Published online before print January 15, 2008
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* Departamento de Imunologia, Instituto de Microbiologia,
Núcleo de Pesquisas de Produtos Naturais,
|| Instituto de Bioquímica Médica, Universidade Federal do Rio de Janeiro, Brazil;
Departamento de Fisiologia e Farmacodinâmica, IOC, FIOCRUZ, Rio de Janeiro, Brazil; and
Departamento de Imunobiologia, Instituto de Biologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil
2 Correspondence: Departamento de Imunologia, Instituto de Microbiologia, Universidade Federal do Rio de Janeiro, Av. Carlos Chagas Filho, 373, CCS, Bl.I, sala I2-051, Cidade Universitária, CEP:21941-902, Rio de Janeiro, Brazil. E-mail: afnobrega{at}gmail.com
ABSTRACT
Lymphopoiesis and myelopoiesis continuously generate mature cells from hematopoietic cell progenitors during the lifetime of the organism. The identification of new endogenous or exogenous substances that can act specifically on the differentiation of distinct cell lineages is of relevance and has potential therapeutical use. Kalanchoe brasiliensis (Kb) is a medicinal plant from the Crassulaceae family, used in folk medicine to treat inflammatory and infectious diseases. Here, we show that short-term treatment of naïve mice with Kb led to a strong and selective inhibition of lymphopoiesis, affecting B and T cell lineages without reduction of the myeloid lineage development. Similar effects were observed after treatment with the highly purified compound kalanchosine dimalate (KMC), obtained from Kb. Numbers of mature lymphocytes in secondary lymphoid organs were preserved in Kb(KMC)-treated mice. The effect of Kb(KMC) was not a result of secondary augmentation of plasma levels of endogenous corticoids; neither involves TNF-
, type-I IFN, or TLR2/TLR4 ligands, which have all been described as selective inhibitors of lymphopoiesis. Flow cytometry analysis of the phenotypes of T and B cell precursors indicate a blockade of maturation on IL-7-dependent, proliferative stages. In vitro, Kb(KMC) inhibited the IL-7-dependent proliferation of pre-B cells and does not induce massive apoptosis of B and T cell precursors. These results suggest that Kb(KMC) is selectively blocking lymphopoiesis through a mechanism that does not involve the previously characterized substances, possibly acting on the IL-7 signaling pathway, opening new perspectives for a potential therapeutic use of Kb-derived drugs.
Key Words: Kalanchoe brasiliensis • corticosteroids • TLR • IFN • TNF
INTRODUCTION
Hematopoiesis continuously generates blood cell types during the lifetime of the organism from hematopoietic stem cells (HSCs) that undergo massive proliferation and differentiation into specific cell types. The differentiation of HSCs into lineage-specific progenitors is a hierarchical process involving a genetic network of transcription factors that control the commitment to different lineages, which results in a major dichotomy segregating lymphopoiesis from myelopoiesis [1 ]. In the adult mouse, mature myelomonocytic cell types have been proposed to derive from a common myelomonocitic cell progenitor, whereas lymphocytes would mainly stem from a common lymphoid progenitor (CLP) [2 ]. Alternative proposals to this scheme have been suggested, where differentiation of HSCs would lead to early segregation of a megakariocyte/erythroid progenitor from a lymphoid-primed, multipotent progenitor, followed by further branching into a granulocyte-macrophage progenitor and CLP [3 ]. Additional progenitor cell types, such as early lymphocyte progenitors and early T cell progenitors (ETPs), have also been characterized, which would give rise to lymphocytes or T cells only, respectively [4 ]. Although ETPs seem to retain some residual myeloid potential, the general picture of lineage-committed progenitors resulting in the segregation of lymphopoiesis from myelopoiesis still applies to alternative schemes suggested for the generation of blood cell types from HSCs.
