Stimulatory effect of Eucalyptus essential oil on innate cell-mediated immune response

Abstract

Besides few data concerning the antiseptic properties against a range of microbial agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence of Eucalyptus oil (EO) extract on the monocytic/macrophagic system, one of the primary cellular effectors of the immune response against pathogen attacks. The activities of this natural extract have mainly been recognized through clinical experience, but there have been relatively little scientific studies on its biological actions. Here we investigated whether EO extract is able to affect the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression induced by the chemotherapeutic agent 5-fluorouracil (5-FU).
Morphological activation of human MDMs was analysed by scanning electron microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit.
EO is able to induce activation of MDMs, dramatically stimulating their phagocytic response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines and requires integrity of the microtubule network, suggesting that EO may act by means of complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune response was also observed in vivo after EO administration, mainly involving the peripheral blood monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly decreased by the chemotherapic.
Our data, demonstrating that Eucalyptus oil extract is able to implement the innate cell-mediated immune response, provide scientific support for an additional use of this plant extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate further investigations also using single components of this essential oil. This might drive development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-suppressive pathologies, in infectious disease and after tumour chemotherapy.

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BMC Immunology
Open Access
Research article
Stimulatory effect of Eucalyptus essential oil on innate cell-mediated
immune response
Annalucia Serafino*1, Paola Sinibaldi Vallebona2, Federica Andreola1,
Manuela Zonfrillo1, Luana Mercuri1, Memmo Federici3, Guido Rasi1,
Enrico Garaci2 and Pasquale Pierimarchi1
Address: 1Institute of Neurobiology and Molecular Medicine – ARTOV, CNR, Via Fosso del Cavaliere 100, 00133 Rome, Italy, 2Department of
Experimental Medicine and Biochemical Science, University of Rome "Tor Vergata", Via Montpellier 1, 00133, Rome, Italy and 3INAF-IASF, Via
Fosso del Cavaliere 100, 00133 Rome, Italy
Email: Annalucia Serafino* - annalucia.serafino@artov.inmm.cnr.it; Paola Sinibaldi Vallebona - sinibaldi-vallebona@med.uniroma2.it;
Federica Andreola - federica.andreola@artov.inmm.cnr.it; Manuela Zonfrillo - manuela.zonfrillo@artov.inmm.cnr.it;
Luana Mercuri - luana.mercuri@artov.inmm.cnr.it; Memmo Federici - memmo.federici@iasf-roma.inaf.it;
Guido Rasi - guido.rasi@artov.inmm.cnr.it; Enrico Garaci - presidenza@iss.it; Pasquale Pierimarchi - pasquale.pierimarchi@artov.inmm.cnr.it
* Corresponding author
Abstract
Background: Besides few data concerning the antiseptic properties against a range of microbial
agents and the anti-inflammatory potential both in vitro and in vivo, little is known about the influence
of Eucalyptus oil (EO) extract on the monocytic/macrophagic system, one of the primary cellular
effectors of the immune response against pathogen attacks. The activities of this natural extract
have mainly been recognized through clinical experience, but there have been relatively little
scientific studies on its biological actions. Here we investigated whether EO extract is able to affect
the phagocytic ability of human monocyte derived macrophages (MDMs) in vitro and of rat
peripheral blood monocytes/granulocytes in vivo in absence or in presence of immuno-suppression
induced by the chemotherapeutic agent 5-fluorouracil (5-FU).
Methods: Morphological activation of human MDMs was analysed by scanning electron
microscopy. Phagocytic activity was tested: i) in vitro in EO treated and untreated MDMs, by
confocal microscopy after fluorescent beads administration; ii) in vivo in monocytes/granulocytes
from peripheral blood of immuno-competent or 5-FU immuno-suppressed rats, after EO oral
administration, by flow cytometry using fluorescein-labelled E. coli. Cytokine release by MDMs was
determined using the BD Cytometric Bead Array human Th1/Th2 cytokine kit.
Results: EO is able to induce activation of MDMs, dramatically stimulating their phagocytic
response. EO-stimulated internalization is coupled to low release of pro-inflammatory cytokines
and requires integrity of the microtubule network, suggesting that EO may act by means of
complement receptor-mediated phagocytosis. Implementation of innate cell-mediated immune
response was also observed in vivo after EO administration, mainly involving the peripheral blood
monocytes/granulocytes. The 5-FU/EO combined treatment inhibited the 5-FU induced
myelotoxicity and raised the phagocytic activity of the granulocytic/monocytic system, significantly
decreased by the chemotherapic.
Published: 18 April 2008
BMC Immunology 2008, 9:17 doi:10.1186/1471-2172-9-17
Received: 27 July 2007
Accepted: 18 April 2008
This article is available from: http://www.biomedcentral.com/1471-2172/9/17
© 2008 Serafino et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Conclusion: Our data, demonstrating that Eucalyptus oil extract is able to implement the innate
cell-mediated immune response, provide scientific support for an additional use of this plant
extract, besides those concerning its antiseptic and anti-inflammatory properties and stimulate
further investigations also using single components of this essential oil. This might drive
development of a possible new family of immuno-regulatory agents, useful as adjuvant in immuno-
suppressive pathologies, in infectious disease and after tumour chemotherapy.
Background
The monocytic/granulocytic system as well as differenti-
ated macrophages constitute the primary cellular effectors
of the immune response, playing a pivotal role in the
detection and elimination of foreign bodies such as path-
ogenic microorganisms. Recognition of foreign microor-
ganisms by these cells ultimately results in phagocytosis
and in the eventual destruction of pathogens by lyso-
somal enzymes. Macrophages perform a variety of func-
tions other than phagocytosis [1]. The phagocytic process
is accompanied by intracellular signals that trigger cellular
responses such as cytoskeletal rearrangement, alterations
in membrane trafficking, activation of apoptosis, release
of chemical mediators such as growth factors, pro- and
anti-inflammatory cytokines and chemokines [2]. Such
mediators, produced by activated macrophages, are essen-
tial for microbe killing but also potentiate inflammatory
reactions; thus regulation of this production is therefore
critical to kill pathogens without inducing tissue injury
[3].
Natural oils are extensively used in cosmetics as well as in
folk medicine for the treatment of a growing number of
more or less specific pathologies. Recently the clinical use
of essential oils has expanded worldwide also including
therapy against various kinds of inflammatory diseases,
such as allergy, rheumatism and arthritis. These activities
have mainly been recognized through clinical experience,
but there have been relatively little scientific studies on
biological actions of these natural extracts. For instance,
the tea tree oil as well as Eucalyptus oil have been demon-
strated to possess antiseptic properties against a range of
bacteria [4-8] and have been used in a number of products
against oral pathogens and different forms of infections. It
has also been suggested that tea tree [9,10] and lavender
oils [11] are able to suppress allergic symptoms through
the inhibition of histamine release [12,13] and cytokine
production [14] in in vitro and in vivo. Monoterpenoid
components of aromatic constituents of Eucalyptus oil are
traditionally used as analgesic, anti-inflammatory, and
antipyretic remedies and are commercially available for
the treatment of the common cold and other symptoms of
respiratory infections. Phytochemical analysis has shown
that the profile of the monoterpenoids changes among
the Eucalyptus species, with potential variations in thera-
peutic properties. In Eucalyptus globulus, the major monot-
erpenoid component is eucalyptol (1,8-cineole),
constituting the 60–90% [15], that has been reported to
inhibit the production and synthesis of tumour necrosis
factor-α (TNF-α), interleukin-1β (IL-1β), leukotriene B4,
and thromboxane B2 in human blood monocytes
[15,16]. An anti-inflammatory activity of eucalyptol in
patients with bronchial asthma has been also described in
a double-blind, placebo-controlled trial [17]. These find-
ings provide scientific support at least for some of the tra-
ditionally accepted uses, in folk medicine, of essential
oils, and in particular of Eucalyptus oil, although a direct
relationship is still to be demonstrated.
To our knowledge, actually there are no available data
concerning the influence of Eucalyptus essential oil on the
cellular components of the immune system, the only
exception is for the effect on some cytokine production
[15,16]. The aim of the present study has been to investi-
gate whether essential oil from Eucalyptus globulus (EO) is
able to affect the phagocytic activity of human monocyte-
derived macrophages (MDMs) in vitro and of peripheral
blood monocytes/granulocytes from immuno-competent
rats treated in vivo in absence or in presence of immuno-
suppression induced by administration of the chemother-
apeutic agent 5-fluorouracil (5-FU).
Results
In vitro effect of Eucalyptus oil on morphological features
and phagocytic activity of human MDMs
EO treatment, at both concentrations used in our experi-
ments, did not affect the macrophage viability, as revealed
by the Trypan blue exclusion method (see Additional file
1). Morphological observation of human MDMs (Fig. 1)
showed that after 24 h treatment with 0.008% or 0.016%
EO, cells assumed the typical activated morphology, also
exhibited by LPS-activated MDMs, showing an enlarged
size, more abundant microvillous structures as compared
to the untreated macrophages, a rougher surface with
prominent filopodia, blebs, and rufflings. Confocal
microscopic observation, after fluorescent beads adminis-
tration to cell cultures (Fig. 2), showed that EO is able to
dramatically increase, in a dose dependent manner, the
phagocytic activity of MDMs, to higher extent compared
to the LPS treatment. In fact, as reported in Fig. 2h, in
untreated control cultures 13.7% of cells were phagocytic,
with a mean of 11 beads phagocytosed per cell. In cultures

