Selective blockade of lymphopoiesis induced by kalanchosine dimalate: inhibition of IL-7-dependent...

Selective blockade of lymphopoiesis induced by kalanchosinedimalate: inhibition of IL-7-dependent proliferation

Luciana S. de Paiva,*,1 Alberto Nobrega,*,1,2 Giany O. De Melo,† Elize A. Hayashi,*Vinicius Carvalho,‡ Patricia M. Rodrigues e Silva,‡ Maria Bellio,* Gerlinde P. Teixeira,§

Vivian Rumjanek,�� Sonia S. Costa,† and Vera Lucia G. Koatz��

*Departamento de Imunologia, Instituto de Microbiologia, †Nucleo de Pesquisas de Produtos Naturais, ��Instituto deBioquımica Medica, Universidade Federal do Rio de Janeiro, Brazil; ‡Departamento de Fisiologia eFarmacodinamica, IOC, FIOCRUZ, Rio de Janeiro, Brazil; and §Departamento de Imunobiologia, Instituto deBiologia, Universidade Federal Fluminense, Rio de Janeiro, Brazil

Abstract: Lymphopoiesis and myelopoiesis con-tinuously generate mature cells from hematopoi-etic cell progenitors during the lifetime of the or-ganism. The identification of new endogenous orexogenous substances that can act specifically on thedifferentiation of distinct cell lineages is of relevanceand has potential therapeutical use. Kalanchoe bra-siliensis (Kb) is a medicinal plant from the Crassu-laceae family, used in folk medicine to treat inflam-matory and infectious diseases. Here, we show thatshort-term treatment of naıve mice with Kb led toa strong and selective inhibition of lymphopoiesis,affecting B and T cell lineages without reduction ofthe myeloid lineage development. Similar effectswere observed after treatment with the highly pu-rified compound kalanchosine dimalate (KMC), ob-tained from Kb. Numbers of mature lymphocytesin secondary lymphoid organs were preserved inKb(KMC)-treated mice. The effect of Kb(KMC)was not a result of secondary augmentation ofplasma levels of endogenous corticoids; neither in-volves TNF-�, type-I IFN, or TLR2/TLR4 ligands,which have all been described as selective inhibi-tors of lymphopoiesis. Flow cytometry analysis ofthe phenotypes of T and B cell precursors indicatea blockade of maturation on IL-7-dependent, pro-liferative stages. In vitro, Kb(KMC) inhibited theIL-7-dependent proliferation of pre-B cells anddoes not induce massive apoptosis of B and T cellprecursors. These results suggest that Kb(KMC) isselectively blocking lymphopoiesis through amechanism that does not involve the previouslycharacterized substances, possibly acting on theIL-7 signaling pathway, opening new perspectivesfor a potential therapeutic use of Kb-deriveddrugs. J. Leukoc. Biol. 83: 000–000; 2008.

Key Words: Kalanchoe brasiliensis � corticosteroids � TLR � IFN � TNF

INTRODUCTION

Hematopoiesis continuously generates blood cell types duringthe lifetime of the organism from hematopoietic stem cells

(HSCs) that undergo massive proliferation and differentiationinto specific cell types. The differentiation of HSCs into lin-eage-specific progenitors is a hierarchical process involving agenetic network of transcription factors that control the com-mitment to different lineages, which results in a major dichot-omy segregating lymphopoiesis from myelopoiesis [1]. In theadult mouse, mature myelomonocytic cell types have beenproposed to derive from a common myelomonocitic cell pro-genitor, whereas lymphocytes would mainly stem from a com-mon lymphoid progenitor (CLP) [2]. Alternative proposals tothis scheme have been suggested, where differentiation ofHSCs would lead to early segregation of a megakariocyte/erythroid progenitor from a lymphoid-primed, multipotent pro-genitor, followed by further branching into a granulocyte-mac-rophage progenitor and CLP [3]. Additional progenitor celltypes, such as early lymphocyte progenitors and early T cellprogenitors (ETPs), have also been characterized, which wouldgive 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 resultingin the segregation of lymphopoiesis from myelopoiesis stillapplies to alternative schemes suggested for the generation ofblood cell types from HSCs.

