchic,E. Bonifacioa and L. Piemontia,∗

aImmunology of Diabetes Unit, bPathology Department,cMedicine I Department and eBone MarrowTransplantation and Haematology Unit, San RaffaeleScientific Institute, via Olgettina 60, 20132 Milan, ItalydDepartment of Pharmacology, University of Milan, ViaVanvitelli 32, 20129 Milan, Italy∗Corresponding author: Lorenzo Piemonti,piemonti.lorenzo@hsr.it

The immunosuppressive activity of rapamycin (RAPA)and its efficacy as an anti-rejection agent in organtransplantation have been ascribed principally to itsanti-proliferative effects on T cells, while the activ-ity on monocytes is partially unknown. In vitro, RAPAreduced monocyte survival by inducing a caspase-independent cell death. RAPA-induced monocyte celldeath (RAPA-CD) was impeded by activation of gran-ulocyte macrophage-colony stimulating factor familyreceptors or toll-like receptor 4, and by exposure toinflammatory cytokines. In vivo, in patients who re-ceived RAPA monotherapy as part of pre-conditioningfor islet transplantation, RAPA affected survival ofmyeloid lineage cells. In the peripheral blood, CD33+

and CD14+ cells decreased, whereas lymphocytesappeared unaffected. In the bone marrow, myeloidprecursors such as CD15+ and CD15+/CD16+ were se-lectively and significantly decreased, but no major cy-totoxic effects were observed. The RAPA-CD suggestsa dependence of monocytes on mammalian target ofRAPA pathways for nutrient usage, and this featureimplies that RAPA could be selectively useful as atreatment to reduce monocytes or myeloid cells inconditions where these cells negatively affect patient,suggesting a potential anti-inflammatory action of thisdrug.

Key words: Apoptosis, inflammation, rapamycin

Received 27 September 2005, revised 31 January 2006and accepted for publication 15 February 2006

Introduction

Rapamycin (RAPA) is a macrocyclic triene antibiotic pro-

duced by the actinomycete streptomyces hygroscopicus

(1). Although RAPA was originally isolated for its antifungal

properties, it is now considered an immunosuppressive

agent and used in transplantation as a result of its effi-

cacy in down regulating interleukin-2 (IL-2) receptor func-

tion (2–7) and prolonging allograft survival in various animal

models (8–11). In humans, it has been used successfully

in islet (12), combined kidney–pancreas (13), renal (14) and

liver (15) transplantation, and as rescue therapy in lung and

heart transplantation (16).

The immunosuppressive activity of RAPA and its efficacy

as an anti-rejection agent in organ transplantation have

been ascribed principally to its anti-proliferative effects on

T cells. The intracellular target of RAPA is the mammalian

target of RAPA (mTOR), a 290-kDa member of the phos-

phatidylinositol 3′-kinase-like family with serine/threonine

kinase activity that regulates protein translation, cell cycle

progression and cellular proliferation (17,18). In response

to growth factors, hormones, mitogens and amino acids,

mTOR is activated through phosphorylation by Akt (19).

Downstream events include the activation of p70 S6K and

inhibition of the 4E-binding protein-1, which result in tran-

scriptional regulation of a subset of proteins involved in cell

proliferation and survival such as the cyclin-dependent ki-

nase inhibitor p27kip1, retinoblastoma protein, cyclin D1,

c-myc or STAT 3 (20–22). The mTOR pathway acts as a cen-

tral sensor for nutrient/energy availability. In the presence

of mitogenic stimuli and sufficient nutrients and energy,

mTOR relays a positive signal to the translational machin-

ery, facilitating events that drive cell growth.

Confirming the general relevance of mTOR in regulating

cell proliferation, the study of anti-proliferative activity of

RAPA was extended to other cell types, and RAPA has

found a role as a tool to suppress neointimal hyperplasia

of coronary stents in humans with coronary artery disease

(23,24) and, due to the ability to inhibit in vitro and in vivo

proliferation of a broad range of human tumor cell lines

(20,25,26), it is classified as a new class of ‘cytostatic’ anti-

cancer agents (22,27–31).

Together with an anti-proliferative action, an activity of

RAPA in inducing cell death was also described. RAPA

causes cell death by apoptosis in BKS-2 lymphoma cell

lines (32) and in the Rh1 and Rh30 rhabdomyosarcoma

cell lines. RAPA enhances apoptosis and increases sensi-

tivity to cisplatin in the human promyelocytic leukemia cell

line HL-60, in the human ovarian cancer cell line SKOV3

(25); and it has been reported to induce differentiation or

Mercalli et al.

apoptosis in a variety of leukemia cells (25,33–37), sug-

gesting that likely mTOR has a critical role also in cell death

(26,38).

Recently, we and others have suggested that cells of the

immune system other than proliferating lymphocytes are

the targets of RAPA action (39–46). In particular, RAPA was

shown to be a good candidate for pharmacological suppres-

sion of dendritic cell functions (43–46) and the generations

of dendritic cell with immunoregulatory activity (41). While

the activity of RAPA on dendritic cell is now well described,

its effects on monocytes/macrophages are relatively un-

known (46).

In the present study, we have analyzed the effect of RAPA

in vitro and in vivo on survival of monocytes.