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treated for 6 h with LPS, the percentage of phagocytic cells
is lightly increased (18.26%), while no increase were
recorded in the mean number of beads/cell. Conversely,
in sample 24 h treated with 0.008% EO, the percentage of
phagocytic cells increases to 27.1% and the average
number of phagocytosed beads/cell raises to 24. The treat-
Morphological features of human MDMs after 24 h of in vitro treatment with Eucalyptus oilFigure 1
Morphological features of human MDMs after 24 h of in vitro treatment with Eucalyptus oil. a-f, phase-contrast
microscopy after Wright Giemsa staining: a, d, untreated control; b, e, MDMs stimulated with 0.1 µg/ml of bacterial lipopoly-
saccharide (LPS); c, f, MDMs treated with 0.016% Eucalyptus oil; a, b, c, original magnification: 20×;d, e, f, original magnification:
40×. g-l, Scanning electron microscopy of untreated (g, j), LPS treated (h, k) and Eucalyptus oil treated MDMs (i, l). Bars: 20
µm.

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ment with the highest concentration of EO (0.016%),
increased so dramatically the phagocytic activity of
MDMs, showing up to 64.8 phagocytosed beads/cell, that
the majority of cells engulfed with indigestible beads pos-
sibly died, leading to a registration of only 10% of phago-
cytic cells. Moreover, pre-treatment with EO 24 h before
the addition of LPS to cell culture was able to increase,
without any cytotoxicity and in a dose-dependent man-
ner, the phagocytic activity of human MDMs in compari-
son with the treatment with LPS alone (Fig. 2j).
We excluded a non specific effect on macrophage phago-
cytic activity caused by the oil preparation, by testing
other two oil extracts, Lavender oil (LavO) and the Tea
Tree oil (TeaTreeO) in parallel with EO. The results
obtained (see the Additional file 2), showed that treat-
In vitro effect of Eucalyptus oil treatment on phagocytic activity of human MDMsFigure 2
In vitro effect of Eucalyptus oil treatment on phagocytic activity of human MDMs. The phagocytic activity of treated
and untreated MDMs was tested by adding to cultures 2 × 107 beads/ml of fluorescent polystyrene beads as described in Meth-
ods. a-g, confocal microscopy images, showing the beads uptake (yellow-green hue) in control (a, b), LPS treated (c, d) and
EOtreated MDMs (e-g); cells were counter-stained with 1 µg/ml propidium iodide (red hue). a, c, e, Original magnification:
40×; b, d, f, g, original magnification: 100×. h, j, Evaluation of phagocytic activity performed counting the number of phagocytic
MDMs, reported as percent of phagocytic cells (solid bars), as well as the number of beads per cell (■). A minimum of 500 cells
per sample were observed. Evaluation of percentage of phagocytic cells in controls maintained at 0°C, to block beads internal-
ization, is also reported (yellow solid bars).