The differentiation of HSCs into myeloid progenitors andlymphoid progenitors depends on multiple cytokines and fac-tors secreted by hematopoietic cells or stromal cells and alsoon the direct engagement of membrane receptors of HSCs withtheir respective ligands on the surface of stromal cells orextracellular matrix. Interestingly, the differentiation processof HSCs can be modulated by inflammatory cytokines (e.g.,TNF-�, type I IFNs) and glucocorticoid or estrogen steroidhormones, which have no role as constitutive growth factor ordifferentiation factor for progenitors. These substances havebeen shown to interfere selectively on the process of HSC

1 These authors have contributed equally to this work.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 Universitaria, CEP:21941-902, Rio de Janeiro,Brazil. E-mail: [email protected]

Received July 2, 2007; revised November 28, 2007; accepted November 29,2007.

doi: 10.1189/jlb.0707441

0741-5400/08/0083-0001 © Society for Leukocyte Biology Journal of Leukocyte Biology Volume 83, April 2008 1

Uncorrected Version. Published on January 15, 2008 as DOI:10.1189/jlb.0707441

Copyright 2008 by The Society for Leukocyte Biology.

differentiation, blocking the development of the lymphoid lin-eage [5–10]. Detailed studies have shown that these moleculesact at different steps of B or T lymphocyte development,directly promoting apoptosis of precursors, inhibiting stromalcell functions, blocking the IL-7-dependent proliferation, orhindering the lymphoid potential of early progenitors, thuschanneling the commitment of multipotent progenitors exclu-sively to the myeloid lineage [11]. More recently, TLR2 andTLR4 receptors have also been shown to interrupt early stepsof lymphopoeisis, sparing the myeloid lineage [12], but theexistence of endogenous ligands for these receptors is stilldebated.

The identification of new endogenous or exogenous sub-stances selectively acting on myelopoiesis and lymphopoiesisis of great interest because of their potential therapeutic use.Natural products obtained from medicinal plants constitute animportant source of new biological agents with therapeuticvalue [13, 14]. Our group has been investigating the propertiesof compounds derived from plants from the genus Kalanchoe,traditionally used in folk medicine to treat inflammatory andinfectious diseases [15]. Previously, we demonstrated that juiceprepared from the fresh leaves of Kalanchoe brasiliensis (Kb)and Kalanchoe pinnata have immunomodulatory and immuno-suppressive properties in murine models of leishmaniasis andarthritis, respectively [16, 17]. Recently, we characterized ahighly 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 highlypurified fraction KMC on the development of B and T lympho-cytes. We show that short-term treatment of naıve mice withKb(KMC) led to a strong and selective inhibition of lympho-poiesis, affecting B and T cell lineages without reduction of themyeloid lineage development. This is the first description of aplant-derived natural product with these properties. The re-sults suggest that the mode of action of Kb(KMC) differs fromthe mechanisms pertaining to previously characterized endog-enous and exogenous substances that also promote the selec-tive ablation of lymphopoiesis. Phenotypic analysis of T and Bcell precursors in Kb(KMC)-treated mice indicates a blockadeof maturation on IL-7-dependent, proliferative stages in vivo;in vitro, the proliferative response of B cell precursors to IL-7is inhibited by Kb(KMC). These results suggest that Kb(KMC)may be selectively acting on the IL-7 proliferative response ofB and T cell progenitors/precursors, opening new perspectivesfor therapeutical use of Kb and its derived products as immu-nomodulatory agents.

MATERIALS AND METHODS

Animals

C57BL/10 and C57BL/10ScCr mice (male, 25 g) were obtained from Univer-sidade Federal Fluminense (Rio de Janeiro, Brazil). Mice genetically targetedfor TLR2�/� [19], TNF-� type I receptor (TNF-RI�/�) [20], and C57BL/6wild-type used as background were obtained from Fiocruz Minas Gerais andRio de Janeiro (Brazil). Mice genetically targeted for IFN type I receptor(IFN-RI�/�) [21] on background (129�C57BL/6) and the wild-type(129�C57BL/6) were obtained from the Department of Immunology, Institutode Ciencias Biomedicas-Universidade de Sao Paulo (Brazil). Animals werehoused in a temperature-controlled room, receiving water and food ad libitum.

All the procedures involving animal experiments were performed in accor-dance to the International Guidelines for Animal Use. The “Committee forAnimal Studies” of the Institute of Microbiology, Federal University of Rio deJaneiro (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 collectedin the morning hours at Rio de Janeiro State (Brazil). A voucher specimen304627 was deposited at the Botanical Garden of Rio de Janeiro (Brazil). Thefresh aerial parts of Kb (24 kg) were washed with a solution of sodiumhypochlorite at 100 ppm, rinsed, and dried at room temperature. Twenty-fourhours later, the fresh plant material was triturated in an industrial foodprocessor to obtain a green juice (19 L), which was clarified by decantation at5°C for 24 h, resulting in a yellow preparation (17.3 L), named Kb juice, whichwas lyophilized for further manipulations. Observation: The name Kb juice isused here instead of extract, as the material was obtained without the help ofany 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. Afterthis period, the white-flake precipitate was recovered by paper filtration. Thefilters were washed with pyrogen-free water (1.0 L), and the filtrates obtainedwere reprecipitated with ethanol (1.5 L) and filtered again. The collected solidmaterial obtained was partially dried at 40°C, dissolved in pyrogen-free water,frozen, and lyophilized. The 1H nuclear magnetic resonance analysis showedthat 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), containingnonethanol-precipitated material, was collected and named SN. The ethanolwas further evaporated, and the SN was lyophilized and kept at 5°C until used.The chemical composition of all Kb preparations was systematically monitoredby using HPLC with the column Asahipak GS-51OH and the Diodearraydetector (Shimadzu, Japan), eluted with 20 mM Tris-HCl buffer, pH 8.0, in aflow rate of 0.4 ml/min.