Methods

Monocyte culturePBMC, highly enriched monocytes (>95% CD14+) and lymphocytes were

obtained from buffy coats of blood donors (through the courtesy of Cen-

tro Trasfusionale, Ospedale San Raffaele, Milan, Italy) by Ficoll and Percoll

gradients and purified by adherence. Monocytes were cultured (maximum

7 days) at 106/mL in six-well multiwell tissue culture plates (Falcon, Becton

Dickinson, Somerville, NJ, USA) in RPMI (Biochrom, Berlin, Germany) 10%

FCS (Hyclone, Logan, UT, USA) with or without RAPA. For toll-like receptor

4 (TLR4) activation, LPS 10 ng/mL was added for at least 24 h of culture. All

cultures were tested for the presence of endotoxin (<0.03 U/mL; Lymulus

Test).

Splenic monocytes were obtained by Ficoll and Percoll gradients obtaining

a greater than 80% CD14+ population. Monocytes were then seeded at

106/mL in six-well multiwell tissue culture plates and cultured for 7 days in

RPMI 1640 with 10% FCS.

Cytokines and reagentsHuman recombinant granulocyte macrophage-colony stimulating factor

(GM-CSF) (specific activity 1.1 × 104 U/mg) was from Novartis (Basel,

Switzerland). Human recombinant IL-1b, IL-3, IL-5, TNFa, IFNc , and M-CSF

were from Pepro Tech EC Ltd. (London, England). RAPA was from Sigma-

Aldrich Chemical Co. (St. Louis, MO, USA). Cyclosporine A was from San-

doz Pharma Ag (Basel, Switzerland). Tacrolimus (FK 506) was from Fujisawa

Pharmaceutical (Osaka, Japan).

Cell morphology, viability, apoptosis and cell cycle analysisTo assess morphology, monocytes were cultured in six-well multiwell tissue

culture plates, detached without enzymatic treatment and cytospin prepara-

tions were made. Alternatively, monocytes were cultured in a chamber slide

system (Falcon). Morphologic analysis was performed by differential inter-

ference microscopy. To assess viability, intact cells were recovered, stained

with propidium iodide (PI) and evaluated by flow cytometry for plasma mem-

brane permeability to PI. To assess apoptosis, phosphatidylserine exposure

was determined using an annexin V-FITC Kit (Bender MedSystems, San

Bruno, CA, USA) in combination with PI (Sigma-Aldrich Chemical Co). Cells

were recovered, washed, labeled, with annexin V-FITC for 30 min on ice

and subsequently with 1 mg/mL PI. Annexin V/PI staining was analyzed on

a BD FACScanTM using Cell Quest software (BD Biosciences, San Jose,

CA, USA). Alternatively, apoptotic cells were identified on the basis of hy-

podiploid DNA content.

SDS-PAGE and Western blot analysisCells (13 × 106) were washed twice with cold PBS and lysed in 600 lL of

lysis buffer (50 mM Hepes-KOH, pH 7.4, 150 mM NaCl, 15 mM MgCl2,

1 mM EGTA, 1% Triton X-100 and protease inhibitors: 1 mg/mL chymo-

statin, 1 mg/mL leupeptin, 1 mg/mL antipain, 1 mg/mL pepstatin A and 1

mM phenylmethyl-sulfonyl fluoride, pH 7.5) for 45 min on ice. Lysates were

centrifuged at 13 000 rpm for 10 min at 4◦C and supernatants analyzed by

10% SDS-PAGE and transferred to nitrocellulose membrane (Amersham

Biosciences, Piscataway, NJ, USA). After 1 h incubation in blocking solu-

tion (5% dried milk in PBS/0.1% Tween 20), filters were incubated with

the appropriate antibodies: anti-bcl-2 (N-19), anti-Bax (N-20) and anti-bcl-xl

(S-18) rabbit polyclonal antibodies (Santa Cruz Biotechnology, Santa Cruz,

CA, USA); anti-p53 (Ab-2) mouse monoclonal antibody (Oncogene Research

Product, Calbiochem, San Diego, CA, USA); anti-Caspase-3 rabbit policlonal

antibody (Pharmingen International, San Diego, CA, USA). Proteins were

visualized with peroxidase-coupled secondary antibody (Amersham Bio-

sciences), using enhanced chemiluminescence for detection (Amersham

Biosciences).

Analyses of caspase activity and inhibition of caspasesThe activation of caspase-3, -7, -9, -8 was analyzed using Carboxyfluorescein

FLICA Assay Kits (B-Bridge International, Inc., Sunnyvale, CA, USA) accord-

ing to the manufacturer’s instructions. For caspase inhibition, cells received

RAPA 24 h before subG1 or annexin V positive fraction was determined. Irre-

versible caspase-3 inhibitor (fluoromethyl ketone-(fmk)-derivatized oligopep-

tides, zDEVD-fmk) was applied simultaneously with RAPA induction. It binds

to the active site of caspase-like proteases and thereby inhibits the enzy-

matic activity: Inhibitor was dissolved in DMSO at 100 mM and diluted with

growth medium to final concentrations of 10, 50 and 100 lM.

RAPA-treated patientsPatients with type 1 diabetes who had reduced awareness of hypoglycemia,

brittle diabetes or progressive complications, despite optimization of insulin

therapy, were candidates for solitary islet transplants (12). Nine patients re-

ceived RAPA (0.1 mg/kg/day) for 37–189 days prior to islet transplantation.