The differentiation of HSCs into myeloid progenitors and lymphoid progenitors depends on multiple cytokines and factors secreted by hematopoietic cells or stromal cells and also on the direct engagement of membrane receptors of HSCs with their respective ligands on the surface of stromal cells or extracellular matrix. Interestingly, the differentiation process of HSCs can be modulated by inflammatory cytokines (e.g., TNF-, type I IFNs) and glucocorticoid or estrogen steroid hormones, which have no role as constitutive growth factor or differentiation factor for progenitors. These substances have been shown to interfere selectively on the process of HSC differentiation, blocking the development of the lymphoid lineage [5
6
7
8
9
10
]. Detailed studies have shown that these molecules act at different steps of B or T lymphocyte development, directly promoting apoptosis of precursors, inhibiting stromal cell functions, blocking the IL-7-dependent proliferation, or hindering the lymphoid potential of early progenitors, thus channeling the commitment of multipotent progenitors exclusively to the myeloid lineage [11
]. More recently, TLR2 and TLR4 receptors have also been shown to interrupt early steps of lymphopoeisis, sparing the myeloid lineage [12
], but the existence of endogenous ligands for these receptors is still debated.
The identification of new endogenous or exogenous substances selectively acting on myelopoiesis and lymphopoiesis is of great interest because of their potential therapeutic use. Natural products obtained from medicinal plants constitute an important source of new biological agents with therapeutic value [13 , 14 ]. Our group has been investigating the properties of compounds derived from plants from the genus Kalanchoe, traditionally used in folk medicine to treat inflammatory and infectious diseases [15 ]. Previously, we demonstrated that juice prepared from the fresh leaves of Kalanchoe brasiliensis (Kb) and Kalanchoe pinnata have immunomodulatory and immunosuppressive properties in murine models of leishmaniasis and arthritis, respectively [16 , 17 ]. Recently, we characterized a highly purified product from Kb, kalanchosine dimalate (KMC), which exhibited a potent, anti-inflammatory activity [18 ].
Here, in this paper, we describe the effect of Kb or its highly purified fraction KMC on the development of B and T lymphocytes. We show that short-term treatment of naïve mice with Kb(KMC) led to a strong and selective inhibition of lymphopoiesis, affecting B and T cell lineages without reduction of the myeloid lineage development. This is the first description of a plant-derived natural product with these properties. The results suggest that the mode of action of Kb(KMC) differs from the mechanisms pertaining to previously characterized endogenous and exogenous substances that also promote the selective ablation of lymphopoiesis. Phenotypic analysis of T and B cell precursors in Kb(KMC)-treated mice indicates a blockade of maturation on IL-7-dependent, proliferative stages in vivo; in vitro, the proliferative response of B cell precursors to IL-7 is inhibited by Kb(KMC). These results suggest that Kb(KMC) may be selectively acting on the IL-7 proliferative response of B and T cell progenitors/precursors, opening new perspectives for therapeutical use of Kb and its derived products as immunomodulatory agents.
MATERIALS AND METHODS
Animals
C57BL/10 and C57BL/10ScCr mice (male, 25 g) were obtained from Universidade Federal Fluminense (Rio de Janeiro, Brazil). Mice genetically targeted for TLR2–/– [19
], TNF- type I receptor (TNF-RI–/–) [20
], and C57BL/6 wild-type used as background were obtained from Fiocruz Minas Gerais and Rio de Janeiro (Brazil). Mice genetically targeted for IFN type I receptor (IFN-RI–/–) [21
] on background (129xC57BL/6) and the wild-type (129xC57BL/6) were obtained from the Department of Immunology, Instituto de Ciências Biomédicas-Universidade de São Paulo (Brazil). Animals were housed in a temperature-controlled room, receiving water and food ad libitum. All the procedures involving animal experiments were performed in accordance to the International Guidelines for Animal Use. The "Committee for Animal Studies" of the Institute of Microbiology, Federal University of Rio de Janeiro (Brazil) approved the animal studies.
Preparation of juice from aerial parts of Kb
The aerial parts of cultivated and not bloomed specimens of Kb were collected in the morning hours at Rio de Janeiro State (Brazil). A voucher specimen 304627 was deposited at the Botanical Garden of Rio de Janeiro (Brazil). The fresh aerial parts of Kb (24 kg) were washed with a solution of sodium hypochlorite at 100 ppm, rinsed, and dried at room temperature. Twenty-four hours later, the fresh plant material was triturated in an industrial food processor to obtain a green juice (19 L), which was clarified by decantation at 5°C for 24 h, resulting in a yellow preparation (17.3 L), named Kb juice, which was lyophilized for further manipulations. Observation: The name Kb juice is used here instead of extract, as the material was obtained without the help of any type of solvent.