Treatment with Kb and fractions

Just before use, lyophilized Kb juice, KMC, or SN was resuspended inpyrogen-free water and filtered through a 0.22-�m Millipore filter. Groups ofthree to five mice were daily i.p.-treated with an injection of 200 �l/mousecontaining 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]. Controlmice received pyrogen-free water. At the end of treatment, mice were killed,and bone marrow, spleen, lymph node, and thymus were used to preparesingle-cell suspensions.

Cell staining and flow cytometry

Fresh single-cell suspensions from bone marrow, spleen, lymph node, andthymus were stained with fluorochrome-conjugated mAb and monitored by flowcytometry. The mAb used for staining were as follow: FITC-goat anti-mouseIgM, 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 Annex-inV-FITC (BD PharMingen, San Diego, CA, USA). Cells were incubated withthe mAb in FACS buffer (PBS, 1% FCS, 0.05% Na-azide) for 20 min on iceand washed twice with FACS buffer. Data were acquired on a FACSCalibur™(BD Biosciences, San Jose, CA, USA) and analyzed using CELLQuest™software (BD Biosciences). Staining with AnnexinV-FITC was done in an-nexin-binding buffer, following the protocol indicated by the manufacturer.

MACS

For purification of B cell precursors, bone marrow cells were incubated inMACS buffer (PBS containing 2 mM EDTA, 5% FCS) with anti-mouse IgMMicroBeads (Miltenyi Biotec, Germany) for 20 min on ice and passed througha nylon mesh. Cells were then applied to a magnetic column (Miltenyi Biotec),and the nonadherent cells were collected; cells were then washed and resus-pended in MACS buffer with anti-mouse CD19 MicroBeads (Miltenyi Biotec)

2 Journal of Leukocyte Biology Volume 83, April 2008 http://www.jleukbio.org

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. Collectedcells were analyzed for purity by flow cytometry and were described asB220�CD19�IgM–, 99.8% purity.

Cell cultures

B cell precursors from bone marrow obtained by positive selection using MACSanti-CD19 MicroBeads, as described above, were cultivated in RPMI 1640supplemented 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 cellline transfected with IL-7 plasmid. After 120 h, cells were recovered, washedextensively to remove IL-7, and recultured for 72 h in the absence or presenceof IL-7, Kb, or KMC, and cell proliferation was measured at the end of theculture period. Single-cell suspensions from spleen (106 cell/ml) were culturedfor 72 h in RPMI (Sigma Chemical Co., St. Louis, MO, USA) as above in theabsence or presence of 12 �g/ml LPS, Kb, or KMC, and cell proliferation wasmeasured at the end of the culture period.

Proliferation assay

Cells (2�105 cells/well) were cultivated in RPMI supplemented with 10% FCSin 96-well, flat-bottom plates in a humidified atmosphere of 5% CO2 at 37°C.In the last 18 h of cell culture, 1 �Ci [3H] thymidine (Sigma Chemical Co.) wasadded to each culture well. Cells were harvested onto glass microfiber filterpaper 934AH (Whatman, Maidstone, UK), and radioactivity was assessed byscintillation counting.

Corticosterone detection

Plasmatic corticosterone concentration was determined by radioimmunoassayusing the kit from ICN-Biomedicals (Costa Mesa, CA, USA), following theinstructions 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 nonparametrictest and were considered statistically significant when P � 0.05.

RESULTS

Selective blockade of B cell development in the bone marrowafter short-term administration of Kb C57BL/10 mice, three tofive animals per group, was i.p.-injected daily with 480 mg/kgKb juice for 4 consecutive days. Twenty-four hours after thelast injection, single-cell suspensions of bone marrow cellswere prepared and analyzed by flow cytometry. Although bonemarrow total cell counts from control mice did not differsignificantly from those of Kb-treated animals, the forward-scatter/side-scatter plot indicated a profound reduction ofevents in the lymphoid region of the plot in the Kb-treatedanimals (Fig. 1, A and B, indicated with arrows). Thephenotypic analysis of bone marrow cell populations for thesurface expression of Mac1 and B220 confirmed that B celllineage was strongly diminished in Kb-treated mice (Fig. 1,C and D), and total numbers of Mac1� myeloid lineage wereessentially preserved (Fig. 1E). Kinetic experiments re-vealed that the major ablation of B cell lymphopoiesisincreased progressively from 1 to 3– 4 days, with no furthersignificant impact on numbers of CD19/B220 bone marrowcells beyond that, until 7 days; therefore, all the subsequentexperiments were done using the 4-day treatment protocol(data not shown).

