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Effect over time of in-vivo administration of the polysaccharide arabinogalactan on immune and hemopoietic cell lineages in murine spleen and bone marrow *.

Summary

Current evidence indicates an immunostimulating role for complex carbohydrates, i.e., polysaccharides, from several plant sources. In the present work, we determined the specific in vivo effects, with time of administration, of one such compound, a neutral arabinogalactan from larch not only on immune (lymphoid) cells, but also on natural killer (NK) lymphoid cells, as well as a variety of other hemopoietic cells in both the bone marrow and spleen of healthy, young adult mice. The latter were injected daily (i.p.) with arabinogalactan (500 [micro]g in 0.1 ml pH 7.2 phosphate buffered saline-PBS) for 7 or 14 days. Additional, aged (1 1/2-2 yr) mice were similarly injected for 14 days only. Control mice were given the PBS vehicle in all cases, following the above injection regimen. Animals from all groups were sampled 24 h after the final injection and the immune and hemopoietic cell populations in the bone marow and spleen were assessed quantitatively. The results indicated that immediately following either 7 or 14 days of arabinogalactan administration to young, adult mice, lymphoid cells in the bone marrow were significantly decreased (p < 0.004; p < 0.001, respectively) relative to controls but remained unchanged at both time intervals in the spleen. NK cells, after 7 days of arabinogalactan exposure, were also decreased significantly in the bone marrow (p < 0.02), but unchanged in the spleen. After 14 days' exposure to the polysaccharide, NK cells in the bone marrow had returned to normal (control) levels, but were increased in the spleen (p <0.004) to levels greater than 2-fold that of control. Among other hemopoietic cell lineages, none was influenced in the bone marrow or spleen by one-week administration of arabinogalactan; however, after two-week exposure, precursor myeloid cells and their mature (functional) progeny (granulocytes), were significantly reduced in the spleen (p <0.043; p <0.006, respectively), as were splenic monocytes (p < 0.001). These lineages in the bone marrow, however, remained steadf astly unaltered even after 14 days of continuous exposure to the agent. Of the vast cascade of cytokines induced in the presence of this polysaccharide, it appears that immunopoiesis- and hemopoiesis-inhibiting ones are most prevalent during at least the first two weeks of daily exposure.

Key words: arabinogalactan, NK cells, spleen, bone marrow, hemopoiesis

Introduction

Considerable evidence exists which has identified immunostimulating compounds within plant species such as Echinacea (Roesler et al., 1991a, b; Steinmuller et al., 1993; Muller-Jakic et al., 1994; Bauer, 1996). One such compound is the acidic complex carbohydrate, arabinogalactan (Wagner et al., 1988; Wagner et al., 1999). In the present study, we aimed to determine the in vivo effect of injecting arabinogalactan, not only on natural killer (NK) cells, i.e., cells representing the first line of defense in vivo against virus infections and developing neoplasms, but also on a wide range of other immune and hemopoietic cell populations in the bone marrow and spleen of mice. Macrophages, a fundamentally important hemopoietic cell in vivo for the functional activity of NK cells and other immune and non-immune cells, release numerous cytokines upon stimulation with purified polysaccharides such as and including arabinogalactan, as demonstrated in vitro (Stimpel et al., 1984; Luettig et al., 1989; Bauer, 1996). The cytokine cascade produced by the stimulated macrophages includes several that are powerful NK cell enhancers, such as interferon and TNF-[alpha] (Luettig et al., 1989; Hauer and Anderer, 1993; Kelly, 1999; Stein et al., 1999; Rininger et al., 2000). Thus we hypothesized that administering arabinogalactan in vivo would also trigger host macrophages to produce a similar battery of cytokines. Furthermore, 7 days, and especially 14 days, would be ample time to reveal changes in population numbers in any other hemopoietic and/or immune cell lineage in the bone marrow and spleen. We have already shown (Sun et al., 1999) that dynamic changes occurred in the above-mentioned populations when whole root extract of E. purpurea, a plant known to contain arabinogalactan, was administered in vivo daily for 7 and 14 days. Our present work did indeed indicate a positive response, in an organ-specific manner, by NK cells, during exposure to this polysaccharide.