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ment with both Lavender and Tea Tree oil extracts do not
affect MDMs phagocytic activity, also confirming the
dose-dependent stimulatory effect of Eucalyptus oil. Cell
viability (see the Additional file 3) showed that LavO,
similarly to EO, resulted not toxic, indicating that the lack
of the effect on MDMs phagocytic ability observed is not
ascribable to oil cytotoxicity. Instead, a moderate effect on
cell viability of the TeaTreeO extract has been recorded.
Scanning electron microscopy observation showed, after
beads addition to EO treated cultures, the presence of
numerous polarised cells exhibiting elongated lamellopo-
dia and filopodia (arrows in Fig. 3d, e), indicative of a
pseudopodial activity. In cell cytoplasm of the EO acti-
vated MDMs, a higher number of phagocytosed beads
(arrowheads in Fig. 3f, h, i), compared to the untreated
control (Fig. 3c) were observed, also distributed at the
filopodial and lamellopodial structures, suggesting that
an active cell motility might contribute to the increased
phagocytic ability towards the foreign bodies.
Morphological features of 24 h EO treated human MDMs after in vitro administration of polystyrene beadsFigure 3
Morphological features of 24 h EO treated human MDMs after in vitro administration of polystyrene beads.
Scanning electron microscopy of untreated (a-c) and 0.008% Eucalyptus oil treated MDMs (d-i) showing the presence, in EO
treated cultures, of numerous polarised cells exhibiting elongated lamellopodia and filopodia (arrows in panels d and e), indica-
tive of a pseudopodial activity. Arrowheads in panels f, h, i point to phagocytosed beads, more numerous respect to the
untreated control (c). Bars: a, d = 20 µm; remaining panels = 10 µm.

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Effect of Eucalyptus oil on production of pro-
inflammatory and immune-modulating cytokines by
human MDMs
In spite of the morphological features typical of activated
macrophages, and of the increased phagocytic ability
acquired by EO-treated or pre-treated MDMs, the release
in the extracellular medium of pro-inflammatory and
immune-modulating cytokines was significantly lower
compared to that recorded for LPS treatment alone (Fig.
4). This effect is particularly evident for the pro-inflamma-
tory cytokines conspicuously produced by MDMs under
LPS stimulation, such as IL-4, IL-6 and TNF-α. In detail, in
absence of LPS stimulation, EO activated macrophages
produced very low levels, comparable to the untreated
control, of IL-4, IL-6 and TNF-α (Fig. 4a2, a3, a5). Moreo-
ver, in accordance with the anti-inflammatory properties
ascribed to EO, pre-treatment with EO 24h before the
addition of LPS to cell culture was able to significantly
decrease the LPS-induced cytokines production (Fig. 4a2,
a3, a5, b). No significant effect on the production of the
other immune-modulating cytokines tested (IL-2, IL10
and IFNγ) was recorded for both EO or LPS treatments
(Fig. 4a1, a4, a6).
Cytoskeletal elements mediating the Eucalyptus oil
stimulated internalization
We also investigated whether the observed differences in
LPS- or EO-induced internalization and cytokine produc-
tion were associated with differences in the cytoskeletal
elements that mediate phagocytosis. In particular, we ver-
ified if the phagocytic ability of LPS- or EO- stimulated
MDMs required integrity of the microtubular network. To
this purpose, control cells and macrophages, pre-treated
with LPS or EO, were treated with the microtubule-desta-
bilizing agent nocodazole and the phagocytic ability was
tested after fluorescent beads administration. Confocal
microscopic observation (Fig. 5) showed that, after dam-
age of the microtubular network by nocodazole treatment
Th1/Th2 cytokines produced by human MDMs after 24 h of in vitro treatment with Eucalyptus oilFigure 4
Th1/Th2 cytokines produced by human MDMs after 24 h of in vitro treatment with Eucalyptus oil. a, Analysis by
cytometric bead array of IL-2 (a1), IL-4 (a2), IL-6 (a3), IL-10 (a4), TNF-α (a5), and IFN-γ (a6) production in untreated controls,
LPS stimulated MDMs and cells treated with 0.008% and 0.016% EO or pre-treated with EO before LPS stimulation. b,
Cytofluorimetric dot plots showing the modification of cytokines profile in culture medium of MDMs LPS-stimulated (b2) or
pre-treated with EO before LPS stimulation (b3); b4, merged dot plot showing the decrement in IL-4, IL-6 and TNF-α concen-
tration induced by EO pre-treatment; b1, cytokines profile in standard. **P <0.001 vs LPS stimulation; ***P < 0.0001 vs LPS
stimulation.