Obtention of KMC from Kb juice
Lyophilized Kb juice was dissolved in pyrogen-free water plus ethanol (50% v/v), and the mixture was maintained at 5°C under agitation for 5 days. After this period, the white-flake precipitate was recovered by paper filtration. The filters were washed with pyrogen-free water (1.0 L), and the filtrates obtained were reprecipitated with ethanol (1.5 L) and filtered again. The collected solid material obtained was partially dried at 40°C, dissolved in pyrogen-free water, frozen, and lyophilized. The 1H nuclear magnetic resonance analysis showed that the precipitate (0.319% yield w/w from the fresh plant) is a pure complex, formed by the new compound kalanchosine and malic acid in a ratio 1:2, thereafter referred to as KMC [18
]. The remaining supernatant (SN), containing nonethanol-precipitated material, was collected and named SN. The ethanol was further evaporated, and the SN was lyophilized and kept at 5°C until used. The chemical composition of all Kb preparations was systematically monitored by using HPLC with the column Asahipak GS-51OH and the Diodearray detector (Shimadzu, Japan), eluted with 20 mM Tris-HCl buffer, pH 8.0, in a flow rate of 0.4 ml/min.
Treatment with Kb and fractions
Just before use, lyophilized Kb juice, KMC, or SN was resuspended in pyrogen-free water and filtered through a 0.22-µm Millipore filter. Groups of three to five mice were daily i.p.-treated with an injection of 200 µl/mouse containing 480 mg/kg Kb, 160 mg/kg KMC, or 320 mg/kg SN during 4 days. The concentrations used in this study were determined before [17
]. Control mice received pyrogen-free water. At the end of treatment, mice were killed, and bone marrow, spleen, lymph node, and thymus were used to prepare single-cell suspensions.
Cell staining and flow cytometry
Fresh single-cell suspensions from bone marrow, spleen, lymph node, and thymus were stained with fluorochrome-conjugated mAb and monitored by flow cytometry. The mAb used for staining were as follow: FITC-goat anti-mouse IgM, anti-mouse IgM-Alexa 647 (Caltag Laboratories, Burlingame, CA, USA), anti-B220-FITC, anti-B220-PE, CD4-FITC, CD8-PE, CD11b-PE, c-kit-PE, CD25-PE, CD25-allophycocyanin, CD44-biotin, avidin-PE-Cy7, and AnnexinV-FITC (BD PharMingen, San Diego, CA, USA). Cells were incubated with the mAb in FACS buffer (PBS, 1% FCS, 0.05% Na-azide) for 20 min on ice and washed twice with FACS buffer. Data were acquired on a FACSCaliburTM (BD Biosciences, San Jose, CA, USA) and analyzed using CELLQuestTM software (BD Biosciences). Staining with AnnexinV-FITC was done in annexin-binding buffer, following the protocol indicated by the manufacturer.
MACS
For purification of B cell precursors, bone marrow cells were incubated in MACS buffer (PBS containing 2 mM EDTA, 5% FCS) with anti-mouse IgM MicroBeads (Miltenyi Biotec, Germany) for 20 min on ice and passed through a nylon mesh. Cells were then applied to a magnetic column (Miltenyi Biotec), and the nonadherent cells were collected; cells were then washed and resuspended in MACS buffer with anti-mouse CD19 MicroBeads (Miltenyi Biotec) for 20 min on ice and subsequently applied to a second magnetic column. Magnetic-retained cells were eluted and washed in PBS, 2 mM EDTA, 5% FCS. The procedure with CD19 MicroBeads was repeated twice. Collected cells were analyzed for purity by flow cytometry and were described as B220+CD19+IgM–, 99.8% purity.