Materials and Methods

Experimental animals

DBA/2 strain male mice (Charles River Laboratories, St. Constant, QC, Canada), aged 10 weeks, were used for all studies involving young adults, while studies involving aged animals were carried out on healthy, 18--24 month old mice of the C57B1/6 strain (Jackson Laboratories, Bar Harbor, ME, USA). All experimental and control mice were housed under micro-isolator conditions in the Animal Care Facility of McGill University. All mice were received in the Facility at 6 weeks of age. They were housed and remained undisturbed in the Facility until the time of treatment. Sentinel mice resident therein regularly demonstrated the absence of all common mouse pathogens throughout the duration of all experiments.

Agent administration

The water-soluble polysaccharide, arabinogalactan (Sigma Chemical Co., St. Louis, MO, USA), derived from Larch wood (LARCOLL: L-arabino-D-galactans), is a highly branched molecule with branched backbone chains of (1-3/6)-linked [beta]-D-galactopyranosyl residues, to which are attached side-chains containing L-arabinofuranosyl and L-arabinopyranosyl residues. Arabinogalactan was injected intraperitoneally to experimental mice of both age groups, at the same time each day for 7 or 14 days at 500 [micro]g in 0.1 ml in pH 7.2 phosphate buffered saline (PBS)/mouse. Control mice were identically injected with only the PBS vehicle.

Preparation of bone marrow and spleen cells for analysis

Twenty four hours after the final arabinogalactan (experimental) or PBS vehicle (control) injection, mice were killed by cervical dislocation (euthanasia method approved by McGill University and the Canadian Council on Animal Care for mice), and their spleens and femurs (bone marrow source) removed. Free cell suspensions of the spleen were prepared by pressing each through stainless steel screens into ice-cold RPMI (Roswell Park Memorial Institute) medium containing 10% millipore filtered FCS (fetal calf serum). Free cell suspensions of the bone marrow were obtained by removing both femurs and repeatedly flushing the marrow plug from the bones into ice-cold medium. The expressed spleen cells and bone marrow cells were then gently pipetted to obtain single-cell suspensions containing predominantly hemopoietic and immune cells. Further clearing of non-cellular debris was achieved by layering each suspension in RPMI + 10% FCS onto 1 ml pure FCS (denser than medium + FCS). The resulting combination was allowed to stand for 5 mm, during which time larger, non-cellular fibers, remnants of vascular cells walls, etc., settled into the pure FCS. The resulting, single-cell suspension in the medium, now debris-free, was siphoned off and washed by centrifugation for 5 mm at 4 [degrees]C at 1100 rpm (250 x G), to produce a cell pellet. This pellet for each organ (bone marrow, spleen) for each mouse was re-suspended in fresh medium + FCS (above) and washed once more as above. From the final re-suspension, the total numbers of nucleated (hemopoietic and immune) cells in the spleen and femurs of each mouse were counted using an electronic particle counter (Coulter Electronics, Hialeah, FL, USA).

Immunoperoxidase labeling of NK cells in the bone marrow and spleen cells of young, adult mice

Mature and maturing NK cells, all of which bear the surface molecule ASGM-1 (Kasai et al., 1980; Beck et al., 1982; Stout et al., 1987) were processed for microscopic visualization by an indirect immunoperoxidase method in standard use in our laboratory (Christopher et al., 1991; Dussault and Miller, 1993, 1994; Currier and Miller, 1998; Mahoney et al., 1998; Whyte and Miller, 1998; Sun et al., 1999; Currier and Miller, 2000a, b, 2002). Although activated (blast) T cells may also bear the AS GM-i surface molecule, such cells are readily distinguishable, morphologically, from NK(ASGM-1+) cells. Moreover, animals maintained under microisolator conditions contain no infections meritorious of T-cell reactions, resulting in a virtual absence of T blasts which may bear ASGM-1.