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Effect of microtubule destabilization by nocodazole on phagocytic activity of Eucalyptus oil stimulated human MDMsFigure 5
Effect of microtubule destabilization by nocodazole on phagocytic activity of Eucalyptus oil stimulated human
MDMs. a, d, Confocal microscopy images, showing the beads uptake (yellow hue) in control (a1, d1), LPS pre-treated (a2, d2)
and EO pre-treated MDMs (a3, d3), in absence (a) or in presence (d) of nocodazole treatment; cells were counter-stained with
PI (red hue). Merged images with differential interference contrast, used to visualize cell morphology, are shown. b, c, Confocal
microscopy images, showing the microtubular network (green hue) in nocodazole treated and untreated cells: b1, c1, controls;
b2, c2, LPS pre-treated MDMs; b3, c3, EO pre-treated MDMs.

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(Fig. 5c), while controls and LPS-stimulated MDMs were
still able to internalize beads (Fig. 5d1, d2), in EO-stimu-
lated cells bead uptake was almost completely inhibited
(Fig. 5d3). These results clearly suggest that LPS- and EO-
induced phagocytosis occur possibly by means of differ-
ent mechanisms, since only the EO-stimulated internali-
zation requires integrity of the microtubule network.
Moreover, as shown in Fig. 5b, EO stimulation is able to
provoke a more dramatic reorganization of microtubular
filaments respect to LPS, strongly suggesting that tubulin
plays an important role in the EO-induced macrophage
activation.
In vivo effect of Eucalyptus oil on activation of peripheral
blood mononuclear cells from immuno-competent rats
We firstly assessed the effect of EO on peripheral blood
mononuclear cells (PBMCs) after in vivo administration to
immuno-competent animals, following the scheme of
treatment reported in Fig. 6a. The evaluation of haemato-
logical parameters at T15 revealed that EO treatment was
able to significantly increase the percentage of circulating
monocytes in peripheral blood of treated rats compared
to the untreated controls, while no significant modifica-
tion in the percentage of circulating granulocytes and lym-
phocytes was evidenced (Fig. 6b). Concomitantly, an
increment in the phagocytic activity of granulocytes, and
at higher extent of monocytes, obtained from treated ani-
mals, was recorded (Fig. 6c, d).
We also examined the monocytic/granulocytic fraction for
the expression of surface molecules that may be indicative
of leucocytes activation. In particular we evaluated the
expression of CD44, the receptor for the extracellular
matrix component hyaluronan that mediates the leuko-
cyte/endothelial interactions leading to extravasation
[18], and of CD25, a marker of circulating monocyte acti-
vation [19,20]. Cytofluorimetric analysis showed that at
T15 EO treatment induced a significant increase of the
expression of CD44, as well as the percentage of CD25+
cells in circulating monocytes compared to the untreated
control rats, and this effect also persisted at T20, after 5
days from the end of EO administration (Fig. 6e, f). No
significant modification in the expression of CD44 or in
the percentage of CD25+ cells was evidenced in circulating
granulocytes and lymphocytes (not shown).
Ex vivo evaluation of the phagocytic activity of monocytes
derived macrophages (MDMs) obtained from treated and
untreated animals at T15, revealed that the stimulatory
effect recorded in circulating monocytes was also retained
in differentiated macrophages (Fig. 7a, c). The ex vivo
phagocytic ability of MDMs obtained from EO treated rats
was increased not only towards a non-specific foreign
bodies such as fluorescent beads, but also towards micro-
biological pathogens, as observed after infection with Sta-
phylococcus aureus (Fig. 7b).
In vivo effect of Eucalyptus oil on phagocytic activity of
peripheral blood mononuclear cells from immuno-
suppressed rats
Finally we verified whether EO treatment could be also
able to induce a recovery of peripheral blood mononu-
clear cells activity after bone marrow suppression induced
by 5-fluorouracil (5-FU), a chemotherapeutic agent exten-
sively used in the treatment of different types of cancer
[21,22] producing myelotoxicity as side effect [23]. The
schematic diagram of treatment is reported in Fig. 8a. The
evaluation of haematological parameters at T15 revealed
that EO treatment was able to induce a recovery of the per-
centage of circulating granulocytes that was significantly
reduced by 5-FU treatment, while no modification was
recorded in the percentage of circulating monocytes, that,
on the other hand, was not influenced by the chemother-
apeutic agent (Fig. 8b).
5-FU treatment also induced a decrease of phagocytic
activity of peripheral blood granulocytes/monocytes, but
in EO/5-FU treated animals the granulocytes/monocytes
phagocytic ability was restored to values near to that of
untreated immuno-competent rats (Fig. 8c, d). Finally,
the ex vivo experiments revealed that the recovery of
phagocytic activity was also present in differentiated mac-
rophages obtained from 5-FU/EO treated animals, com-
pared to rats treated with 5-FU alone (Fig. 8e).
Discussion
Some biological effects of Eucalyptus oil (EO) extract such
as its antiseptic properties against a range of microbial
agents [6,7] and, recently, its anti-inflammatory potential,
both in vitro [15,24] and in vivo [16,17,25], mainly con-
cerning the effect on some cytokine production, have
been demonstrated. However, rather little is known about
the influence of this essential oil on the cellular compo-
nents of the immune system, and in particular on the
monocytic/macrophagic system constituting one of the
primary cellular effectors of the immune response against
foreign particles and pathogen attacks.
In this study we present findings indicating that EO from
Eucalyptus globulus is able to induce morphological and
functional activation of human MDMs in vitro, dramati-
cally stimulating the phagocytic response of these effec-
tors of the innate immune defence. The acquisition of
morphological features of EO activated macrophages and
the increased phagocytic ability is coupled to a low release
of IL-4, IL-6 and TNF-α pro-inflammatory cytokines, in
contrast to that recorded under LPS stimulation.

