(Journal of Leukocyte Biology. 2001;70:274-276.)
© 2001
by Society for Leukocyte Biology
Deleterious effects of Echinacea purpurea and melatonin on myeloid cells in mouse spleen and bone marrow
N. L. Currier,
M. Sicotte and
S. C. Miller
Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec, Canada
Correspondence: S. C. Miller, Ph.D., Department of Anatomy & Cell Biology, McGill University, 3640 University Ave., Rm 2/28, Montreal, Quebec H3A 2B2, Canada. E-mail: smiller{at}med.mcgill.ca
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ABSTRACT
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The neurohormone, melatonin, a product of the pineal gland, is a potent
immune cell stimulant. Phytochemicals contained in root extracts of the
plant species Echinacea purpurea are also potent as immune
cell stimulants. Both agents are potent stimulants of T, B, and/or
natural killer cells, but little is known of their effect on other
hemopoietic cells, specifically granular leukocytes, also participants
in a wide variety of disease defense processes. Given their current
popularity and availability for amelioration of a) jet lag and sleep
disorders (melatonin) and b) virus-mediated respiratory infections
(E. purpurea), we investigated the effects of these agents
on granular leukocytes and their precursors, myeloid cells. Mice
received these agents daily for 7 or 14 days via the diet, thus
mimicking human administration, after which spleens and bone marrow
were removed and assessed for mature, differentiated granulocytes and
their myeloid progenitors. The influence of these agents was directly
related to the stage of cell maturity. Administration of both agents
together resulted in significantly elevated levels of myeloid
progenitor cells in both bone marrow and spleen and significantly
reduced levels of mature, functional granulocyte progeny in both
organs, suggesting a) increased precursor proliferation, b)
antiapoptosis among the progenitors, and/or c) inhibition of precursor
maturation—the latter readily explaining the paucity of mature
granulocyte progeny. In conclusion, individual administration of either
the herbal derivative and melatonin was either without effect (E.
purpurea) or even advantageous (melatonin) to cells of this
lineage, but when administered together, these agents significantly
perturbed myelopoiesis.
Key Words: granulocytes
Melatonin (MLT), a pineal gland-derived neurohormone, is produced
almost exclusively during the hours of darkness and coordinates
circadian biological rhythms [1
, 2
]. MLT is
also an immunostimulant [3
4
5
6
]. Its current popularity
derives from its value in resetting disrupted sleep rhythms instigated
by the phenomenon of "jet lag," as well as in the correction of
transient or chronic sleep disorders.
A second immunostimulant, primarily targeting natural killer cells
[7
, 8
], is a phytocompound-containing root
extract from the plant Echinacea purpurea. These extracts
have become extremely popular recently for their reported health
benefits, and they are in use both prophylactically and for abatement
of virus-mediated infections and assorted inflammations
[8
9
10
]. To date, however, aside from our own recent
studies [6
, 7
], there exists no information
on the in vivo influences of MLT and ,f46E. purpurea on other
(nonimmune) cells acting in the disease defense process, i.e., the
granular leukocytes.
Male, young-adult DBA/2 mice, housed under microisolator conditions to
control airborne pathogens, were administered MLT (0.0142 mg/mouse/day)
and E. purpurea root extract (0.45 mg/mouse/day) via the
diet, precisely according to our established protocols
[6
, 7
]. Doses were derived from
manufacturer "daily recommended doses" for consumption by humans.
All treated and control animals were killed 1 day after the last
feeding, i.e., days 8 and 15. Free-hemopoietic-cell suspensions were
prepared from their spleens (representative of a peripheral lymphoid
organ) and the bone marrow from both femurs (the central generating
sites of cells of the myeloid lineage) by our well-established methods
[11
12
13
14
]. Briefly, free-cell suspensions were obtained
by gently pipetting the minced, fresh organs in RPMI 1640 medium +
5% fetal calf serum, followed by centrifugation at 250
g to obtain a cell pellet. This procedure was
repeated three times to produce a clean cell suspension, free of
connective tissue, etc. From the known total organ cellularity (spleen
and both femurs/mouse) obtained with an electronic counter of
individual cells (Coulter Electronics, Hialeah, FL) and from the
percentages of both precursor (proliferating) myeloid cells
(myeloblasts and myelocytes) and their maturing or mature cell progeny
(metamyelocytes, band forms, and polymorphonuclear granulocytes)
recorded from differential counts on hematologically strained
cytospots, the absolute numbers of the two major subgroups of myeloid
cells (progenitors and differentiated cells) were obtained. This
morphological categorization among cells in the myeloid lineage is well
established in our lab [6
, 7
,
11
, 13
] and clearly distinguishes precursors
from differentiated/mature cells. MacNeal’s tetrachrome stain
allowed clear four-color contrast of the subcellular components, which,
together with the unique features of each of the five maturational
stages (listed above) within the myeloid lineage, permitted ready
definition of all developmental stages thereof.