At transplant, patients received 1 mg/kg dacluzimab every 2 weeks (five

doses), RAPA with target levels of 12–15 ng/mL for the first 3 months and

7–10 ng/mL thereafter, and FK506 at a target level of 3–6 ng/mL (12,47). Pa-

tients were aged 35 ± 7 years, had diabetes for 21 ± 8 years, an hemoglobin

A1c (HbA1c) of 8.53 + 0.95%, serum creatinine of 0.8 + 0.11 mg/dL, insulin

requirement of 43 + 16 U/day, and 5 were male. All patients gave informed

consent for the investigations. The protocols were approved by the ethical

committee of the Istituto Scientifico Ospedale San Raffaele.

Hematopoietic analysesRelative and absolute numbers of myeloid and lymphoid populations were

analyzed in peripheral blood at day 0, 7, 14, 21 after RAPA treatment. Bone

marrow (BM) aspirate and biopsy were taken before and 21 days after

RAPA treatment. Flow cytometry analysis was performed on aspirates. BM

biopsies were fixed with Bouin’s solution, decalcified with an EDTA-based

solution, paraffin embedded and cut at 4 lm thick sections. Biopsies were

stained with hematoxylin and eosin, Giemsa and reticulin silver impreg-

nation. Reticulin content was evaluated as previously described (48). Im-

munohistochemistry was performed by the avidin–biotin peroxidase com-

plex method and visualized with 3,3′-diaminobenzidine tetrahydrochloride

cromogen method, using a DAKO automated immunostainer. For antigen

retrieval, slides were placed in 0.01 M EDTA buffer at pH 8 and pH 9 for

CD34 and phosphoglucomutase (PGM1), and underwent three 4-min and

two 5-min 780 W cycles at 90◦C in a microwave oven before immunos-

taining, respectively. Staining of blasts and micro-vessels were performed

using anti-CD34 (QBEnd/10, Novocastra, Newcastle upon Tyne, UK; work-

ing dilution 1:100), and for monocytes and macrophages using anti-CD68R

(PGM1, Dako, Copenhagen, Denmark; dilution 1:150).

1332 American Journal of Transplantation 2006; 6: 1331–1341

Inhibiting mTOR Induces Monocyte Death

Microvascular density (MVD) was measured by visual micro-vessel scoring

as previously described (42). All fields of the section were counted at 200×microscopic magnification and a final average MVD value for the whole

section was registered. Hypocellular areas as well as areas with serous fat

atrophy were excluded from MVD estimation.

Statistical analysisData are expressed as mean ± SD or median and interquartile interval and

compared by Student’s t-test or Wilcoxon signed rank test. Comparisons of

time 0 versus 7, 14 and 21 days of RAPA treatment were performed using

the non-parametric Wilcoxon signed rank test for paired samples. For all

analyses, a two-tailed p-value of 0.05 was considered significant. Statistical

analyses were performed using the Statistical Package for Social Science

(SPSS 11.0; SPSS, Chicago, IL, USA).

Results

RAPA affects monocyte viability by inducingcell deathThe effect of RAPA on cell survival was assessed in vitro on

PBMC. Human PBMC from healthy donors were cultured

in the presence of RAPA (0.1–10 ng/mL). A statistically sig-

nificant (p < 0.01, n = 24) dose-dependent decrease of

PBMC recovery was detected starting from drug concen-

trations of 1 ng/mL (Figure 1A). In the presence of RAPA, a

preferential loss of monocytes was clearly present with a

lower number of myeloid cell surviving after 5 days of cul-

ture (Figure 1B). Consistent with a selective monocytes

cell death, RAPA significantly increased double positive

0 2 3 5

10510.1

Time (days)

RAPA(5 ng/ml)CtrlRAPA (ng/ml)

- + - +

Annexin V- Annexin V+

- + - +

CD14+ CD3+

Day 1 Day 2 Day 1 Day 2

0 1023

Parameter 1

0 1023

Parameter 1

0 1023

Parameter 1

0 1023

Parameter 1

Figure 1: In vitro effect of RAPA on PBMC. (A) Dose-dependent cell death of PBMC with RAPA. Cell recovery after 2, 3 and 5 days in the

presence of increasing concentrations of RAPA was determined by counting viable cell using the Burker chamber on the basis of Trypan

blue exclusion. Data are presented as means ± SE, n = 24. The absolute number of PBMC in control group was 700, 667 ± 50, 388 ± 95

and 304 ± 100 × 103/mL, respectively, for days 0, +2, +3 and +5 of culture. (B) Forward-side scatter profiles of RAPA (5 ng/mL)-treated

PBMC after 1, 2 and 5 days of culture. The panel is a representative experiment of 24 performed. The lymphocyte (Ly) and the monocyte

(Mo) fractions are outlined. (C) Annexin V/CD3 or CD14 double positive cells in PBMC after treatment with RAPA (5 ng/mL) after 1 and

2 days of culture. Data are presented as median, n = 4. ∗<0.05 versus control.

CD14+/annexinV+ cells (but not CD3+/annexinV+ cells) in

PBMC after a 48-h culture period (Figure 1C). A great vari-

ability was present between PBMC from different donors

in terms of control cell survival and monocyte susceptibility

to RAPA action.