Immunoperoxidase labeling of AS GM-1 surface molecules was carried out as follows: the monoclonal primary antibody, rabbit anti-ASGM-1 (Wako Pure Chemicals, Dallas, TX, USA) was conjugated to a biotinylated secondary antibody, goat anti-rabbit IgG (Sigma BioSciences, St. Louis, MO, USA). Briefly, 100 [micro]1 of each cell suspension (bone marrow and spleen from each mouse) were incubated with 100 [micro]l of primary antibody (concentration 1:40), or normal rabbit serum (control) for 30 mm on ice. The cells were then washed twice by centrifugation (5 minutes, 1100 rpm, 4 [degrees]C), the supernatant was discarded, and the resulting cell pellet was re-suspended in 100 [micro]1 of RPMI + 10% FCS. These cells were then incubated with the biotinylated secondary antibody (concentration 1:100) for 30 minutes on ice, washed as above and re-suspended in cytospotting medium (0.009% NaCI, 0.001% EDTA, and 0.05% bovine serum albumin in distilled water). The cells were then cyto-centrifuged (Shandon Cytospin, Pittsburg, P A, USA) onto albumin-coated glass microscope slides which were then air-dried, fixed in ice-cold absolute methanol for 30 mm and rehydrated in a 1:1 methanol:PBS (pH 7.2) solution for 10 min followed by 100% PBS (pH 7.2) for 5 mm. The slides containing the cellular cytospots were then bathed in 3% [H.sub.2][O.sub.2] (Fisher Scientific, Whitby, ON, Canada) for 10 mm at room temperature to block endogenous peroxidase activity, washed in PBS and incubated with the avidin-biotin complex (Vector Laboratories, Burlingame, CA, USA) containing biotinylated-peroxidase enzymes for 45 mm in a humidity chamber. The slides were then washed once more as above, and finally bathed in a solution of the chromogenic agent, diaminobenzidine (DAB) (150 mg of DAB, 300 ml PBS at pH 7.6, and 100 [micro]1 of 30% 11202) for 13 mm, then washed twice more in PBS (pH 7.2). The slides were then air-dried, and finally stained with MacNeal's tetrachrome hemotologic stain to permit the identity of all other hemopoietic and immune cells in bo th organs.

Identification of all hemopoietic and immune cells

Immunoperoxidase labeling of the AS ASGM-1 surface molecule, together with MacNeal's tetrachrome staining of the whole cytospots, permitted identification of NK cells as lymphocytes of small and medium size, well-established criteria for the identity of these cells (Herberman et al., 1975; Djeu et al., 1979; Senik et al., 1979; Christopher et al., 1991; Dussault and Miller, 1993, 1994; Miller, 1994; Currier and Miller, 1998) in mice. The proportions of several other morphologically identifiable hemopoietic and immune cell populations (non-NK lymphocytes, nucleated erythroid cells, monocytes and myeloid (granulocytic) cells, in both their precursor and mature forms) were also obtained by means of light microscopy (x100) 00) by well-established lineage identification techniques (Miller and Osmond, 1974, 1975; Miller et al., 1978; Christopher et al., 1991; Miller, 1992; Dussault and Miller, 1994, 1996; Currier and Miller, 1998; Mahoney et al., 1998; Whyte and Miller, 1998; Sun et al., 1999; Currier and Miller, 2 000a, b, 2002), which are in continuous use in our laboratory.

From the known total organ cellularity, the proportions of NK cells were converted to absolute numbers of NK cells/organ/mouse in each arabinogalactan-injected and control animal. Similarly, the proportions of each of several morphologically distinct subgroups of hemopoietic cells (non-NK lymphocytes, nucleated erythroid, myeloid cells, including precursors and mature granulocytes, and monocytes), were recorded from a total of 2000 individual cells from 2-Scytospots/organ/mouse in experimental and control groups. From these percentages, the absolute numbers of each of these cell subgroups could be determined via the known total cellularity of each organ/mouse obtained previously using the electronic cell counter.

Although the method of analysis is labor-intensive, no equipment available to date can accurately classify 6 different lineages of hemopoietic and immune cells, including precursor and mature forms, especially since in some cases, sub-populations are present only in very low levels.

Immunophenotypic staining and flow-cytometric analysis of NK and B lymphocytes in aged mice

To label surface molecules which identify specifically NK cells, bone marrow and spleen cells were incubated with both PE-conjugated DX5 (pan-NK cells) monoclonal antibody, and FITC-conjugated anti-NK 1.1 (PK 136). To identify B lmyphocytes, bone marrow and spleen cells were incubated with FITC-conjugated anti-mouse B220 (RA3-6B2) (PharMingen, San Diego, CA, USA). Incubations were carried out for 30 min at 4 [degrees]C, as described (Miyake et al., 1991). In all cases, other samples were stained with isotypematched monoclonal antibodies of irrelevant specificity, to serve as negative controls.

Using a FACScan flow cytometer equipped with a doublet discrimination module, FITC and PE were excited with the 488-nm line of an argon laser and emissions were detected at 520 and 576 nm, respectively. A minimum of 10,000 events was collected for each sample, gated on forward scatter vs side scatter to exclude debris and cell clumps. Results were analyzed using FACScan LYSYS II software (Becton Dickinson, Mountainview, CA, USA).