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In vivo effect of Eucalyptus oil on activity of peripheral blood mononuclear cells from immuno-competent ratsFigure 6
In vivo effect of Eucalyptus oil on activity of peripheral blood mononuclear cells from immuno-competent rats.
a, Schematic diagram of rats treatment, detailed in Methods. b, Evaluation by flow cytometry of the percentage of circulating
granulocytes, monocytes and lymphocytes in untreated control (yellow solid bar) and in rats after 15 days of EO administration
(red solid bars). c, d, Cytofluorimetric evaluation, by phagotest kit, of phagocytic activity of granulocytes and monocytes from
untreated control (yellow solid bar) and from rats after 15 days of EO administration (red solid bars). The number of internal-
ized bacteria has been recorded as Mean Fluorescence Intensity (MFI): mean values of 12 animals/group are reported as per-
cent of increment of MFI at T15 respect to T0. Representative plots (d) of untreated controls and Eucaliptus oil treated animals
are also shown; R1: granulocyte population, R2: monocyte population; phagocytic activity in controls maintained at 0°C, to
block E. coli bacteria internalization, is also reported (grey lines). e, Cytofluorimetric analysis of expression of CD44, reported
as MFI, in treated and untreated rats at the end (T15) and after 5 days from the end of EO administration (T20). f, Cytofluori-
metric evaluation of percentage of CD25+ monocytes in treated and untreated rats at T15 and at T20. *P < 0.01 vs control; **P
< 0.001 vs control; ***P < 0.0001 vs control; ns = P not significative.

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The differences in LPS- or EO-induced cytokine produc-
tion were associated with differences in the cytoskeletal
elements that mediate phagocytosis, since EO-stimulated
internalization requires integrity of the microtubule net-
work, while LPS-stimulation does not. This suggests,
almost preliminary, that LPS and EO might act by means
of different mechanisms, possibly involving different
phagocytic receptors. It is actually known that internaliza-
tion by macrophages occurs by a restricted number of
phagocytic receptors present on their surface [26]. Specif-
ically, infectious agent are mainly phagocytosed by com-
plement receptors (CR), after relatively non specific
opsonization with complement, and by Fc receptors (FcR)
after specific opsonization with antibodies. Important dif-
ferences in the molecular mechanisms underlying phago-
cytosis by these two different receptors are actually
recognized. These include differences in the cytoskeletal
elements that mediate ingestion, differences in vacuole
maturation, and differences in inflammatory responses.
In particular, FcR-mediated phagocytosis is tightly cou-
pled to the production and secretion of pro-inflammatory
mediators such as cytokines and reactive oxygen interme-
diates [27], while CR-mediated phagocytosis is not. Fur-
thermore, only the CR-mediated internalization requires
integrity of the microtubular network [28]. On this con-
text, our data indicate that EO might implement patho-
gens internalization stimulating the CR-mediated
phagocytosis. EO-induced implementation of innate cell-
mediated immune response, coupled to reduced produc-
tion of pro-inflammatory cytokines and toxic oxygen
intermediates, might have profound implications for the
inflammatory response during pathogen infections.
The presence in the EO treated cultures of numerous
phagocyting polarised cells, exhibiting elongated lamello-
podia and filopodia, indicates that stimulation of pseu-
Ex vivo evaluation of the phagocytic activity of MDMs obtained from EO treated and untreated ratsFigure 7
Ex vivo evaluation of the phagocytic activity of MDMs obtained from EO treated and untreated rats. Confocal
microscopy images showing the uptake of fluorescent beads (a) or of S. aureus bacteria (b) by MDMs obtained from EO
treated and untreated rats at T15; (b) S. aureus bacteria were visible in cell cytoplasm after PI staining; original magnification:
100×. (c) Evaluation of phagocytic activity of rat MDMs performed ex vivo counting the number of phagocytic MDMs, reported
as percent of phagocytic cells (solid bars), as well as the number of beads per cells (■). The percentage of phagocytic cells in
controls maintained at 0°C, to block beads internalization, is also reported (yellow solid bars).

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In vivo effect of EO on activity of peripheral blood mononuclear cells from 5-FU immuno-suppressed ratsFigure 8
In vivo effect of EO on activity of peripheral blood mononuclear cells from 5-FU immuno-suppressed rats. a,
Schematic diagram of rats treatment, detailed in Methods. b, Evaluation at T15 of the percentage of circulating granulocytes and
monocytes in untreated control (yellow solid bar), in 5-FU immuno-suppressed rats (cyan solid bar) and in rats after 5-FU/EO
combined administration (red solid bars). c, d, Cytofluorimetric evaluation of phagocytic activity of granulocytes (c) and mono-
cytes (d) from untreated control, 5-FU immuno-suppressed rats and rats after 5-FU/EO combined administration; the percent-
age of phagocytic cells (solid bars), as well as the MFI (■), indicative of the number of internalized bacteria/cells, are reported.
e, Evaluation of phagocytic activity of rat MDMs performed ex vivo counting the number of phagocytic MDMs, reported as per-
cent of phagocytic cells (solid bars), as well as the number of beads per cells (■).*P < 0.01; **P < 0.001; ns = P not significative.