To define the characteristics (49–52) of the cell death in-

duced by RAPA, purified monocytes were assessed using

morphology, DNA staining, annexin V/PI staining, caspase

activation. Consistent with apoptotic cell death, RAPA sig-

nificantly increased annexin V positive cells and cells with

hypodiploid DNA content in purified monocytes during a

48-h culture period (Figure 2A–2C). Not all characteris-

tics were consistent with apoptosis, however. Optical mi-

croscopy showed that 48-h culture with RAPA reduced

the percentage of monocytes with a morphology of liv-

ing cells (53 ± 25% vs 75 ± 9% in control cultures, p =0.04), and significantly increased the percentage of cells

with a morphology of dying cell (but not apoptotic cell)

characterized by cell swelling, marked vacuolization of the

cytoplasm, and membrane blebbing (44 ± 12% vs 22 ±8.5%; p = 0.038; Figure 2D). Moreover, despite hyploidia,

and annexin V positivity, RAPA treatment did not appear

to affect the protein expression of apoptosis promoter and

suppressor genes like Bcl-2, p53, Bax, Bcl-xl and Caspase-3

(Figure 3A).

To confirm that RAPA-induced cell death (RAPA-CD)

without caspase activation, a time course analysis of

American Journal of Transplantation 2006; 6: 1331–1341 1333

Mercalli et al.

Figure 2: RAPA-induced cell death in monocytes. (A) Purified monocytes were incubated for 48 h with or without RAPA (5 ng/mL)

and subsequently analyzed by annexin V/PI staining. The panel is a representative experiment of 6 performed. (B) Cell cycle analysis

and hypoploid fraction (M1) on purified monocytes cultured in the presence of 5 ng/mL of RAPA for 1, 2 or 5 days. The panel profiles

are a representative experiment of 16 performed. The values reported are the medians of 16 experiments. (C) Time course analysis of

RAPA-induced cell death. Purified monocytes were cultured with or without RAPA (5 ng/mL) and evaluated for DNA content and annexin

V/PI staining at 1, 3, 6, 12, 24 and 48 h. Data are presented as mean ± SD. n = 3. (D) Monocyte morphology in the presence of RAPA.

Left: differential interference microscopy showing monocytes cultured in a chamber slide system in the presence of 5 ng/mL of RAPA.

Black arrows point to the dying cells. Left: monocytes were cultured for 48 h in the presence of 5 ng/mL of RAPA and a cell counting was

performed. Data are expressed as percentage of cells/field. At least 20 fields for each experiment were counted. The data represent the

median of six experiments.

caspase-3/7, -8 and -9 activation using fluorochrome-

labeled inhibitors of caspase (FLICA) was performed

(Figure 3B). Monocytes cultured with or without RAPA

(5 ng/mL) for 1, 3, 6, 12, 24 and 48 h were evalu-

ated for FLICA. Even if RAPA significantly increased

the sub G1 fraction and the annexin V positive cells

starting from 12-h culture (Figure 2C), simultaneously it

did not affect FLICA+ cells. Moreover, double staining

with FLICA (FAM-DEVD-FMK to detect caspase-3 and

caspase-7) and PI performed 12 and 24 h after RAPA

exposure demonstrated that there was an increase in

the FLICA−/PI+ subpopulation without change in the

FLICA+/PI− and FLICA+/PI+ subpopulations, confirming

the induction of cell death without the caspase-3/7 activa-

tion (Figure 3C). Finally, the RAPA-CD was not reversed

by the irreversible caspase-3 inhibitor (fluoromethyl

ketone-(fmk)-derivatized oligopeptides, zDEVD-fmk)

(Figure 3D).

The effect of RAPA was also investigated on differenti-

ated macrophage and human spleen-derived monocytes,

as an alternative cell source. RAPA did not significantly af-

fect the survival after in vitro maturation of monocytes to

macrophages, suggesting that the sensitivity to RAPA-CD

is lost after differentiation (Figure 4A), while RAPA-CD in

freshly isolated spleen monocytes (data not shown).

GM-CSF family receptor activation prevents RAPA-CDCytokines of the IL-3, IL-5 and GM-CSF family are important

regulators of hematopoiesis through the modulation of pro-

liferation, differentiation and survival of various hematopoi-

etic cell lineages and their precursors (53,54). Since it is

known that a major function of IL-3/IL-5/GM-CSF cytokines

is the inhibition of cell death in their target cells (55), we

tested the ability of these cytokines to modulate RAPA-

CD in monocytes. GM-CSF and IL-3 prevented RAPA-CD,

whereas IL-5 that specifically targets eosinophils did not.

The effect was dose dependent, starting from 1 ng/mL

(Figure 4B).

TLR4 activation and inflammatory cytokines preventRAPA-CDIt is known that monocyte viability is regulated by biologi-

cally active peptides released during inflammation (56,57)

and by LPS-mediated TLR4 activation. We therefore eval-

uated the effect of IL-1b, LPS and TNFa on RAPA-CD

bcl-xl

caspase-3

0 .5 5

RAPA (ng/ml)A

0 24 48 0 24 48 0 24 48FLIC

ositiv

Caspase 3/7 Caspase 9 Caspase 8

BRAPACtrl

FLICA (Caspase 3/7)

Ctrl +RAPA19% 7%

32% 9%

26% 12%

38% 14%

RAPA (ng/ml) - 10- 10 10 - 10- 10 10

zDEV-fmk (μM) 100- 100- 10 100- 100- 10

SubG1 AnnexinV+

Figure 3: RAPA-induced cell death in monocytes is caspase independent. (A) Western blot analysis of bcl-2, Bax, bcl-xl, p53, caspase-