Cell cultures
B cell precursors from bone marrow obtained by positive selection using MACS anti-CD19 MicroBeads, as described above, were cultivated in RPMI 1640 supplemented with 10% FCS (Giboc, Grand Island, NY, USA). Cells (106/ml) were cultivated in the presence of IL-7-enriched SN produced by a J558 cell line transfected with IL-7 plasmid. After 120 h, cells were recovered, washed extensively to remove IL-7, and recultured for 72 h in the absence or presence of IL-7, Kb, or KMC, and cell proliferation was measured at the end of the culture period. Single-cell suspensions from spleen (106 cell/ml) were cultured for 72 h in RPMI (Sigma Chemical Co., St. Louis, MO, USA) as above in the absence or presence of 12 µg/ml LPS, Kb, or KMC, and cell proliferation was measured at the end of the culture period.
Proliferation assay
Cells (2x105 cells/well) were cultivated in RPMI supplemented with 10% FCS in 96-well, flat-bottom plates in a humidified atmosphere of 5% CO2 at 37°C. In the last 18 h of cell culture, 1 µCi [3H] thymidine (Sigma Chemical Co.) was added to each culture well. Cells were harvested onto glass microfiber filter paper 934AH (Whatman, Maidstone, UK), and radioactivity was assessed by scintillation counting.
Corticosterone detection
Plasmatic corticosterone concentration was determined by radioimmunoassay using the kit from ICN-Biomedicals (Costa Mesa, CA, USA), following the instructions of the manufacturer. Radioactivity was measured in a 
-counter (Isomedic-ICN 4/600).
Statistical analysis
The data were analyzed by the Student’s t-test or Mann-Whitney nonparametric test and were considered statistically significant when P < 0.05.
RESULTS
Selective blockade of B cell development in the bone marrow after short-term administration of Kb
C57BL/10 mice, three to five animals per group, was i.p.-injected daily with 480 mg/kg Kb juice for 4 consecutive days. Twenty-four hours after the last injection, single-cell suspensions of bone marrow cells were prepared and analyzed by flow cytometry. Although bone marrow total cell counts from control mice did not differ significantly from those of Kb-treated animals, the forward-scatter/side-scatter plot indicated a profound reduction of events in the lymphoid region of the plot in the Kb-treated animals (Fig. 1A
and 1B
, indicated with arrows). The phenotypic analysis of bone marrow cell populations for the surface expression of Mac1 and B220 confirmed that B cell lineage was strongly diminished in Kb-treated mice (Fig. 1C
and 1D)
, and total numbers of Mac1+ myeloid lineage were essentially preserved (Fig. 1E)
. Kinetic experiments revealed that the major ablation of B cell lymphopoiesis increased progressively from 1 to 3–4 days, with no further significant impact on numbers of CD19/B220 bone marrow cells beyond that, until 7 days; therefore, all the subsequent experiments were done using the 4-day treatment protocol (data not shown).
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Figure 1. Administration of Kb-impaired B cell lymphopoiesis. C57BL/10 mice received daily i.p. treatment with water or 480 mg/kg Kb for 4 days. On the 5th day, bone marrow cells were analyzed by flow cytometry. The figure shows the side-scatter and forward-scatter parameters of bone marrow-nucleated cells from water (A) and Kb-treated mice (B). The location of lymphoid cells is indicated with an arrow within the closed polygon gate of nucleated cells. Bone marrow cells were stained with mAb to identify the B220+ and membrane-actived complex 1 (Mac1)+ cell lineages in water (C)- and Kb (D)-treated mice. B220+ and Mac1+ cells are enclosed within rectangular gates, and their percentages are indicated. (E) Total numbers of Mac1+ and B220+ cells/femur are shown. The figure shows a typical result obtained from three different experiments with three mice per group. Data in (E) show mean values and SD from all 3 different experiments; *, Mann-Whitney test with P < 0.05; CTR, control.