Statistical analysis

The influence of arabinogalactan administration on NK and other immune cells, as well as on the various hemopoietic cell lienages identified in both the bone marrow and spleen, were analyzed statistically using the student's t-test (two-tailed). Standard error, and/or standard deviation, was derived for all groups, for young adult and aged mice. The difference between the means of the experimental and corresponding control groups were compared and probability values of p < 0.05 were considered statistically significant.

Results

After 7 days of administration of arabinogalactan to healthy, young adult male mice, the absolute number of NK cells (lymphoid in morphology, and bearing the NK cell surface molecule of lineage identity), was decreased significantly (p < 0.02) in the bone marrow to only 7.1% of control levels (Fig. 1). By contrast, NK cell levels in the spleen at this same time period, did not differ from control (Fig. 1).

Table 1 indicates that after 7 days of daily administration of this polysaccharide, the total number of non-NK lymphoid cells in the bone marrow were, like NK cells, decreased significantly (p < 0.004), to only one-third control numbers, while all other hemopoietic cell lineages, in both the bone marrow and the spleen of these 7-day-treated mice, remained unchanged relative to control.

After 14 days of daily arabinogalactan administration, NK cell numbers in the bone marrow rose to control levels (Fig. 2). In the spleen, however, NK cells were increased significantly (p < 0.004) at this period, to levels greater than 2-fold control (Fig. 2). The slight but not statistically significant reduction in the absolute number of NK cells in the control mice at 7 and 14 days (Figs. 1 and 2), reflects a consistent observation in our lab and others, indicating that handling of mice on a daily basis is adequate to reduce, with time, the extremely stress-sensitive NK cells. Hence the need for parallel controls in all in vivo studies involving NK cells.

Table 1 indicates, however, that even after 2 weeks of daily exposure to the polysaccharide, non-NK lymphoid cells in the bone marrow were still decreased significantly (p < 0.001), while the absolute numbers of all other hemopoietic cells in the bone marrow remained steadfastly at control levels. In the spleen, however, after 14 days of arabinogalactan administration (Table 1), the absolute numbers of precursor myeloid cells and their progeny (mature granulocytes) were decreased significantly (p < 0.043 and p <0.006, respectively). Monocytes, cells which share a common stem cell with myeloid cells, were also reduced significantly (p < 0.001) after 14 days exposure to the polysaccharide (Table 1). Finally, when arabinogalactan was administered to very old mice (18-24 mo), for 14 days, NK cells in the bone marrow and spleen did not differ from control (Table 2). In these old mice, 2 weeks of administration of arabinogalactan were similarly ineffective in increasing the absolute numbers of non-NK lymphoid cells , i.e., B220+B-lymphocytes, in both the bone marrow and spleen (Table 2).

Discussion

The results indicate that arabinogalactan administration in vivo has dynamic, organ-dependent, and exposure time-related effects on NK cells. The subsequent significant increase in NK cell number after 2 weeks in the spleen, the primary destination of newly produced, bone marrow-derived NK cells (Miller, 1982; Pollack and Rosse, 1987), suggests a super-normal (rapid) exit of bone marrow-derived, new NK cells, during the arabinogalactan exposure. Since NK cells uni-directionally traffic to the spleen (Miller, 1982), the super-normal numbers of NK cells found in that organ at 2 weeks would reflect the rapid delivery to it of super-normal export from the bone marrow during the second week of arabinogalactan exposure. The shifts in time among the negative, neutral and positive effects of arabinogalactan on NK cells, from the bone marrow to the spleen, would support this interpretation. We found, similarly (Sun et al., 1999), that the absolute number of NK cells in the spleen was also unaffected by administering t he arabinogalactan-containing, herb, Echinacea purpurea, for 1 week, but that NK cell numbers subsequently became significantly super-normal in that organ by 2 weeks after E. purpurea exposure. Given the similar dynamics in response of NK cells to both arabinogalactan and E. purpurea (Sun et al., 1999), it is possible that the polysaccharide alone, or in large measure, may account for the NK-enhancing and immunostimulating properties assigned to the whole extract of E. purpurea (Steinmuller et al., 1993; Melchart et al., 1995; Rininger et al., 2000; Currier and Miller, 2000a, b, 2002). Nevertheless, it is also possible that actual new NK cell production occurs in the spleen under the influence of sustained (2 week) arabinogalactan exposure. The spleen is capable of generating NK cells de novo (Miller and Shatz, 1991) and, moreover, will take on this activity under non-physiological conditions (Biron et al., 1983), i.e., 2 weeks' sustained exposure to arabinogalactan.