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dopodial activity and cell motility might contribute to the
augmented phagocytic capability. The in vitro stimulatory
effect was achieved using concentrations of the essential
oil ranging from 0.008% and 0.016% (v/v), doses that
resulted absolutely non toxic and up to 100 fold lower
than those used in vitro by other authors [24,29].
Implementation of innate cell-mediated immune
response has been observed not only in in vitro experi-
ments but also in treated BDIX rats after 15 days EO
administration. The stimulatory effect in treated animals,
mainly involving the peripheral blood monocytes/granu-
locytes, led to a raise in the percentage of circulating
monocytes as well as to an increment in the phagocytic
activity of granulocytes and mostly of monocytic cells.
These latter cells also exhibited an enhanced potential of
extravasation ability, as suggested by increased expression
of CD44 on their cellular membrane [18], as well as char-
acteristics of activated monocytes [19,20], as indicated by
the higher percentage of CD25+ cells in circulating mono-
cytes, compared to the untreated control rats.
The stimulatory effect on phagocytic ability recorded in
circulating monocytes was also retained in monocyte-
derived macrophages from treated animals, as showed by
the ex vivo experiments. The EO dose used in the in vivo
experiments (12 mg/Kg/die) did not produce evident
toxic effect on treated rats, as suggested by the lack of body
weight reduction and of animals mortality. This dose was
comparable to doses recommended for oral therapy in
humans and much more lower than dose that have been
described to exhibit sub-acute toxicity in rats (about 300
mg/kg/die) [30].
Besides the effect of EO on the cell-mediated immune
response in immuno-competent rats, this essential oil is
also able to induce a dramatic recovery of granulocytes/
monocytes activity after bone marrow suppression pro-
duced by 5-FU administration. In fact, combined treat-
ment 5-FU/EO not only inhibited myelotoxicity, as
resulted by the restoration of granulocytes number to nor-
mal values, but also raised the phagocytic activity of gran-
ulocytes and monocytes as well as of the ex vivo analyzed
monocyte-derived macrophages, significantly decreased
in animals treated with the chemotherapic alone. Since
myelosuppression continues to be a major dose-limiting
side effect for most chemotherapeutic agent such as 5-FU
[23], our results suggest that the combination of EO with
5-FU might be taken into account for the prevention of
immunotoxicity and myelotoxicity caused by 5-FU
administration, even if additional studies should be car-
ried out to demonstrate that the antitumor activity of 5-
FU is not influenced by this combined therapeutic strat-
egy.
Moreover, further experiments are in progress to deter-
mine whether the immuno-stimulatory effect, observed
both in vitro and in vivo, could be due to the major monot-
erpenoid component eucalyptol (1,8-cineole), constitut-
ing the 60–90% of Eucalyptus oil [15], or is related to its
synergic cooperation with the other minor components
present in the crude extract; preliminary results seem to be
indicative of this later hypothesis.
Conclusion
Most knowledge of the therapeutic use of plant essential
oils is acquired through folklore, and only some activities
of these natural extracts are actually supported by scien-
tific studies. But plants are an important source for drug
discovery, and investigations on biological actions of
plant medicinal extracts, as well as the understanding of
the mechanisms underlying these actions drive the search
for novel drugs.
Overall, our data, demonstrating that Eucalyptus essential
oil from Eucalyptus globulus is able to implement the
innate cell-mediated immune response, provide scientific
support for an additional use of this plant extract, besides
those concerning its known antiseptic and anti-inflamma-
tory properties. Thus, the present study stimulates further
investigations also using single components of essential
oil extracts from various species of Eucalyptus for develop-
ment of a possible new class of immuno-regulatory agents
useful as adjuvant in immuno-suppressive pathologies, in
infectious disease as well as in tumour chemotherapy.
Methods
Human monocyte derived macrophage (MDMs) cultures
Peripheral blood mononuclear cells (PBMCs) were iso-
lated from buffy coats of healthy donors by density gradi-
ent centrifugation using Lympholyte-H (Cederlane,
Hornby, Ontario, Canada). The lymphocytic/monocytic
fraction was then re-suspended in RPMI 1640 medium
(Hyclone Labs Inc. Logan, UTAH) supplemented with
10% (v/v) heat-inactivated fetal calf serum (FCS)
(Hyclone), L-glutamine (2 mM), penicillin (100 IU/ml)
and streptomycin (100 mg/ml) and cells were seeded on
175 cm2 flasks and maintained at 37°C in 5% CO2, to
generate adhering MDMs. After 1 h of culture, non-adher-
ing cells were removed and the residual adhering MDMs
were maintained in culture for 7 days to obtain partially
differentiated macrophages. At this time, cells were
detached using cold PBS, seeded at a density of 2.5 × 104
cells/cm2 in 35 mm culture plates or on cover-slips (∅ 10
mm) and allowed to adhere for 4–5 days before treat-
ments.
MDMs in vitro treatments
Essential oil from Eucalyptus globulus was purchased from
Cruciani Prodotti Crual® Srl, (Rome, Italy). To exclude