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Figure 2. B cell precursors and immature B cells are preferentially diminished in mice treated with Kb. Bone marrow cells were obtained as described in Figure 1
, stained with anti-IgM and anti-B220 mAb, and analyzed by flow cytometry for the presence of different B cell lineage populations: pro-B and pre-B cells (B220+IgM–), immature B cells (B220+IgM+), and mature B cells (B220+high IgM+). In the plot, the location of pro-B and pre-B cells is indicated as Gate (I), immature B cells as Gate (II), and mature B cells as Gate (III). Mice were injected with water (A) or with Kb (B). (C) The total numbers of B220+, pro-B/pre-B, B220+CD25+IgM– (preB II), B220+c-kit+IgM– (preB I), and immature B cells per femur are plotted as the bar graph, showing mean values and SD; *, P < 0.05. (D) The percentages of pre-BII B220+CD25+IgM–, pre-BI B220+c-kit+IgM–, and immature B cells within bone marrow B220+ cells are plotted, as the bar graph, showing mean values and SD; *, P < 0.05. Mature B cells were excluded from the analysis. The data presented in the figure are representative of three different experiments with three mice per group.
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Table 1. Administration of Kb Selectively Reduced Lymphocytes in Primary Lymphoid Organs
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Figure 3. Impairment of T cell development in mice treated with Kb. Thymus from mice treated as described in Figure 1
were removed, thymocytes were stained with anti-CD4, and anti-CD8 mAb were analyzed by flow cytometry for the presence of different T cell lineage populations: CD4–/CD8–, CD4+/CD8–, CD4–/CD8+, and CD4+/CD8+ cells. Mice were treated with water (A) or Kb (B). The percentages of the different T cell populations are indicated in each quadrant. (A and B) A typical result obtained from three different experiments with three mice per group. Total cell numbers per thymus of each population (C) or percentages of each population (D) are plotted as bar graphs showing mean values and SD; *, P < 0.05. DN, Double-negative; SP, single-positive.
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Figure 4. Differential effects of Kb/KMC on DN T cell precursors. Thymus from mice treated as described in Figure 1
were removed, and thymocytes were analyzed by flow cytometry for the presence of different T cell lineage subpopulations. (A and B) The cytometry dot-plot of the subpopulations DN1 CD4–CD8–CD44+CD25–, DN2 CD4–CD8–CD44+CD25+, DN3 CD4–CD8–CD44–CD25+, and DN4 CD4–CD8–CD44–CD25– in control (A) and Kb/KMC-treated mice (B). This figure is representative of two different experiments with five mice per group. The percentages of DP CD4+CD8+ and DN CD4–CD8– thymocytes are shown (C). Percentages of DN1, DN2, DN3, and DN4 within DN are shown (D); both are plotted as bar graphs showing mean values and SD; *, P < 0.05.
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Figure 5. KMC purified from Kb has the major inhibitory activity on B cell development. Groups of mice were injected i.p. daily with 480 mg/kg Kb, 320 mg/kg SN, or 160 mg/kg KMC, as described in Materials and Methods. On the 5th day, bone marrow cells were prepared and stained with anti-IgM and anti-B220 mAb and analyzed for the presence of pro-B and pre-B cells (B220+,IgM–) and immature B cells (B220+,IgM+) by flow cytometry. The data show median values and SD from three different experiments (Mann-Whitney test); *, P < 0.05.
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Figure 6. Blockade of lymphopoiesis by Kb does not depend on a specific increase of endogenous corticoids. Groups of five C57BL/10 mice were treated i.p. daily with water or Kb for 4 days. On the 5th day, levels of seric corticosterone of treated and nonmanipulated mice (None) were measured by radioimmune assay. Horizontal bars represent the mean value for each group, and * indicate that water and Kb-treated mice were significantly different from the values obtained with nonmanipulated animals (Mann-Whitney test).
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Figure 7. Limited entry into apoptosis of T and B cell precursors in the presence of Kb/KMC in vitro. Purified B220+CD19+IgM– bone marrow cells (see Materials and Methods) were cultured for 18 h and assayed for apoptosis by flow cytometry staining with AnnexinV and propidium iodide (PI): (A) control; (B) dexamethasone 10–8 M; (C) Kb 30 µg/ml; (D) Kb 100 µg/ml. Thymocytes were cultured for 18 h and assayed for apoptosis by flow cytometry staining with AnnexinV and propidium iodide: (E) control; (F) dexamethasone 10–8 M; (G) Kb 30 µg/ml; (H) Kb 100 µg/ml.