B220� cells in the bone marrow comprise B cells precur-sors (pro-B/pre-B), immature B cells, and mature B lympho-cytes. Analysis of cell populations for the simultaneous expres-sion of B220 and surface IgM showed that the pro-B/pre-B andimmature B cell numbers were significantly diminished in micetreated with Kb, with lesser effect on recirculating, mature Blymphocytes (Fig. 2, A and B). A more detailed analysis of theeffect of Kb treatment in B cell precursors revealed that pre-BIIcells (B220�CD25�IgM–) and immature B cells were the

Fig. 1. Administration of Kb-impaired B cell lymphopoiesis. C57BL/10 mice received daily i.p. treatment with water or480 mg/kg Kb for 4 days. On the 5th day, bone marrow cells were analyzed by flow cytometry. The figure shows theside-scatter and forward-scatter parameters of bone marrow-nucleated cells from water (A) and Kb-treated mice (B). Thelocation of lymphoid cells is indicated with an arrow within the closed polygon gate of nucleated cells. Bone marrow cellswere stained with mAb to identify the B220� and membrane-actived complex 1 (Mac1)� cell lineages in water (C)- andKb (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 threedifferent experiments with three mice per group. CTR, Control.

de Paiva et al. Blockade of lymphopoiesis by kalanchosine dimalate 3

populations whose numbers were dramatically reduced, withlesser impact on the pre-BI (B220�c-kit�IgM–) population.(Fig. 2C shows total bone marrow cell numbers; Fig. 2Dindicates percentages of each cell type within bone marrowB220� cells.)

T cell development is affected by treatmentwith Kb

The impact of Kb treatment in peripheral lymphoid organsand thymus was also investigated. Cell numbers of spleenand peripheral lymph nodes were little or not affected inKb-treated animals (Table 1), with no effect in the T or Blymphocyte compartment (data not shown). However, the

analysis of thymus revealed a profound reduction of cellnumbers in this organ (Table 1). The flow cytometry pheno-typic analysis of thymocytes showed that the population ofdouble-positive (DP) CD4�/CD8� immature T cells wasmassively reduced in mice treated with Kb. Figure 3, A andB, shows a typical result obtained from two different exper-iments with five mice per group. Total cell numbers perthymus of each population (Fig. 3C) or percentages of eachpopulation (Fig. 3D) are also shown. These results suggestthat the main targets of Kb action would be the primarylymphoid organs, with a severe compromise of B cell and Tcell development. It is important to note that although SPthymocytes are percentually enriched in the thymuses of

Fig. 2. B cell precursors and immature B cells arepreferentially diminished in mice treated with Kb. Bonemarrow cells were obtained as described in Figure 1,stained with anti-IgM and anti-B220 mAb, and analyzedby flow cytometry for the presence of different B celllineage 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 andpre-B cells is indicated as Gate (I), immature B cells asGate (II), and mature B cells as Gate (III). Mice wereinjected with water (A) or with Kb (B). (C) The totalnumbers of pro-B/pre-B, B220�CD25�IgM– (preB II),B220�c-kit�IgM– (preB I), and immature B cells perfemur are plotted as the bar graph, showing meanvalues and SD; *, P � 0.05. (D) The percentagesof pre-BII B220�CD25�IgM–, pre-BI B220�c-kit�IgM–, and immature B cells within bone marrowB220� cells are plotted, as the bar graph, showingmean values and SD; *, P � 0.05. Mature B cells wereexcluded from the analysis. The data presented in thefigure are representative of three different experimentswith three mice per group.

TABLE 1. Administration of Kb Selectively Reduced Lymphocytes in Primary Lymphoid Organs

Mouse # Spleen cell numbers Lymph node cell numbers Thymus cell numbers

CTR 1 64.50 29.24 94.50CTR 2 62.70 (64.36 1.60) 28.87 (28.63 0.76) 99.00 (102.17 9.65)CTR 3 65.90 27.78 113.00Kb 1 52.80 27.90 18.92Kb 2 59.10 (62.80 12.28) 27.40 (26.40 2.18) 12.62 (25.53 17.20)Kb 3 76.50 23.89 45.05

C57BL/10 mice received daily i.p. treatment with water (CTR) or 480 mg/kg Kb for 4 days. On the 5th day, cells from spleen, mesenteric lymph nodes, andthymus were obtained. The table shows the total numbers of cells/organ with the respective mean values and standard deviations. The data are representative oftwo different experiments with three mice per group.


Kb-treated animals, their absolute numbers are lower thanfor control mice.