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Page 13 of 16
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that the oil preparation used contained any endotoxins,
we tested the EO extract using the Limulus Amebocyte
Lysate (LAL) test (PYROGENT. Plus – Lonza Walkersville,
Inc., Walkersville MD) The LAL test is a qualitative test for
Gram-negative bacterial endotoxin utilizing a lysate pre-
pared from the circulating amebocytes of the horseshoe
crab (Limulus polyphemus) standardized to detect the
labelled concentration (EU/ml) of the FDA Reference
Standard Endotoxin. Limulus Amebocyte Lysate was
mixed in equal parts with the solution being tested and
incubated 1 h at 37°C. In the presence of endotoxin, a
positive reaction is characterized by the formation of a
firm gel; in the absence of endotoxin, gelation does not
occur. The assay was done as a yes/no test. The EO sam-
ples and controls were diluted in sterile water, performed
in triplicate and run in parallel. Positive controls consisted
in 2 ng/ml (1 EU/ml) E. coli Control Standard Endotoxin
or 0.1 µg/ml LPS; negative control consisted in sterile
water. The LAL test excluded that the EO extract contained
any endotoxins. (see Table S1 in the additional file 4).
Human MDMs were treated with 0.008% v/v in RPMI
1640 medium (corresponding to about 50 µg/ml) or
0.016% v/v (about 100 µg/ml) of Eucalyptus oil (EO) for
24 h. Concentrations were selected on the basis of the
lowest, non toxic, effective doses found in a preliminary
dose-response experiment (see Additional file 1). Cell via-
bility after EO treatment was determined by the Trypan
blue dye exclusion method. MDMs stimulated with 0.1
µg/ml of bacterial lipopolysaccharide (LPS, Sigma-Aldrich
Co., St. Louis, Mo, USA) for 6 h were used as positive con-
trol of macrophage activation. The effect of EO 24 h pre-
treatment before stimulation with LPS was also analysed.
Essential oils from Lavender oil and the Tea Tree oil (Cru-
ciani Prodotti Crual® Srl), were used as controls to exclude
a non specific effect on macrophages phagocytic activity
caused by the oil preparation. For any microscopic analy-
sis cells were grown on cover-slips.
Optical and Scanning Electron Microscopy (SEM)
Analysis of morphological feature of activated MDMs, was
carried out by phase-contrast microscopy after Wright
Giemsa staining and by scanning electron microscopy
(SEM). For SEM observation, MDMs were fixed with 2.5%
glutharaldehyde in 0.1 M Millonig's phosphate buffer
(MPB) at 4°C for 1 h. After washing in MPB, cells were
post-fixed with 1% OsO4 in the same buffer for 1 h at 4°C
and dehydrated using increasing acetone concentrations.
The specimens were critical-point dried using liquid CO2
and sputter-coated with gold before examination on a
Stereoscan 240 scanning electron microscope (Cambridge
Instr., Cambridge, UK).
Evaluation of phagocytic activity of human MDMs by
confocal microscopy
The phagocytic activity of treated and untreated MDMs
was tested by adding to cultures 2 × 107 beads/ml of yel-
low-green fluorescent polystyrene beads (∅ 1 µm, at a
ratio of at least 10 beads/cell) with excitation/emission
wavelengths of approximately 495 nm/515 nm (Molecu-
lar Probes, Inc., Eugene, OR). After 30 min, cells were
fixed with paraformaldehyde, counter-stained with 1 µg/
ml propidium iodide (PI – Sigma-Aldrich) and observed
by the confocal microscope LEICA TCS SP5 (Leica Instru-
ments, Heidelberg, Germany). The excitation/emission
wavelengths employed for PI staining were 568 nm/590
nm. A minimum of 500 cells per sample were observed,
the number of phagocytic MDMs (reported as percent of
phagocytic cells), as well as the number of beads per cell,
were counted. MDMs subjected to beads administration
and maintained at 0°C to block internalization, was used
as negative control of uptake. Quantitative assessment
was done in a blinded fashion. The experiment was
repeated three times and the mean values were plotted.
We analyzed at least 20 buffy coats from different healthy
donors.
Evaluation of cytokines production
The concentration of IL-2, IL-4, IL-6, IL-10, TNF-α, and
IFN-γ secreted into the culture media by human macro-
phages after in vitro EO treatment were determined by
using the BD Cytometric Bead Array human Th1/Th2
cytokine kit (BD Pharmingen) according to the manufac-
turer's protocol [31]. Flow cytometry analysis was carried
out using a FACSCalibur flow cytometer (Becton Dickin-
son, Mountain View, CA). The effect of EO pre-treatment
on the pro-inflammatory and immune modulating
cytokines production by macrophages stimulated with
LPS was also evaluated.
In vitro inhibitory study
To depolymerize microtubules, control cells and macro-
phages, pre-treated with LPS or EO, were treated with 2
µg/ml nocodazole for 30 min. For phagocytic activity test-
ing, after nocodazole treatment, fluorescent polystyrene
beads were added to the cell culture, samples were proc-
essed as described above and analysed by confocal micro-
scopy. The effect of nocodazole on microtubular network
was analysed after immuno-staining, using an anti-
human tubulin mouse monoclonal antibody (Molecular
Probes) revealed with the secondary Alexa Fluor 488-con-
jugated anti-mouse IgG (Molecular Probes), by confocal
microscopy. Cells were counter-stained with PI (Sigma-
Aldrich).
Animals and in vivo treatments
Inbred male BDIX rats (Charles River, Calco, Italy), 8
weeks old and weighing 220–250 g, were held for 7 days,