3 in 24-h RAPA-treated monocytes. An equal loading of proteins on to each lane was performed. (B) Purified monocytes cultured 48 h

with or without RAPA were incubated at time 1, 3, 6, 12, 24 and 48 h with FLICA (FAM-DEVD-FMK to detect caspase-3 and caspase-7;

FAM-LEHD-FMK to detect caspase-9, FAM-LETD-FMK to detect caspase-8). The data represent the mean of three experiments. (C)

Purified monocytes cultured 12 and 24 h with or without RAPA were incubated with FLICA (FAM-DEVD-FMK to detect caspase-3 and

caspase-7), subsequently briefly exposed to PI and analyzed by flow cytometry. Scattergrams represent binding of FLICA by untreated

cells (control) or cells treated with RAPA (5 ng/mL). Four distinct subpopulations could be distinguished based on differences in FLICA and

PI fluorescence. The subpopulations represented the sequential transition from cells that were both FLICA and PI negative (FLICA−/PI−:

living cell), to early caspase activation (FLICA+/PI−: early apoptosis), to loss of plasma membrane ability to exclude PI (FLICA+/PI+: late

apoptosis), and finally when the cell propensity to bind FLICA was eliminated (FLICA−/PI+: very late apoptotis or necrosis). The panel

is a representative experiment of 3 performed. (D) Treatment with zDEVD-fmk (10–100 lM), a caspase-3 specific inhibitor, for 24 h was

used to evaluate the involvement of caspase-3 in RAPA-CD. zDEVD-fmk was applied simultaneously with RAPA for 24 h before subG1 or

annexin V positive fraction was determined. The data represent the mean of two experiments.

(Figure 4B). TLR4 activation by LPS completely prevented

RAPA-CD starting from 1 ng/mL, and IL-1b at concentra-

tions up to 100 ng/mL only partially inhibited RAPA-CD.

TNFa alone (0.1–10 ng/mL) did not modulate RAPA-CD,

whereas the simultaneous exposure to TNFa and INFccompletely prevented RAPA-CD. It is reported that mono-

cytes are able to produce GM-CSF when activated, sug-

gesting both autocrine and paracrine control of cell survival

after activation. As predicted, LPS stimulated GM-CSF re-

lease by monocytes to concentrations that were able to

prevent RAPA-CD (>1 ng/mL). Despite this, the addition of

blocking anti-GM-CSF antibodies did not revert LPS action

on preventing RAPA-CD, indicating that GM-CSF autocrine

production was not solely responsible for this effect of LPS

(data not shown).

In vivo effect of RAPA on peripheral blood leukocyteand BMIn view of the in vitro effect of RAPA, we examined mono-

cytes counts in peripheral blood of type 1 diabetic patients

who were treated with RAPA as part of their immuno-

suppression therapy after pancreatic human islet trans-

plant. Nine patients who received RAPA (0.1 mg/kg/day)

as monotherapy prior to islet transplant were analyzed

prospectively. Analysis of monocyte counts pre- and post-

RAPA treatment showed a persistent reduction in mono-

cytes after drug administration (Figure 5A and 5B). Also

platelet counts decreased significantly in the first 21 days

of treatment, whereas red blood cell counts appeared un-

affected (Table 1). Peripheral blood leukocytes were eval-

uated by flow cytometry in 7 of 9 patients at day 0, 7,

14, 21 after RAPA treatment (Table 1). With respect to

the myeloid lineage, RAPA treatment resulted in a sig-

nificant rapid decrease in CD33+ blood cells and a de-

layed decrease of CD14+ blood cells (Figure 5C). With

respect to the lymphoid lineage, peripheral CD2+, CD3+,

CD19+, CD16+ cells were unaffected (Figure 5C) and a

transient increase in CD4+CD45RO+, CD8+CD45RO+ and

CD19+CD22+ cells was evident (Table 1). All nine patients

had BM biopsies before and 21 days after RAPA ther-

apy. Flow cytometry analysis of BM confirmed an effect

of RAPA on the myeloid lineage (Table 2). Some myeloid

precursors (CD15+, CD15+/CD16+) significantly decreased

during RAPA treatment. BM CD14+ cells were not sig-

nificantly reduced. The lymphoid lineage appeared gen-

erally unaffected. Changes in the overall cellularity were

Mercalli et al.

Annexin V

Ctrl RAPA (10 ng/ml)

rentia

IL-1 β LPS TNF α

IL-3 IL-5 GM-CSF

0.1 110

** * * *

Figure 4: RAPA-induced monocyte death modulation: macrophage differentiation, GM-CSF family receptors and TLR4 activation.(A) Sensitivity to RAPA-induced cell death of freshly isolated monocytes and of macrophages. Human monocyte cells were separated

through Ficoll/Percoll gradients and adherence. Freshly isolated monocytes (CD14+/CD68−) and mature macrophages (CD14−/CD68+,

obtained over 4 days, cultured in the presence of M-CSF 20 ng/mL) were incubated for 3 days with RAPA (10 ng/mL) and subsequently

analyzed by annexin V/PI staining. RAPA-induced cell death in freshly isolated monocytes but not in mature macrophages. The panel is a

representative experiment of 4 performed. (B) GM-CSF family receptors and TLR4 activation prevents RAPA-induced monocyte death.