A more detailed analysis of early T cell development, eval-uating maturation of DN cells, showed an enrichment for theDN2 CD25�CD44� subpopulation in Kb-treated mice, with asimultaneous reduction of DN3 CD25�CD44– and DN4CD25–CD44– and no effect on DN1 CD25–CD44�. Figure4, A and B, shows a typical result obtained from three differ-ent experiments with three mice per group; percentages of DPand DN are shown in Figure 4C; Figure 4D shows percentagesof DN1, DN2, DN3, and DN4 within total DN cells.

Characterization of the substance promoting theblockade of lymphopoiesis

Fractionation of Kb juice with ethanol precipitation yielded twofractions: a SN and a highly purified precipitate formed by acomplex salt between kalanchosine (3,6-diamino-4,5-dihy-droxyoctanedioic acid) and malic acid, with 1:2 ratio, recentlycharacterized [18] and named KMC. To test the selective effectof these fractions, animals were treated with 480 mg/kg Kb, 160mg/kg KMC, or 320 mg/kg SN to keep the proportion betweenKMC (33%) and SN (67%), found in the original juice. Asshown in Figure 5, the administration of KMC induced aprofound depletion of T and B cell precursors similar to theeffect of whole Kb juice; SN-treated animals exhibited a partialinhibitory effect.

Inhibition of B and T cell development followingadministration of Kb does not involveendogenous corticoids or massive apoptosis oflymphocyte precursors

The strong effect of Kb in decreasing T and B cell lymphopoi-esis while preserving mature lymphocytes presents similaritywith the effect of in vivo glucocorticoid treatment of mice [9].Therefore, we investigated whether the inhibitory effect of Kbwas associated with the release of endogenous glucocorticoidsthat could be induced by Kb(KMC). As shown in Figure 6,mice treated with Kb juice have similar levels of serum corti-costerone as animals treated with water, therefore excludingthe hypothesis of a selective increase of this hormone inKb-injected animals. Interestingly, both groups have equallyhigher corticosterone levels than nonmanipulated animals, in-dicating corticoid release resulting from the stress as a result ofmanipulation of the injected animals.

It has been shown that the ablation of lymphopoiesis ob-served after corticoid treatment is a result of induction ofmassive apoptosis of lymphoid precursors, which can also beobserved in vitro. We then investigated if Kb(KMC) couldinduce apoptosis of lymphoid precursors in vitro. As expected,in vitro treatment of pro-B/pre-B cells with increasing doses ofdexamethasone led to massive apoptosis of precursors (Fig. 7,A–D); however, only a marginal effect was observed in thepresence of increasing doses of Kb(KMC). Similar results wereobtained with T cell precursors (Fig. 7, E–H). Also, the total

Fig. 3. Impairment of T cell development in micetreated with Kb. Thymus from mice treated as describedin Figure 1 were removed, thymocytes were stainedwith anti-CD4, and anti-CD8 mAb were analyzed byflow cytometry for the presence of different T celllineage populations: CD4–/CD8–, CD4�/CD8–, CD4–/CD8�, and CD4�/CD8� cells. Mice were treated withwater (A) or Kb (B). The percentages of the different Tcell populations are indicated in each quadrant. (A andB) A typical result obtained from three different exper-iments with three mice per group. Total cell numbersper thymus of each population (C) or percentages ofeach population (D) are plotted as bar graphs showingmean values and SD; *, P � 0.05. DN, Double-negative;SP, single-positive.


number of live cells after 72 h of in vitro culture of bonemarrow cells is equivalent in the absence or presence of Kb(not shown). These results also indicate that Kb does not haveany blatant toxic effect on survival/mortality of cells in vitro.

Selective blockade of lymphopoiesis by KMC didnot depend on the action of TNF-�, type I IFN,TLR2, or TLR4

TNF-� and type I IFNs have been shown to selectively inhibitlymphopoiesis, sparing myelopoiesis [5, 10]. To investigatewhether the effect of Kb(KMC) could be associated with theaction of these endogenous mediators known to modulate thelymphopoiesis, we treated gene-targeted mice for TNF-RI�/�

or for the IFN-RI�/� with Kb(KMC). The results obtained inboth mouse lineages show that the treatment with Kb(KMC)led to a profound and selective reduction of lymphopoiesis(Table 2).

It has been shown recently that selective inhibition of lym-phopoiesis can also be obtained by the direct engagement ofTLR2 or TLR4 on early hematopoietic progenitors [12]. There-fore, we also investigated a role for those receptors inKb(KMC)-treated mice, assessing the effect in C57BL/10ScCrmice, a mouse strain naturally null mutant of tlr4, and intlr2�/� gene-targeted mice. The results obtained do not sup-port a role for these TLRs on the mechanism of action of

Kb(KMC), as the effects on C57BL/10ScCr or TLR2�/� mousestrain were similar to those observed on C57BL/10 wild-typemice (Table 2). These experiments were also informative torule out the possibility that trace amounts of contaminants ofbacterial origin, mainly LPS or lipoproteins, could be presentin our preparation of Kb(KMC) and be responsible for theeffects observed on lymphopoiesis, directly or through theaction of secreted cytokines. Additionally, a preparation of Kbjuice was passed through a column of polymyxin B to removeany LPS, confirmed by the limulus assay, and was as effectiveto inhibit lymphopoiesis in wild-type mice as the original Kbjuice (data not shown).