We previously found [7
] that administration of E.
purpurea for 7 or 14 days had no influence on the numbers of
either precursor or differentiated cells in the myeloid lineage, in
either the spleen or the bone marrow. On the other hand, we found in
both organs [6
] that 7 or 14 days of dietary MLT
slightly but significantly increased the absolute number of cells in
both categories (precursors and differentiated cells).
In the present study, we found (Fig. 1a
b
) that regardless of duration of exposure of the mice to the
combination treatment, the absolute numbers of maturing and mature
cells (granulocytes) in the myeloid lineage, in both the spleen and
bone marrow, were profoundly reduced relative to mice receiving
untreated chow or, as we have already shown, relative to mice receiving
either E. purpurea [7
] or MLT
[6
] for 7 or 14 days. In contrast (Fig. 2a
b
), the number of proliferating precursors in this lineage was
significantly greater than in the control group; consequently, these
precursor cells were significantly more numerous than we had previously
found when each of these agents was administered alone
[6
, 7
].
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Figure 1. Absolute (total) numbers of mature (functional) granulocytes in the
spleen (a) and bone marrow (b) of mice consuming untreated (vehicle
control) or treated (MLT + E. purpurea extract) diet
chow for 7 or 14 days. Data are means ± SE for 9–10
mice/group. P < 0.01 (control vs. both
treatment intervals—spleen); P < 0.008 and 0.0004
(control vs. 7- and 14-day treatment groups, respectively—bone
marrow). No significant difference between 7- and 14-day treatment
groups was detected in either the spleen or the bone marrow.
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Figure 2. Absolute (total) numbers of precursor (proliferating) myeloid cells in
the spleen (a) and bone marrow (b) of mice consuming untreated (vehicle
control) or treated (MLT + E. purpurea extract) diet
chow for 7 or 14 days. Data are means ± SE for 9–10
mice/group. P < 0.00001 and 0.0003 (control vs. 7- and 14-day treatment groups—spleen); P <
0.00005 and 0.00003 (control vs. 7- and 14-day treatment groups—bone
marrow). No significant difference was detected between 7- and 14-day
treatment groups in either the spleen or the bone marrow.
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The effect of coadministration of the two agents appeared to be
directly related to the level of cell maturity within the lineage.
MLT-enhanced granulocytopoiesis has been demonstrated by our laboratory
and others in vitro [6
, 15
].
The abundance of precursor myeloid cells did not result in the
anticipated augmentation in mature cell progeny. Increased precursor
proliferation, maturation inhibition, or antiapoptosis in the precursor
myeloid population would explain their elevated numbers concomitant
with the paucity in progeny (mature granulocytes). MLT binds to
receptors on T-helper 1 cells resulting in increased production of the
proliferation-inducing, antiapoptotic cytokines interleukin (IL)-2, -4,
and -6 and interferon 
[5
, 16
] and
granulocyte macrophage-colony stimulating factor (GM-CSF) release from
bone marrow stromal cells [17
]. Although more limited
information is available on the interplay of E. purpurea
with cells of the hemopoietic system, it is known that macrophages, in
the presence of E. purpurea extracts, are stimulated to
produce the cytokine IL-1 [18
]. IL-1 is a fundamental
cytokine in vivo and may well be defined as a "systems regulator,"
given that it modulates many hemopoietic and immune cell lineages by
interaction with a host of other proliferation and differentiation
factors, i.e., tumor necrosis factor; kit ligand; IL-4, -6, -7, and -8;
granulocyte-colony stimulating factor; and GM-CSF
[18
19
20
21
22
].
Thus, these observations demonstrating significant disturbances in
myeloid population dynamics in two key organs might reflect the net
result of multiple cytokine interplays triggered by the two types of
agents together. Regardless of the mechanism, a halt in the
differentiation of cells in this lineage toward functional end cells
ultimately compromises this important part of the nonspecific, disease
defense mechanism.
The significance of this study pertains to its relevance in combination
therapy/prophylaxis whereby nonprescription, commercially available
neutriceuticals, etc., with proven or presumed health benefits, are
administered in combinations or together with prescribed
pharmacological agents. We have shown in this study that
coadministration of at least the two types of immunostimulants can be
considerably deleterious to other cells in the disease defense process,
i.e., the myeloid cells.
Received January 22, 2001;
revised April 11, 2001;
accepted May 23, 2001.
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