Monocytes were cultured with RAPA (5 ng/mL) in the absence (filled column) or presence of different concentrations of IL-3, IL-5, GM-CSF,

IL-1b, LPS and TNFa for 72 h and cell cycle analysis was performed. The proportion of hypodiploid cells remaining in culture (ordinate

axis) is shown. The data represent the mean ± SE of three independent experiments. ∗p < 0.05, Student’s t-test.

observed in two patients: one (#2) had a slightly hypocel-

lular marrow after RAPA treatment, and the second (#7)

had a transition from hypercellular pre-treatment to nor-

mocellular BM after RAPA treatment. Two patients with

slightly hypocellular marrow (#3, #6) did not modify this

feature after RAPA treatment. Myeloid-erythroid ratios in

the BM were within physiological limits in all patients pre-

and post-treatment. All patients had normal maturation

in pre-treatment samples and four patients (#2, #4, #6,

#8) displayed slight maturation defects involving both ery-

throid and myeloid lineages in post-RAPA evaluation. In-

creased numbers, or a non-homogeneous distribution of

CD34+ precursors was not appreciable in these BM. Re-

active lymphoid aggregates were observed in two patients

(#1, #9), and in one of these, this aggregate was present

only after RAPA treatment. Slight fibrosis was present in

three patients: in one patient (#4), a focal reticulin increase

was present after RAPA treatment, while in two patients

(#1, #7), the biopsy taken after RAPA treatment showed a

reduction and the disappearance of fibrosis, respectively.

Two patients (#2, #3) had areas of gelatinous transforma-

tion of the BM, a phenomenon reported in type 1 dia-

betes (58). Micro-vessel density and macrophage distribu-

tion were unaffected by RAPA treatment (Table 2).

In view of the effect of RAPA, we also retrospectively ex-

amined monocyte counts in the peripheral blood of pa-

tients with type 1 diabetes, who were treated with RAPA

plus FK506 as part of their immunosuppression therapy

after pancreatic human islet transplant (Edmonton proto-

col) (12). In seven patients, due to the side effects, RAPA

(0.1 mg/kg/day) was replaced by mycophenolate mofetil

(1–2 g/day) without any change of the other immunosup-

pressive drugs. Monocyte counts increased persistently

following RAPA replacement (Figure 6A and 6B).

Moreover, retrospective analysis of monocyte counts pre-

and post-transplantation evidenced a persistent monocyte

decrease in patients who received islet transplantation

with FK506 plus RAPA maintenance therapy, but not in pa-

tients who received transplants under maintenance thera-

pies that did not include RAPA (Figure 6C).

-100 -50 0 50 100M

RAPA (0.1 mg/kg/day)

pre post

P=0.004

Absolu

0 7 14 21

Figure 5: In vivo effect of RAPA on peripheral blood leukocytes. (A and B) Circulating blood monocyte numbers 100 days before

and 100 days after drug treatment in 9 patients with type 1 diabetes who received RAPA (0.1 mg/kg/day) as monotherapy prior to islet

transplant. The absolute numbers for each patient are shown (A) and the means ± SD of circulating blood monocytes before and after

RAPA treatment were compared using Student’s t-test for paired samples. (C) The in vivo effect of RAPA was studied prospectively in

seven of nine patients receiving RAPA (0.1 mg/kg/day) for at least 30 days prior to islet transplant. Peripheral blood leukocytes (CD2+,

CD3+, CD16+, CD19+, CD33+, CD14+) were evaluated at day 0, 7, 14, 21 by flow cytometry. The absolute numbers for each patient are

shown. ∗p < 0.05 versus day 0, Wilcoxon Signed Rank test for paired samples.

Table 1: Peripheral blood population before and after RAPA treatment in humans

Pre-RAPA1 +7 +14 +21

WBC 6700 (5950–7700) 6400 (4950–7550) 5800 (4050–6250) 5100∗ (4550–6950)Myeloid/Erythroid

CD33 4527 (4014–5340) 4316 (3027–5340) 2722∗∗ (2479–4325) 3294∗ (2256–3977)

CD14 451 (348–727) 527 (394–650) 472 (325–657) 375∗ (265–446)

PLT 253 (219–321) 215∗ (180–322) 194∗∗ (163–239) 222∗∗ (167–267)

RBC (1012/L) 4.58 (4.28–5.07) 4.66 (4.24–4.92) 4.68 (4.28–5.12) 4.53 (4.30–4.92)

HCT (%) 43.5 (35.7–43.9) 43.5 (38.6–44.6) 42.5 (34.7–45.2) 41.8 (35.1–44.2)

Hb (g/dL) 14.5 (12.3–14.9) 14.5 (12.37–14.77) 14.4 (11.3–15.4) 14.3 (11.5–14.9)Lymphoid

CD2 1246 (993–1927) 1240 (1097–2036) 1205 (1048–1657) 1267 (942–1585)

CD19 274 (132–447) 419 (146–617) 384 (155–593) 265 (140–461)

CD19/CD22 131 (93–298) 330∗ (121–484) 253∗ (155–417) 235 (117–443)

CD19/CD25 67 (27–134) 77 (27–181) 70 (43–227) 70 (33–164)

CD3 1133 (809–1900) 1181 (999–1917) 1047 (895–1654) 1163 (781–1484)