In vitro treatment with Kb or KMC inhibited theIL-7-dependent proliferation of B cell precursors

The selective effect of Kb(KMC) on B and T cell developmentseems to target the precursor stages with a strong proliferativecapacity in both lineages. We thus investigated a possible roleof Kb(KMC) on the in vitro proliferative response of B cellprecursors to IL-7, the major growth factor for B and T cellprecursors in the adult mouse. The proliferation in the pres-ence IL-7 was measured in short-term cultures of purified Bcell precursors or total bone marrow. As shown in Figure 8,addition of Kb(KMC) to the cultures inhibited the proliferativeresponse of B cell precursors to IL-7 in bone marrow cultures

Fig. 4. Differential effects of Kb/KMC on DN T cellprecursors. Thymus from mice treated as described inFigure 1 were removed, and thymocytes were analyzedby flow cytometry for the presence of different T celllineage subpopulations. (A and B) The cytometrydot-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 twodifferent experiments with five mice per group. Thepercentages 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 areplotted as bar graphs showing mean values and SD; *,P � 0.05.


in a dose-dependent manner (Fig. 8A); similar results wereobtained with purified B cells precursors B220�CD19�IgM–,99% purity (Fig. 8B). Proliferation of mature B cells inresponse to LPS, which is independent of IL-7, was onlymarginally inhibited by Kb/KMC, showing that KMC is notacting as a general antimitotic substance (Fig. 8C).

DISCUSSION

The results described here show that treatment of mice withjuice prepared from leaves of Kb resulted in a profound inhi-bition of B and T cell development, with substantial depletionof immature lymphocytes and their precursors in thymus andbone marrow. All the inhibitory effects of Kb juice on lympho-poiesis in vivo and in vitro could be reproduced by KMC, ahighly purified compound obtained by ethanol precipitationfrom juice. KMC comprises the new metabolite kalanchosine, a3,6-diamino-4,5-dihydroxyoctanedioic acid, and malic acid ina 1:2 stoichiometric ratio; this is the first description of aplant-derived natural product with these properties. Impor-tantly, the potent impact in lymphocyte development was not aresult 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 lymphoidlineage. The percentages of Mac1� bone marrow B cells are

substantially increased, as shown in Figure 1, C and D; how-ever, there is no statistically significant increase in totalMac1� cell numbers, as shown in Figure 1E. The increase inMac1� percentages is mostly explained by the reduction ofB220� cells. A small, stimulatory effect of Kb/KMC onMac1� cannot be discarded; however, this would be a mar-ginal effect when compared with the dramatic ablation of B celldevelopment. Interestingly, the number of mature lymphocytesin lymph nodes and spleen was not altered, demonstrating thespecificity of the Kb(KMC) effect for lymphopoiesis.

A profound inhibition of lymphopoiesis with simultaneouspreservation of mature lymphocyte cell numbers is compatiblewith the normal population dynamics of lymphocytes. B cellsare continuously being produced throughout the life in thebone marrow, with an output of newly formed B lymphocytesaveraging 15–20 � 106 cells/day in the young adult mouse;only 5–10% of the newly formed B cells are incorporated in thecompartment of mature B cells, as a result of antigen-specificselection processes [22–24]. An analogous scenario also ap-plies for T cells, although the thymic output of newly formed Tlymphocytes is much lower (1–2�106 cells/day in the youngadult animal), being compensated by a more intense prolifer-ation of mature T cells in peripheral lymphoid organs. There-fore, for B and T cells, at a given time-point, the normalpercentage of newly formed lymphocytes does not exceed5–10% of the total pool of peripheral lymphocytes. Theseaspects of population dynamics explain why lymphopoiesis canbe reduced dramatically for a few days without much impact onnumbers of mature lymphocytes in secondary lymphoid organs[24], as observed here. The preservation of numbers of mature

Fig. 5. KMC purified from Kb has the major inhibitory activity on B celldevelopment. Groups of mice were injected i.p. daily with 480 mg/kg Kb, 320mg/kg SN, or 160 mg/kg KMC, as described in Materials and Methods. On the5th day, bone marrow cells were prepared and stained with anti-IgM andanti-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. Thedata show median values and SD from three different experiments (Mann-Whitney test); *, P � 0.05.