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, type I IFN, TLR2, or TLR4 and type I IFNs have been shown to selectively inhibit lymphopoiesis, sparing myelopoiesis [5
, 10
]. To investigate whether the effect of Kb(KMC) could be associated with the action of these endogenous mediators known to modulate the lymphopoiesis, we treated gene-targeted mice for TNF-RI–/– or for the IFN-RI–/– with Kb(KMC). The results obtained in both mouse lineages show that the treatment with Kb(KMC) led to a profound and selective reduction of lymphopoiesis (Table 2
). |
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Table 2. Kb Impaired T and B Cell Development in TNF-RI–/– and IFN-RI–/–, C57BL/10ScCr, or TLR2–/– Mice
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In vitro treatment with Kb or KMC inhibited the IL-7-dependent proliferation of B cell precursors
The selective effect of Kb(KMC) on B and T cell development seems to target the precursor stages with a strong proliferative capacity in both lineages. We thus investigated a possible role of Kb(KMC) on the in vitro proliferative response of B cell precursors to IL-7, the major growth factor for B and T cell precursors in the adult mouse. The proliferation in the presence IL-7 was measured in short-term cultures of purified B cell precursors or total bone marrow. As shown in Figure 8
, addition of Kb(KMC) to the cultures inhibited the proliferative response of B cell precursors to IL-7 in bone marrow cultures in a dose-dependent manner (Fig. 8A)
; similar results were obtained with purified B cells precursors B220+CD19+IgM–, >99% purity (Fig. 8B)
. Proliferation of mature B cells in response to LPS, which is independent of IL-7, was only marginally inhibited by Kb/KMC, showing that KMC is not acting as a general antimitotic substance (Fig. 8C)
.
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Figure 8. Kb and KMC inhibited the proliferative response of B cell precursors to IL-7. (A) Total bone marrow cells from naïve mice were cultured for 5 days with IL-7, washed, and recultivated for an additional 72 h with IL-7 in the absence or presence of increasing concentrations of Kb/KMC (µg/ml); proliferation was measured by thymidine uptake as described in Materials and Methods. (B) Purified B220+CD19+IgM– B cell precursors, >99.8% purity, were cultured for 3 days with IL-7 in the absence or presence of increasing concentrations of Kb/KMC (µg/ml); proliferation was measured by thymidine uptake. (C) Spleen cells were cultured for 72 h with LPS in the presence of increasing concentrations of Kb (µg/ml); proliferation was measured by thymidine uptake.
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The results described here show that treatment of mice with juice prepared from leaves of Kb resulted in a profound inhibition of B and T cell development, with substantial depletion of immature lymphocytes and their precursors in thymus and bone marrow. All the inhibitory effects of Kb juice on lymphopoiesis in vivo and in vitro could be reproduced by KMC, a highly purified compound obtained by ethanol precipitation from juice. KMC comprises the new metabolite kalanchosine, a 3,6-diamino-4,5-dihydroxyoctanedioic acid, and malic acid in a 1:2 stoichiometric ratio; this is the first description of a plant-derived natural product with these properties. Importantly, the potent impact in lymphocyte development was not a result of a general toxic effect of Kb or KMC on hematopoiesis, as the generation of myeloid cell types was not disturbed, indicating that Kb(KMC) was selectively affecting the lymphoid lineage. The percentages of Mac1+ bone marrow B cells are substantially increased, as shown in Figure 1C and 1D ; however, there is no statistically significant increase in total Mac1+ cell numbers, as shown in Figure 1E . The increase in Mac1+ percentages is mostly explained by the reduction of B220+ cells. A small stimulatory effect of Kb/KMC on Mac1+ cannot be discarded; however, this would be a marginal effect when compared with the dramatic ablation of B cell development. Interestingly, the number of mature lymphocytes in lymph nodes and spleen was not altered, demonstrating the specificity of the Kb(KMC) effect for lymphopoiesis.