CD3/CD16 34 (15–74) 26 (16–65) 18 (6–47) 20 (7–53)

CD16 186 (159–207) 181 (122–326) 202 (153–273) 148 (135–183)

CD3/CD4 596 (435–884) 664 (552–863) 552 (446–742) 626 (448–771)

CD3/CD8 271 (217–628) 335 (250–691) 259 (185–723) 302 (201–706)

CD4/CD45Ra 289 (138–354) 246 (170–325) 211 (137–317) 257 (130–335)

CD8/CD45Ra 238 (126–434) 245 (166–624) 210 (179–534) 263 (129–547)

CD4/CD45Ro 384 (268–613) 522∗ (331–766) 415 (340–566) 510 (287–605)

CD8/CD45Ro 97 (36–226) 127∗ (72–274) 134 (58–264) 103 (58–312)

CD4/25 331 (162–506) 361 (228–478) 238 (195–415) 257 (141–397)

CD4/DR 51 (30–67) 36 (21–75) 37 (12–56) 38 (24–52)

CD8/DR 34 (13–101) 33 (17–194) 25 (12–71) 34 (20–87)

1Data are expressed as median of cell count (106/L) and interquartile interval.∗p < 0.05; ∗∗p < 0.01; Wilcoxon Signed Rank test for paired samples.

Mercalli et al.

Table 2: Bone marrow immunohistochemistry and phenotype be-

fore and after RAPA treatment

Pre-RAPA Post-RAPA p1

Immunohistochemistry2

CD34+ 4 (3–5) 5 (3–6) 0.47

CD68+ (PGM1) 23 (22–25) 23 (22.5–26) 0.59

MVD (CD105+) 9 (4.6–14.5) 11 (5.4–18.5) 0.26Flow cytometry2

Lymphoid marker

CD19 20 (16–25) 22 (12–27) 0.59

CD22 21 (15–26) 16 (13–27) 0.48

CD19/CD22 19 (15–24) 16 (13–27) 0.85

CD19/CD25 3 (1.5–8.1) 5.4 (2.1–8.3) 0.85

CD16 10.5 (9.4–14) 11 (8.8–13) 0.51

CD3 62 (55–75) 68 (52–75) 0.59

CD16/CD3 2.4 (0.6–5) 2.1 (1.4–3.2) 0.76

CD3/CD4 31 (28–32) 34 (22–36) 0.51

CD3/CD8 29 (18–40) 26 (18–38) 0.21

CD4/CD45Ro 23 (14–27) 18 (17–26) 0.89

CD8/CD45Ro 10 (8–19) 6 (5–13) 0.01

CD4/CD45Ra 9.7 (7–21) 13 (10–18) 0.44

CD8/CD45Ra 18 (13–26) 19 (11–28) 0.95

CD4/DR 3.6 (2.2–7.7) 4.2 (3.1–8.7) 0.77

CD8/DR 4.8 (1.9–13) 4.3 (2.5–5.9) 0.12

CD4/CD25 9.6 (2.3–16.5) 14 (4.8–16) 0.67

CD2 71 (60–79) 72 (67–79) 0.76Myeloid marker

CD33 71 (68–80) 69 (65–73) 0.21

CD14 6 (4.5–7) 6.5 (5.1–6.75) 0.85

CD34 2.5 (1.5–3) 2.6 (2–3.5) 0.03

CD16 39 (28–54) 44 (38–64) 0.5

CD13 31 (22–44) 29 (21–35) 0.23

CD15 62 (59–70) 55 (50–61) 0.01

CD15/CD16 51 (38–56) 35 (29–43) 0.01

CD16/CD13 25 (18–39) 22 (17–26) 0.59

CD16/CD33 35 (29–59) 33 (24–42) 0.07

CD33/CD14 6.2 (5–7) 6.5 (5.7–7.12) 0.4

CD16/CD34 1.6 (0.6–1.8) 0.9 (0.75–2) 0.94

1Statistical analysis was performed by Wilcoxon Signed Rank

test for paired samples.2Data are expressed as median of percentage (interquartile

interval).

Discussion

This is the first study describing the in vitro and in vivo ef-

fects of RAPA on monocyte survival. It demonstrates that

RAPA plays a role in the physiology and survival of mono-

cyte/myeloid lineage cells. In fact, RAPA in vitro induced

monocyte death but spared lymphocytes. RAPA-induced

monocyte death via a caspase-3 independent pathway.

In vivo RAPA induced a significant reduction in peripheral

monocytes, white blood cell CD14+ or CD33+, and platelet

counts. A role for RAPA on myeloid lineage survival was

confirmed also at the level of the BM where, although no

major short-term toxicity was observed, myeloid precur-

sors such as CD15+ and CD15+/CD16+ were selectively

and significantly decreased during RAPA treatment. The in

vivo inhuman study was made possible by the evaluation

of a unique group of patients without any hematologic ab-

normality who were treated in monotherapy with RAPA.

Thus, RAPA appeared highly relevant to normal mono-

cyte survival, suggesting that it may have both a poten-

tial anti-inflammatory action in healthy subjects and a cyto-

static/cytotoxic effect in certain myeloid-monocytic malig-

nancies.