Fig. 6. Blockade of lymphopoiesis by Kb does not depend on a specificincrease of endogenous corticoids. Groups of five C57BL/10 mice were treatedi.p. daily with water or Kb for 4 days. On the 5th day, levels of sericcorticosterone of treated and nonmanipulated mice (None) were measured byradioimmune assay. Horizontal bars represent the mean value for each group,and * indicate that water and Kb-treated mice were significantly different fromthe values obtained with nonmanipulated animals (Mann-Whitney test).


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 factorssecreted by the bone marrow stroma. More advanced lymphoidprecursors that have already started the expression of rear-ranged variable receptors, in thymus or bone marrow, arestrictly dependent on IL-7 to survive, proliferate, and undergoseveral rounds of mitosis, before losing the ability to respond to

this cytokine. As the capacity to respond to IL-7 is lost, latelymphocyte precursors complete the final steps of differentia-tion, culminating with the expression of receptors BcR or TcR,giving rise to the newly formed, immature lymphocytes [25–32]. Here, we found that Kb(KMC) inhibited the proliferativeresponse of B cell precursors to IL-7 in vitro, which couldexplain the ablation of lymphopoiesis observed in vivo, and iscoherent with the preferential reduction of pre-BII and imma-ture B cells, the latter being the immediate developmentalstage of the former. We observed that a minor subpopulation ofc-kit� B cell precursors responded to IL-7 in vitro, even in thepresence of Kb/KMC (data not shown), suggesting that theinhibitory action of Kb/KMC on IL-7 signaling may be re-stricted to a defined developmental stage of the precursor.These data, obtained in vitro, are also consistent with theresults obtained in vivo, where the c-kit� pre-BI precursorswere less affected by Kb/KMC treatment (Fig. 2, C and D). Therelative resistance of these more primitive cell precursorscould reflect a higher capacity of this cell type to expel the drugfrom 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 alsoconsistent with the notion that Kb or KMC would interfere withthe signaling pathway downstream of the IL-7R. IL-7 knockout(KO) and IL-7R KO mice exhibit a blockade in T cell devel-opment at the transition between DN2 and DN3, and theseanimals 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� thymo-cytes in Kb/KMC-treated animals is consistent with the inhi-

Fig. 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 Materialsand Methods) were cultured for 18 h and assayed for apoptosis by flow cytometry staining with AnnexinV and propidium iodide (PI): (A) control; (B) dexamethasone10�8 M; (C) Kb 30 �g/ml; (D) Kb 100 �g/ml. Thymocytes were cultured for 18 h and assayed for apoptosis by flow cytometry staining with AnnexinV and propidiumiodide: (E) control; (F) dexamethasone 10�8 M; (G) Kb 30 �g/ml; (H) Kb 100 �g/ml.

TABLE 2. Kb Impaired T and B Cell Development in TNF-RI–/–

and IFN-RI–/–, C57BL/10ScCr, or TLR2–/– Mice

Cell numbers(% of control)

TNF-RI–/– B220�/IgM– 12.5 3.4CD4�/CD8� 8.12 6.3

�-IFN-RI–/– B220�/IgM– 8.3 0.9CD4�/CD8� ND

C57BL10/ScCr B220�/IgM– 38.7 1.2CD4�/CD8� 14.7 11.4

B6 TLR2–/– B220�/IgM– 15.1 5.5CD4�/CD8� 10.33 2.4

Mice genetically deficient for TNF-RI–/–, IFN-RI–/–, C57BL/10ScCr (TLR4-negative mutant), or TLR2–/– received daily i.p. treatment with water or 480mg/kg Kb for 4 days. On the 5th day, cells from bone marrow and thymus wereobtained. The table shows cell numbers of bone marrow B220�/IgM– andthymocytes CD4�/CD8�; the values are expressed as percentages of cellnumbers obtained in a control group of animals injected with water only(100%). The data are representative of two different experiments with threemice per group. ND, Not determined.


bition of the strong, proliferative capacity of the DN4 subpopu-lation, which is dependent on IL-7 as a trophic factor [34]. Itseems that Kb/KMC is affecting the two subpopulations withhigher mitotic capacity DN2 and DN4, both dependent on IL-7,resulting in the depletion of their immediate progeny, DN3 andDP CD4�CD8� thymocytes, respectively [29]. Interestingly,we found that IL-2-dependent proliferation of mature, activatedT cells is only marginally inhibited by KMC (data not shown).As the receptors for IL-7 and IL-2 share a common �-chain andseveral JAK/STAT members of the intracellular signaling path-way [35, 36], a detailed, comparative analysis of the effects ofKb/KMC on both pathways may help to identify the moleculartarget of Kb/KMC.