A profound inhibition of lymphopoiesis with simultaneous preservation of mature lymphocyte cell numbers is compatible with the normal population dynamics of lymphocytes. B cells are continuously being produced throughout the life in the bone marrow, with an output of newly formed B lymphocytes averaging 15–20 x 106 cells/day in the young adult mouse; only 5–10% of the newly formed B cells are incorporated in the compartment of mature B cells, as a result of antigen-specific selection processes [22 23 24 ]. An analogous scenario also applies for T cells, although the thymic output of newly formed T lymphocytes is much lower (1–2x106 cells/day in the young adult animal), being compensated by a more intense proliferation of mature T cells in peripheral lymphoid organs. Therefore, for B and T cells, at a given time-point, the normal percentage of newly formed lymphocytes does not exceed 5–10% of the total pool of peripheral lymphocytes. These aspects of population dynamics explain why lymphopoiesis can be reduced dramatically for a few days without much impact on numbers of mature lymphocytes in secondary lymphoid organs [24 ], as observed here. The preservation of numbers of mature lymphocytes further documents that KMC is not a general, toxic substance to lymphoid cell types.
In the adult mouse, the growth of the earliest lymphoid-committed precursors is supported by several different factors secreted by the bone marrow stroma. More advanced lymphoid precursors that have already started the expression of rearranged variable receptors, in thymus or bone marrow, are strictly dependent on IL-7 to survive, proliferate, and undergo several rounds of mitosis, before losing the ability to respond to this cytokine. As the capacity to respond to IL-7 is lost, late lymphocyte precursors complete the final steps of differentiation, culminating with the expression of receptors BcR or TcR, giving rise to the newly formed, immature lymphocytes [25 26 27 28 29 30 31 32 ]. Here, we found that Kb(KMC) inhibited the proliferative response of B cell precursors to IL-7 in vitro, which could explain the ablation of lymphopoiesis observed in vivo, and is coherent with the preferential reduction of pre-BII and immature B cells, the latter being the immediate developmental stage of the former. We observed that a minor subpopulation of c-kit+ B cell precursors responded to IL-7 in vitro, even in the presence of Kb/KMC (data not shown), suggesting that the inhibitory action of Kb/KMC on IL-7 signaling may be restricted to a defined developmental stage of the precursor. These data, obtained in vitro, are also consistent with the results obtained in vivo, where the c-kit+ pre-BI precursors were less affected by Kb/KMC treatment (Fig. 2C and 2D) . The relative resistance of these more primitive cell precursors could reflect a higher capacity of this cell type to expel the drug from the cytoplasm through ATP-binding cassette transporters, a general property of HSCs, and more primitive progenitors.
The data obtained for T cell lineage maturation are also consistent with the notion that Kb or KMC would interfere with the signaling pathway downstream of the IL-7R. IL-7 knockout (KO) and IL-7R KO mice exhibit a blockade in T cell development at the transition between DN2 and DN3, and these animals accumulate DN2 cells [33
]. Here, we found that Kb/KMC-treated animals have a larger compartment of DN2 cells. Moreover, the massive reduction of DP CD4+CD8+ thymocytes in Kb/KMC-treated animals is consistent with the inhibition of the strong, proliferative capacity of the DN4 subpopulation, which is dependent on IL-7 as a trophic factor [34
]. It seems that Kb/KMC is affecting the two subpopulations with higher mitotic capacity DN2 and DN4, both dependent on IL-7, resulting in the depletion of their immediate progeny, DN3 and DP CD4+CD8+ thymocytes, respectively [29
]. Interestingly, we found that IL-2-dependent proliferation of mature, activated T cells is only marginally inhibited by KMC (data not shown). As the receptors for IL-7 and IL-2 share a common -chain and several JAK/STAT members of the intracellular signaling pathway [35
, 36
], a detailed, comparative analysis of the effects of Kb/KMC on both pathways may help to identify the molecular target of Kb/KMC.