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Page 14 of 16
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housed in a pathogen-free animal facility with free access
to water and standard food and kept in accordance with
European Community guidelines. Experiments were
approved by the local committee on animal experimenta-
tion, and were performed under strict governmental and
international guidelines. EO was administrated for 15
days per os, at a dose of 12 mg/Kg/day, by adding, every
evening, the essential oil extract directly to drinking water
(7 µl/day of essential oil per animal). Two sets of in vivo
experiments were carried out. In the first one, in which we
tested the effect of EO on peripheral blood mononuclear
cells of immuno-competent rats, animals were divided in
two groups (12 animals/group), an untreated control
group and a group treated for 15 days with EO (see sche-
matic diagram in Fig. 6a). In a second experiment, in
which we tested whether EO treatment could be able to
induce a recovery of peripheral blood mononuclear cells
activity after bone marrow suppression, rats of both
groups (12 animals/group) were intra-peritonally injected
with a single dose of 100 mg/Kg of the chemotherapeutic
agent 5-fluorouracil (5-FU) on day 7 from the beginning
of EO treatment (see schematic diagram in Fig. 8a). In
both sets of experiments, from all animals, anaesthetized
by inhalation of 2-bromo-2-chloro-1,1,1-trifluoroethane
(Fluka, Sigma-Aldrich), blood was collected, by intracar-
diac puncture, before treatment (day 0 – T0), on day 7
(T7), on day 15 (T15) and on day 20 (T20). For evaluation
of haematological parameters, after erythrocytes lysis by
FACS Lysing Solution (Becton Dickinson), mononuclear
cell fractions in RPMI 1640 medium were analyzed for
forward (FSC) and sideward (SSC) scatter patterns in a flu-
orescence-activated cell sorter (FACScan, Becton Dickin-
son). Gates were defined to identify populations of cells
with different FSC/SSC characteristics, corresponding to
granulocytes, lymphocytes or monocytes populations and
results were expressed as percent of total cells. In addition,
rat peripheral blood mononuclear cells (PBMCs) were
isolated by density gradient centrifugation using Lym-
pholyte-H (Cederlane, Hornby, Ontario, Canada). The
lymphocytic/monocytic fraction was then re-suspended
in RPMI 1640 medium and rat adhering macrophages
were obtained as described previously for human MDMs
and used for the ex vivo experiments.
Evaluation of phagocytic activity of rat peripheral
mononuclear cells and macrophages
The evaluation of phagocytic activity of monocytes/gran-
ulocytes from peripheral blood of BDIX rats, after in vivo
EO administration, in absence or in presence of bone mar-
row suppression by 5-FU administration, was carried out
by cytofluorimetric analysis using the phagotest kit
(ORPEGEN Pharma, Heidelberg, Germany), following
the manual's protocol. This test allows the quantitative
determination of leukocyte phagocytosis in heparinized
whole blood. It contains fluorescein (FITC)-labelled
opsonized bacteria (E. coli-FITC) and necessary reagents,
and measures the overall percentage of phagocytic mono-
cytes and granulocytes (that have ingested one or more
bacteria per cell) and the individual cellular phagocytic
activity (number of bacteria per cell) recorded as Mean
Fluorescence Intensity (MFI).
The phagocytic activity of rat MDMs, obtained from
treated and untreated animals and grown on cover-slips,
was evaluated ex vivo by confocal microscopy after fluores-
cent beads administration, as previously described for the
in vitro experiments on human MDMs. To test the phago-
cytic ability towards microbial pathogens, MDMs from
treated and untreated immuno-competent animals were
subjected to an in vitro infection with a suspension of Sta-
phylococcus aureus (1 × 105bacteria/cell), and observed
after 6 h of culture by confocal microscopy.
Evaluation of CD44 expression and CD25+ cells in
circulating monocytes
The expression of CD44 and the percentage of CD25+ cells
in circulating monocytes from treated and untreated rats
were evaluated by cytofluorimetric analysis. Living cells
were incubated with a mouse anti-rat CD44H (clone OX-
49; BD Pharmingen), detected using a PE-conjugated pol-
yclonal anti-mouse IgG (BD Pharmingen) or with a FITC-
conjugated mouse anti-rat CD25 (clone OX-39; BD
Pharmingen), using a FACScan flow cytometer (Becton
Dickinson).
Statistical analysis
For statistical analysis the two-tailed Student's t test was
used. For the in vitro evaluations, at least three independ-
ent experiments have been carried out and data are given
as the mean ± SD. For the in vivo and ex vivo experiments,
results are reported as mean of 12 animals/group ± SD.
For all analyses, significance was calculated with a P value
< 0.01.
Abbreviations
EO: Eucalyptus oil; 5-FU: 5-fluorouracil; LavO: Lavender
oil; LPS: bacterial lipopolysaccharide; MDMs: monocyte-
derived macrophages; MPB: Millonig's phosphate buffer;
PBMCs: peripheral blood mononuclear cells; SEM: scan-
ning electron microscopy; TeaTreeO: Tea Tree oil.
Authors' contributions
AS conceived of the study and designed the experiments,
carried out all microscopic analyses, collected and inter-
preted data, drafted the manuscript. PSV has been
involved in drafting the manuscript and revising it criti-
cally. FA performed the macrophage cultures and treat-
ment, carried out preparation of samples for confocal and
electron microscopy observations, participated in the in
vivo study. MZ carried out the in vivo study, and the prep-

BMC Immunology 2008, 9:17 http://www.biomedcentral.com/1471-2172/9/17
Page 15 of 16
(page number not for citation purposes)
aration of samples for cytofluorimetric analysis, partici-
pated in the macrophage cultures maintenance and
treatment. LM participated in the in vivo study, and in the
macrophage cultures maintenance and treatment. MF par-
ticipated in the design of the study and has been involved
in revising the manuscript critically. GR has been involved
in drafting the manuscript and revising it critically. EG has
been involved in revising the manuscript critically. PP par-
ticipated in the design of the experiments, carried out all
cytofluorimetric analysis, and has been involved in draft-
ing the manuscript and revising it critically.
Additional material
Acknowledgements
The authors would like to thank Rossana Psaila Ph.D for her precious
assistance in macrophage cultures maintenance and treatment, Dr. Paolo
Daniele Siviero for his advises about the Intellectual Property Right, Mar-
tino Tony Miele for linguistic styling of the manuscript and Mariangela Rasi
for her secretarial assistance. This work was partially supported by
National Research Council, INMM-ARTOV.
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Additional file 1
Dose-response experiment performed to select the lowest, non toxic, effec-
tive doses of Eucalyptus oil used in the in vitro studies. The data provided
report the cell survival and the phagocytic activity of MDMs after 24 h
treatment with increasing concentrations of EO
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2172-9-17-S1.pdf]
Additional file 2
In vitro effect of Lavender oil (LavO) or Tea Tree oil (TeaTreeO) treat-
ments on phagocytic activity of human MDMs. The data provided show
the phagocytic activity of MDMs treated for 24 h with 0.008% and
0.016% LavO or TeaTreeO, compared to EO used at the same concen-
trations.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2172-9-17-S2.pdf]
Additional file 3
In vitro effect of LavO or TeaTreeO treatments on viability of human
MDMs. The data provided show the viability of MDMs treated for 24 h
with 0.008% and 0.016% LavO or TeaTreeO, compared to EO used at
the same concentrations.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2172-9-17-S3.pdf]
Additional file 4
Endotoxin detection by Limulus Amebocyte Lysate (LAL) test. The data,
provided in a Table, show the results of LAL test performed on EO extract
used in this study and in negative (sterile water) or positive (E. coli Con-
trol Standard Endotoxin or LPS) controls.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1471-
2172-9-17-S4.pdf]

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