The ultimate molecular configuration of Echinacea arabinogalactan may be different from that of Larch arabinogalactan (employed in this study). Any such differences may not, however, lie in the significant portions of the polysaccharide responsible for NK enhancement, but rather in the numerous side-branches which differ between Larch and Echinacea arabinogalactan. The latter source of acidic arabinogalactan is known to be an NK stimulant, and this study reports, for the first time, that Larch source arabinogalactan is also capable of NK cell enhancement, indicating that indeed, the stimulating properties of the molecule probably lie in more fundamental, structural components common to both Larch-derived and Echinacea-derived arabinogalactan.

Virtually all the lymphocytes in mammalian bone marrow of the young adult are of the B cell lineage, i.e., cells mediating humoral immune responses via the production of specifically targeted immunoglobulin. The bone marrow, under normal conditions, is the central generating center for de novo production of both the B and the NK lymphocyte lineages. NK cells in healthy, young adult mouse bone marrow, however, account for only 2-5% (Roder et al., 1978; Clark et al., 1986; Pollack and Rosse, 1987; Miller and Shatz, 1991) of all the bone marrow lymphocytes. [T.sub.c] lymphocytes (mediators of target cell destruction by cell-cell contact), and [T.sub.h] lymphocytes ("helper" cells, almost always necessary for most immune responses to target), are virtually absent from normal, young adult bone marrow. Such an absence of any T cells is further reinforced in the present study, where all mice were maintained in air-filtered, micro-isolator units. Even in the spleen, an organ normally housing T lymphocytes of both types in relative abundance, T cells would be present in very low numbers simply because of lack of need in this exceptionally clean environment.

Our observations of significantly reduced numbers of lymphocytes in the bone marrow at both 7 and 14 days after beginning arabinogalactan, virtually exclusively represent B lymphocytes. B lymphocytes are normally produced in the total bone marrow organ of young, adult mice at the rate of 16 x [10.sup.6]/day (Osmond and Park, 1987), from whence they are delivered into the circulation for whole body distribution as a major part of the immunosurveillance mechanism. The profound decrease in bone marrow-based lymphocytes (B cells), at both i and 2 weeks after beginning araginogalactan, followed by no increase, even at 2 weeks of arabinogalactan exposure, in B lymphocyte levels in the spleen (normally containing 60-70% of all splenic lymphocytes), indicates actual inhibition of B lymphocyte production in the bone marrow in the presence of this polysaccharide. That this may indeed be the case is supported, at least in vitro, by considerable evidence indicating that certain cytokines, i.e., IFN-[gamma] and TNF[alpha] , released by macrophages in the presence of arabinogalactan, are inhibitory to B lymphocytes (Reynolds et al., 1987; Mongini et al., 1988; Luettig et al., 1989; Hauer and Anderer, 1993; Abed et al., 1994; Rininger et al., 2000).

By contrast, we have demonstrated in vivo (Sun et al., 1999), that the absolute levels of bone marrow lymphocytes in normal, young adult mice given whole E. purpurea, were not changed during either 1 or 2 weeks of daily exposure to the herb. It is possible that arabinogalactan contained in whole E. purpurea was too dilute among all the other compounds to produce any detectable effect on bone marrow lymphocytes.

Our observations that both precursor and mature cells in the myeloid lineage and the monocytes were virtually halved in absolute numbers in the spleen, but not in the bone marrow, with 14 days exposure to arabinogalactan, suggest a proliferation inhibition mediated by this agent acting peripherally, but not centrally, i.e., in the bone marrow, also a well-established generating site for myeloid cells. Because both myeloid cells and monocytes derive originally from a common stem cell, it is not surprising that proliferation interruption affecting one lineage would affect the other. Given the cascade of negative and positive cytokines induced by arabinogalactan-stimulated hemopoietic and immune cells (Wagner et al., 1985; Luettig et al., 1989), it is not unexpected that one or more of them may have acted directly, or indirectly via other cells, to lead ultimately to the net negative effect observed on myelopoiesis (reduction in precursors and, subsequently, their mature granulocyte progeny).