The observation that Kb or KMC suppresses the response of B cell progenitors to IL-7 in vitro is suggestive of their mode of action in vivo. However, it cannot be excluded that Kb/KMC could also be acting through alternative and indirect mechanisms as well. In vivo blockade of lymphocyte development could also result from the action of different cytokines and hormones that have been characterized as selective inhibitors of lymphopoiesis (e.g., glucorticoid, estrogen hormones, TNF-, type I IFN, and its homologue limitin) [37
]. Detailed studies have shown that these molecules act on different stages of lymphocyte development, directly promoting apoptosis of precursors, inhibiting stromal cell functions, blocking the IL-7-dependent proliferation, or orientating the commitment of multipotent progenitors exclusively to the myeloid lineage [5
6
7
8
9
10
11
, 37
38
39
]. Therefore, we thought it would be essential for the characterization of the mode of action of Kb/KMC in vivo to investigate whether the inhibitory effect of Kb/KMC on lymphopoiesis could also depend on the secondary production of any of the above-cited molecules that could be secreted in the presence of Kb/KMC. This is a critical point, as those mediators, hormones, and cytokines have pleiotropic effects on the organism, promoting harmful side-effects in therapeutical protocols; the possibility to obtain a new drug interfering with lymphocyte biology without the effects of hormones and cytokines is of relevance.
We showed that glucocorticoid hormones were not involved as mediatiors of Kb/KMC effects (Fig. 5)
. Furthermore, in the presence of Kb or KMC, T and B cells precursors cultured in vitro did not enter into apoptosis massively, as observed in the presence of glucocorticoids (Fig. 7)
, bringing additional evidence for a distinct mode of action of Kb(KMC) in vivo. B cell development is also impaired by an estrogen steroid hormone through the inhibition of the secretion of factors by stromal cells, such as endogenous IL-7 [8
, 37
], without inducing apoptosis of B cell progenitors. However, male and female animals are equally susceptible to Kb suppression, ruling out the hypothesis of estrogen as a mediator of the Kb effect in vivo. Furthermore, the in vitro proliferation of B cell precursors induced by exogenous IL-7 was strongly inhibited by Kb or KMC, differently to the in vitro effect of estrogen, which does not inhibit the proliferation of pre-B cells in response to that cytokine. Secondary induction of type I IFN or TNF- following the systemic administration of Kb or KMC could also explain the selective, inhibitory effect of Kb on lymphopoiesis [5
, 10
, 38
, 39
]. However, the administration of Kb or KMC to TNF-RI–/– and IFN-RI–/– gene-targeted mice resulted in profound inhibition of T and B cell lymphopoiesis in both mouse strains, strongly suggesting that these cytokines are also not involved in mediating the effects of Kb or KMC in vivo. The experiments with C57BL/10ScCr and TLR2–/– mice also excluded a role for TLR2 and TLR4 signaling on the blockade of lymphopoiesis by Kb/KMC (Table 2)
. Those findings that molecules associated with inflammatory or proinflammatory processes are not involved in mediating the blockade of lymphopoiesis by Kb/KMC treatment are coherent with the anti-inflammatory activity of Kb/KMC described previously [17
]. More recently, IL-6 has also been described as a blocking agent of early lymphopoiesis [40
]. We have not investigated here a role for IL-6; however, it seems to us unlikely that this cytokine would be involved because of the observed anti-inflammatory action of Kb.
Altogether, the experiments described here suggest that the juice of Kb or its purified fraction KMC is blocking lymphopoiesis through a new mechanism that differs from the modes of action described previously for hormones and cytokines that can induce an analogous effect. Evidence is provided to support the notion that Kb/KMC is interfering with the response of lymphoid progenitors to IL-7. These results open new perspectives for a possible therapeutic use of Kb-derived products as immunomodulatory agents, as IL-7 has a central role not only on lymphocyte development but also affects the biology of mature T cells, including regulatory T cells [41 42 43 44 45 ]. Finally, IL-7 has also been shown to be important for the growth of certain T cell lymphomas and breast cancer, and Kb/KMC could be a promising candidate to control these malignant tumors [46 , 47 ].
ACKNOWLEDGEMENTS
This work was supported by the Brazilian Agencies: FAPERJ, FINEP, FUJB, CNPq, and CAPES. We are grateful to Zenildo B. Morais Filho (NPPN, Universidade Federal do Rio de Janeiro) for technical support.
FOOTNOTES
1 These authors contributed equally to this work. 
Received July 2, 2007; revised November 28, 2007; accepted November 29, 2007.
REFERENCES
β T cell development in the thymus of normal and genetically altered mice Curr. Opin. Immunol. 9,263-275[CrossRef][Medline] chain: a functional component of the interleukin-7 receptor Science 262,1877-1880
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