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American Journal of Transplantation 2006; 6: 1331–1341Blackwell Munksgaard
C© 2006 The AuthorsJournal compilation C© 2006 The American Society of
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,[email protected]
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
1331
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
Day 1
Day 2
Day 5
0.75
1
0 2 3 5
10510.1
Ce
llre
co
ve
ry
(% o
f C
trl)
A
Time (days)
B
Mo
Ly
RAPA(5 ng/ml)CtrlRAPA (ng/ml)
C
0
25
50
75
100
- + - +
Annexin V- Annexin V+
- + - +
CD14+ CD3+
Rapa
Day 1 Day 2 Day 1 Day 2
*
%
10
23
0
0 1023
Pa
ram
ete
r 2
10
23
0
0 1023
Pa
ram
ete
r 2
10
23
0
0 1023
Pa
ram
ete
r 2
Parameter 1
10
23
0
0 1023
Pa
ram
ete
r 2
Parameter 1
10
23
0
0 1023
Pa
ram
ete
r 2
Parameter 1
10
23
0
0 1023
Pa
ram
ete
r 2
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
1334 American Journal of Transplantation 2006; 6: 1331–1341
Inhibiting mTOR Induces Monocyte Death
bcl-2
bax
bcl-xl
p53
caspase-3
0 .5 5
RAPA (ng/ml)A
0
25
50
0 24 48 0 24 48 0 24 48FLIC
A p
ositiv
e c
ells
(%)
Caspase 3/7 Caspase 9 Caspase 8
BRAPACtrl
FLICA (Caspase 3/7)
PI
Ctrl +RAPA19% 7%
5%
32% 9%
5%
26% 12%
6%
38% 14%
5%
12h
24h
C
0
15
30
RAPA (ng/ml) - 10- 10 10 - 10- 10 10
zDEV-fmk (μM) 100- 100- 10 100- 100- 10
SubG1 AnnexinV+
D
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
American Journal of Transplantation 2006; 6: 1331–1341 1335
Mercalli et al.
Annexin V
71
24
41
55
29
124
Ctrl RAPA (10 ng/ml)
84 3
76
91 3
24
Fre
shly
isola
ted
Macro
phage
diffe
rentia
ted
PI
A
0
50
100
Ctr
l
RA
PA
Sub G
1 (%
)
*
110
100 1
10
100
10
10
IL-1 β LPS TNF α
+ I
NF
50
0U
* * *
*
ng/ml
IL-3 IL-5 GM-CSF
0.1 110
0.1 110
0.1 110
** * * *
*
B
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).
1336 American Journal of Transplantation 2006; 6: 1331–1341
Inhibiting mTOR Induces Monocyte Death
0
0.5
1.0
-100 -50 0 50 100M
o (
10
12/L
)
RAPA (0.1 mg/kg/day)
Days
A
Mo
(1
01
2/L
)
0
0.5
1.0
pre post
B
P=0.004
Days
Absolu
tenum
ber
(10
12/L
)
0
3.5
7
0 7 14 21
* *
CD33
0
0.5
1
0 7 14 21
*
CD14
0
0.5
1CD19
0
1.5
3CD3
0
0.5
1CD16
0
1.5
3CD2
C
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.
American Journal of Transplantation 2006; 6: 1331–1341 1337
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
1338 American Journal of Transplantation 2006; 6: 1331–1341
Inhibiting mTOR Induces Monocyte Death
IAK
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
ITA
25 50 750
0.5
0 25 50 750M
o (
10
12/L
)
Days Days
A
0
0.5
1.0
-100 -50 0 50 100
RAPA (0.1 mg/kg/day)
MMF(1-2g/day)
0
0.5
1.0
Mo
(1
01
2/L
)M
o (
10
12/L
)
B
C
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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