The lack of effect of arabinogalactan on bone marrow myeloid cells is not unusual. We have shown previously that the myeloid lineage is spared from any influence -- positive or negative -- in the bone marrow, but not in the spleen, of young adult mice given the drug indomethacin for 2 weeks (Miller, 1992). Such resilience may reflect the need for maintaining the stability of the vitally important bone marrow as the fundamental hemopoietic center, in the face of systemic, physiological disturbances in the periphery, i.e., sustained (7, 14 days) administration of at least arabinogalactan and indomethacin, and possibly many other agents.

Given the fact that we have not seen any such influence in the myeloid and monocytoid cell lineages when whole root extract of E. purpurea was administered for 14 days (Sun et al., 1999), the results for the same exposure time for arabinogalactan in the present study may reflect a dilution effect -- arabinogalactan co-existing with a wide range of other compounds in E. purpurea.

Finally, we had demonstrated previously that E. purpurea is capable of quantitatively and functionally rejuvenating the NK cells in aged mice (Currier and Miller, 2000a). Arabinogalactan as a single derivative, however, appears incapable of accomplishing the same augmentations in aged mice. The spleen, the depot for virtually all such newly produced, bone marrow-derived NK cells, reflects this lack of NK cell production-stimulation in the bone marrow in the presence of arabinogalactan. Thus, the marrow stromal cell network, i.e., its microenvironment, appears to be insensitive to NK cell production stimuli during advanced age, when it is undergoing normal, rapid decline (Hotta et al., 1980; Sidorenko et al., 1990). Age-related inefficiency of the specific NK cell-governing stromal cells in the bone marrow has been demonstrated (Miller, 1982; Pollack and Rosse, 1987).

In aged mice, B lymphocytes, i.e., B220+ cells, have not responded significantly to the presence of arabinogalactan, in either the bone marrow or the spleen, even after 14 days of daily administration. This general lack of response by cells of the B lineage would indicate that receptors on the cells in these aged mice are incapable of binding to, or are inefficient binders to, the cytokines produced by arabinogalactan-responsive macrophages. This would support existing, substantial, experimental evidence indicating that surface receptors for cytokines (negative or positive), as well as the intracellular machinery to make cytokines, declines sharply in lymphocytes in both animals (Thoman and Weigle, 1982; Vie and Miller, 1986; Patel and Miller, 1992) and humans (Nagel et al., 1988) of advanced age.

In summary, the present study has provided a systematic, in vivo analysis, under controlled laboratory conditions, of the dynamic effects of an important polysaccharide, on several hemopoietic and immune cell lineages. This study has demonstrated that a significant and positive result of administering arabinogalactan in vivo, is the augmentation of NK cells, an observation we have previously established after administering arabinogalactan-containing, whole extract of E. purpurea (Sun et al., 1999). Nevertheless, this study has also revealed what may be construed as potentially negative effects, i.e., the profound decrease in the levels of myeloid cells (granulocytes) and monocytes. These cells are fundamental elements, functioning in virtually all aspects of pathogen defense, and, in the case of monocytes, even in cancer abatement via their secretion of anti-tumor and immunostimulatory cytokines. This study suggests that, in the long run, it may be more efficacious in terms of prophylaxis and/or therapy, to a dminister whole E. purpurea (or other herb with medicinal or health-sustaining properties). Whole product contains multiple compounds, each serving either single, or synergistically-acting, physiologically significant functions. That the collective whole may indeed be better than any isolated derivative thereof, is supported by other studies (Voaden et al., 1972; Rininger et al., 2000). Further studies are planned to investigate whether arabinogalactan, as an isolated derivative, is as effective in leukemia abatement as whole root extract of the arabinogalactan-containing herb, E. purpurea (Currier and Miller, 2000b, 2002).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1

The effect of arabinogalactan administration to young, adult DBA/2 male
mice on the population sizes of various hemopoietic and immune cell
lineages in the bone marrow and spleen.