Since the target of RAPA action is the inhibition of mTOR,

our findings suggest that mTOR pathways are essential

in monocyte/myeloid lineage survival. A potential role of

mTOR on survival of non-proliferating cells was suggested

by studies on osteoclasts (59,60) and dendritic cells (42–

44,46), cell types that can be derived from monocytes. Our

findings suggest and confirm that myeloid lineage cells are

peculiarly sensitive to RAPA and that the cell death which

occurs as a result of inhibition is consistent with a vital role

of mTOR in nutrient usage by cells. It is important to note

that by classical approaches (hypodiploid DNA content, an-

nexin V staining) cells appeared to be undergoing apoptotic

cell death, but that a more detailed analysis revealed that

death did not appear to affect the typical apoptotic path-

ways. Even if the exact definition and classification of the

RAPA-CD in monocyte could not be done without an ultra-

structural analysis, the absence of caspases-3/7, -8 and -9

activation after RAPA exposure and the evidence that cell

death takes place when caspase activity has been pharma-

cologically neutralized permit us to classify RAPA-CD as a

caspase-independent cell death (49–52).

Monocyte death resulting from RAPA was prevented by

macrophage differentiation and by activation through at

least three different signal pathways: GM-CSF/IL-3 recep-

tors, TLR4 and IL-1b/TNFa/IFNc receptors. This suggests

that monocyte survival is regulated by different intracellular

pathways acting in concert with the differentiation or ac-

tivation state of the cell. The mTOR pathway inhibited by

RAPA, regulating the survival of the myelo-monocyte lin-

eage in the resting state, may provide an important home-

ostatic mechanism for controlling the number of phago-

cytes available in a manner dependent upon basal nutri-

tional status. Consistent with our findings and this hy-

pothesis are the findings that patients with anorexia have

hematological changes (lower total leukocyte, neutrophil,

monocyte and platelet counts, normal hemoglobin and lym-

phocytes count) (61), similar to those found here in patients

treated with RAPA. The prevention of RAPA-CD by activa-

tion via TLR4 or IL-1b/TNFa/IFNc suggests the existence

of mTOR-independent survival pathways that regulate the

monocyte survival after recruitment to a site of inflam-

mation or infection; and this is consistent with previous

work showing that IL-1b and LPS can prevent spontaneous

monocyte-programmed cell death in culture (56,57). Also,

GM-CSF action on monocytes leads to activation of p70S6

kinase via a RAPA-resistant pathway, MAPK-related ki-

nase, and a RAPA-sensitive pathway, mTOR-related kinase

(62). Since inhibition of some type of caspase-independent

cell death strongly correlates with phosphorylation of

RAPA (ng/ml) 0.009.85 (6.2-16.6)

FK506 (ng/ml) 0.003.96 (2.83-4.9)CyA (ng/ml) 235 (151-361)0.00

MMF YesNoSteroid 10 mg/dayNo

ATG/ALG YesNo

anti CD25 NoYes

25 50 750

0 25 50 750M

Days Days

-100 -50 0 50 100

RAPA (0.1 mg/kg/day)

MMF(1-2g/day)

RAPA MMF

P=0.001

Figure 6: In vivo effect of RAPA onperipheral blood leukocytes. (A and

B) Circulating blood monocyte counts

100 days before and 100 days af-

ter RAPA (0.1 mg/kg/day) replacement

with mycophenolate mofetil (MMF) in

seven patients with type 1 diabetes

who received RAPA and FK506 as im-

munosuppressive therapy after islet

transplantation. The absolute numbers

for each patient are shown (A) and

the mean ± SD of circulating blood

monocytes before and after RAPA re-

placement were compared using the

Student’s t-test for paired samples.

(C) Circulating blood monocyte number

(mean) in patients with type 1 diabetes

who received islet transplants (ITA, n =12) or islet after kidney transplant (IAK,

n = 29). The immunosuppressive regi-

men and the median serum concentra-

tion of drugs are shown (B).

ribosomal protein S6 (63), the activation of the RAPA-

resistant pathway could explain the ability of GM-CSF to

prevent monocyte death in the presence of RAPA.

In contrast to our findings, Woltman et al. (46), using a simi-

lar experimental design, reported that monocytes were un-

affected by RAPA. It is commonly experienced that human

peripheral blood monocytes spontaneously and progres-

sively lose viability when cultured in the absence of stimuli

(56). Of note is that the monocytes in the study of Wolt-

man were devoid of cell death after culture, suggesting

that they may have been rendered resistant to cell death

through activation via FCS or via adhesion during the pu-

rification steps, thereby explaining the discrepancies.

In summary, this in vivo and in vitro study found that RAPA

will affect survival of monocyte and myeloid lineage cells.

Thus RAPA treatment could be selectively useful as a treat-

ment to reduce monocytes or myleloid cells, and therefore

could be considered not only a lymphocyte ‘oriented’ ther-

apy but also a potential anti-inflammatory therapy. More-

over, the mechanism of cell death observed through mTOR

inhibition in monocytes identifies an exquisite dependence

of these cells on mTOR pathways for nutrient usage, sug-

gesting that mTOR is important for regulating homeostasis

also of certain non-proliferating cells in conditions of limited

nutrient availability.

Acknowledgments

Valeria Sordi is enrolled as a PhD student at the Ludwig-Maximilians Uni-

versity of Munich, Germany.

This work was supported by Telethon Italy and the Juvenile Diabetes Re-

search Foundation (JT01Y01).

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