The observation that Kb or KMC suppresses the response ofB cell progenitors to IL-7 in vitro is suggestive of their mode ofaction in vivo. However, it cannot be excluded that Kb/KMCcould also be acting through alternative and indirect mecha-nisms as well. In vivo blockade of lymphocyte developmentcould also result from the action of different cytokines andhormones that have been characterized as selective inhibitorsof lymphopoiesis (e.g., glucorticoid, estrogen hormones,TNF-�, type I IFN, and its homologue limitin) [37]. Detailedstudies have shown that these molecules act on different stagesof lymphocyte development, directly promoting apoptosis ofprecursors, inhibiting stromal cell functions, blocking the IL-7-dependent proliferation, or orientating the commitment ofmultipotent progenitors exclusively to the myeloid lineage[5–11, 37–39]. Therefore, we thought it would be essential forthe characterization of the mode of action of Kb/KMC in vivo toinvestigate whether the inhibitory effect of Kb/KMC on lym-phopoiesis could also depend on the secondary production ofany of the above-cited molecules that could be secreted in the

presence of Kb/KMC. This is a critical point, as those media-tors, hormones, and cytokines have pleiotropic effects on theorganism-promoting, harmful side-effects in therapeutical pro-tocols; the possibility to obtain a new drug interfering withlymphocyte biology without the pleiotropic effects of hormonesand cytokines is of relevance.

We showed that glucocorticoid hormones were not involvedas mediatiors of Kb/KMC effects (Fig. 5). Furthermore, in thepresence of Kb or KMC, T and B cells precursors cultured invitro did not enter into apoptosis massively, as observed in thepresence of glucocorticoids (Fig. 7), bringing additional evi-dence for a distinct mode of action of Kb(KMC) in vivo. B celldevelopment is also impaired by an estrogen steroid hormonethrough the inhibition of the secretion of factors by stromalcells, such as endogenous IL-7 [8, 37], without inducing apo-ptosis of B cell progenitors. However, male and female animalsare equally susceptible to Kb suppression, ruling out the hy-pothesis of estrogen as a mediator of the Kb effect in vivo.Furthermore, the in vitro proliferation of B cell precursorsinduced by exogenous IL-7 was strongly inhibited by Kb orKMC, differently to the in vitro effect of estrogen, which doesnot inhibit the proliferation of pre-B cells in response to thatcytokine. Secondary induction of type I IFN or TNF-� follow-ing the systemic administration of Kb or KMC could alsoexplain the selective, inhibitory effect of Kb on lymphopoiesis[5, 10, 38, 39]. However, the administration of Kb or KMC toTNF-RI�/� and IFN-R�/� gene-targeted mice resulted in pro-found inhibition of T and B cell lymphopoiesis in both mousestrains, strongly suggesting that these cytokines are also notinvolved in mediating the effects of Kb or KMC in vivo. Theexperiments with C57BL/10ScCr and TLR2�/� mice also ex-cluded a role for TLR2 and TLR4 signaling on the blockade of

Fig. 8. Kb and KMC inhibited the proliferative response of B cell precursors to IL-7. (A) Total bone marrow cells from naıve mice were cultured for 5 days withIL-7, washed, and recultivated for an additional 72 h with IL-7 in the absence or presence of increasing concentrations of Kb/KMC (�g/ml); proliferation wasmeasured by thymidine uptake as described in Materials and Methods. (B) Purified B220�CD19�IgM– B cell precursors, 99.8% purity, were cultured for 3days with IL-7 in the absence or presence of increasing concentrations of Kb/KMC (�g/ml); proliferation was measured by thymidine uptake. (C) Spleen cells werecultured for 72 h with LPS in the presence of increasing concentrations of Kb (�g/ml); proliferation was measured by thymidine uptake.


lymphopoiesis by Kb/KMC (Table 2). Those findings that mol-ecules associated with inflammatory or proinflammatory pro-cesses are not involved in mediating the blockade of lympho-poiesis 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 blockingagent of early lymphopoiesis [40]. We have not investigatedhere a role for IL-6; however, it seems to us unlikely that thiscytokine would be involved because of the observed anti-inflammatory action of Kb.

Altogether, the experiments described here suggest that thejuice of Kb or its purified fraction KMC is blocking lympho-poiesis through a new mechanism that differs from the modesof action described previously for hormones and cytokines thatcan induce an analogous effect. Evidence is provided to sup-port the notion that Kb/KMC is interfering with the response oflymphoid progenitors to IL-7. These results open new perspec-tives for a possible therapeutic use of Kb-derived products asimmunomodulatory agents, as IL-7 has a central role not onlyon lymphocyte development but also affects the biology ofmature T cells, including regulatory T cells [41–45]. Finally,IL-7 has also been shown to be important for the growth ofcertain T cell lymphomas and breast cancer, and Kb/KMCcould be a promising candidate to control these malignanttumors [46, 47].

ACKNOWLEDGMENTS

This work was supported by the Brazilian Agencies: FAPERJ,FINEP, FUJB, CNPq, and CAPES. We are grateful to ZenildoB. Morais Filho (NPPN, Universidade Federal do Rio de Ja-neiro) for technical support.

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