 Total Mature
 Lymphoid Cells Granulocytes
 (X[10.sup.6]) (X[10.sup.6])

Treatment

7 days
ara (a) 0.286 [+ or -] 0.067 (c,d) 2.98 [+ or -] 0.33
vehicle (b) 0.999 [+ or -] 0.037 2.99 [+ or -] 0.51


7 days
ara 56.07 [+ or -] 5.26 4.96 [+ or -] 0.80
vehicle 57.42 [+ or -] 1.94 4.71 [+ or -] 1.78


14 days
ara (a) 0.299 [+ or -] 0042 (c,d) 3.26 [+ or -] 0.38
vehicle (b) 0.515 [+ or -] 0.05 2.50 [+ or -] 0.32


14 days
ara 58.25 [+ or -] 3.80 2.48 [+ or -] 0.41
vehicle 52.84 [+ or -] 4.81 4.40 [+ or -] 0.39

 Precursor Nucleated
 Myeloid Cells Erythroid Cells
 (X[10.sup.6]) (X[10.sup.6]

Treatment Bone marrow

7 days
ara (a) 0.504 [+ or -] 0.052 4.14 [+ or -] 0.050
vehicle (b) 0.511 [+ or -] 0.115 2.97 [+ or -] 0.38

 Spleen
7 days
ara 0.580 [+ or -] 0.093 6.94 [+ or -] 1.79
vehicle 0.546 [+ or -] 0.125 8.57 [+ or -] 1.61

 Bone marrow
14 days
ara (a) 0.476 [+ or -] 0.052 3.87 [+ or -] 0.33
vehicle (b) 0.389 [+ or -] 0.049 4.16 [+ or -] 0.46

 Spleen
14 days
ara 0.620 [+ or -] 0.149 17.83 [+ or -] 2.04
vehicle 1.320 [+ or -] 0.290 25.14 [+ or -] 3.48

 Monocytes

 (X[10.sup.6])

Treatment

7 days
ara (a) 0.047 [+ or -] 0.005
vehicle (b) 0.081 [+ or -] 0.026


7 days
ara 0.068 [+ or -] 0.045
vehicle 0.102 [+ or -] 0.043


14 days
ara (a) 0.037 [+ or -] 0.007
vehicle (b) 0.046 [+ or -] 0.015


14 days
ara 0.119 [+ or -] 0.052
vehicle 0.605 [+ or -] 0.088

(a)injected daily (500 [mu]g in 0.1 ml 7.2 PBS) intraperitoneally.

(b)injected daily (0.1 ml 7.2 PBS) intraperitoneally.

(c)Mean [+ or -] s.e.: 6-7 mice.

(d)determined from differential (percentage) counts of 2,000 total cells
in each organ from each mouse, on stained cytospot preparations, and
converted, via the known total numbers of nucleated
cells/organ/enumerated by means of an electronic cell counter), to
absolute numbers of cells in each morphologically identifiable category
indicated.

Table 2

The effect of arabinogalactan administration to aged (a) male C57B/6J
mice on the population sizes of 2 distinct immune cell lineages in the
bone marrow and spleen.

Treatment Organ B220+ cells (d)

 (X[10.sup.6)

arabinogalactan (b) bonemarrow 10.4 [+ or -] 0.41 (f)
vehicle (c) bonemarrow 9.2 [+ or -] 1.30
arabinogalactan spleen 20.1 [+ or -] 10.46
vehicle spleen 23.1 [+ or -] 0.07

Treatment NK1.1+/
 DX5+ cells (e)
 (X[10.sup.6])

arabinogalactan (b) 2.50 [+ or -] 1.07
vehicle (c) 1.70 [+ or -] 0.06
arabinogalactan 1.44 [+ or -] 0.52
vehicle 2.16 [+ or -] 0.76

(a) mice ranged from 18-24 months of age

(b) given daily (500 [mu]g in 0.1 ml pH 7.2 PBS) intraperitoneally, for
14 days

(c) given daily (0.1 ml pH 7.2 PBS) intaperitoneally, for 14 days

(d) cells labeled for the B220 surface molecule specifically identify
lymphocytes of the B lineage.

(e) cells labeled for the NK1.1 and the DX5 surface molecules,
specifically identify lymphocytes of the NK lineage.

(f) Mean [+ or -] s.d.: 3 mice (Standard deviation has been retained
(vs. s.e.) because of the small sample size).


* This work was supported by a grant from the Cancer Research Society, Inc., to S. C. Miller

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S. C. Miller, Department of Anatomy & Cell Biology, McGill University, 3640 University Ave., Montreal, Quebec, Canada H3A 2B2
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Author:Currier, N.L.; Lejtenyi, D.; Miller, S.C.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Geographic Code:1USA
Date:Mar 1, 2003
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