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
Abstract: 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. J. Leukoc. Biol. 70: 274 –276; 2001. 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– 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. 274
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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 –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 [11–14]. 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
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:
[email protected] Received January 22, 2001; revised April 11, 2001; accepted May 23, 2001.
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Fig. 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.
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 signif-
icantly more numerous than we had previously found when each of these agents was administered alone [6, 7]. 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
Fig. 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.
Currier et al. Deleterious effects of E. purpurea and melatonin on myeloid cells
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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 –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.
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