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Institut National de la Santé et de la Recherche Médical,
* Unité 268 and
Unité 506, Institut André Lwoff, Hôpital Paul-Brousse, Villejuif, France
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The spleen is not a site of hemopoiesis, even though under pathologic situations, notably certain myeloproliferative disorders, production of hemopoietic cells can be demonstrated, probably due to the proliferation of displaced bone marrow precursors stored in the spleen (2). Recently, we have shown that stromal fibroblasts established in culture from the spleen of patients with such an abnormal myeloid activity, directly contributed to myeloid proliferation and differentiation of patients’ blood hemopoietic progenitors (3). This raised the possibility that regulatory effects of splenic fibroblasts on blood progenitors might similarly occur under physiologic conditions. We thus investigated the influence of fibroblasts isolated from human adult spleen on the proliferation and differentiation of resting blood CD34+ progenitors. Given the broad repertoire of adhesion molecules, surface ligands, and cytokines/chemokines that human fibroblasts have been shown to express and produce (4, 5, 6, 7), we also analyzed the phenotypic characteristics of spleen-derived fibroblasts with respect to their effects on hemopoietic cells.
Our study provides the first evidence that stromal fibroblasts from human adult spleen can regulate NK cell development, and that constitutive expression of IL-15 on their cell surface plays a functional role in the process of NK cell genesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Normal adult spleen samples (n = 4) were obtained from both male and female donors, aged 49–70 years, from whom consent for multiple organ donation had been granted (Etablissement Français des Greffes, Kremlin-Bicêtre, France). Fibroblasts were isolated by gentle grinding of splenic tissue in a sterile glass homogenizer. Tissue homogenates were then centrifuged at 1000 rpm for 5 min. Cellular aggregates present in the pellet were washed three times with PBS and then transferred to culture flasks in a small volume of RPMI 1640 medium (Life Technologies, Cergy-Pontoise, France) supplemented with 15% FCS (Life Technologies) to adhere. The medium was changed every 3–4 days until a substantial outgrowth of cells was observed, at which point cells were trypsinized (Life Technologies) and subsequently subcultured at a 1:2 split ratio for up to three passages before analysis. The fibroblastic nature of the cultures was assessed by their homogeneous staining for anti-smooth muscle-actin with a typical stress fiber distribution characteristic of myofibroblasts (8). All cells also showed strong staining for other mesenchymal Ags, including vimentin, fibronectin, and collagen type I and III. Homogeneous cell surface expression of Ag ASO2, specific for fibroblasts (9), was also detected by flow cytometry, whereas Ag CD31, characteristic of endothelial cells, was missing.
Isolation of human CD34+ cells from adult resting peripheral blood
Mononuclear cells obtained from the peripheral blood of healthy donors (n = 9) were prepared by centrifugation over a Ficoll solution (specific gravity, 1.077; Pharmacia Biotech, Orsay, France). After overnight plastic adherence in culture medium, the nonadherent mononuclear CD34+ cell fraction was isolated using a supermagnetic microbead selection kit and varioMACS columns according to the manufacturer’s instructions (Miltenyi Biotec, Paris, France). Purity was >90%.
Culture of CD34+ cells with human spleen-derived fibroblasts
Fibroblasts were grown to confluence in 60-mm petri dishes (Falcon; BD Biosciences, Le Pont de Claix, France) or six-well and 0.4-µm pore size Transwell plates (PolyLabo, Strasbourg, France) and incubated for 3 wk with CD34+ cells (3:10 ratio with fibroblasts) in standard culture medium supplemented with 10% FCS; no exogenous cytokines were added. Half-medium changes were performed once a week. In some experiments cultures were also treated with various combinations of neutralizing Abs as follows. Anti-stem cell factor (anti-SCF3; 1 µg/ml) goat polyclonal Ab, 60 ng/ml anti-Flt3 ligand (anti-Flt3L) mAb 40416.111, and 100 ng/ml anti-IL-15 mAb M111 (R&D Systems, Abingdon, U.K.) were added at the start of experiments and subsequently at each medium change. At the indicated times nonadherent cells were harvested by washing and counted, and cell viability was determined by the trypan blue dye exclusion test. Analysis of monolayer-derived fibroblasts was performed after a 3-min trypsinization at 37°C.
Cell immunophenotyping
Cells were stained with various combinations of mAbs as listed
in Table I
according to conventional
direct and indirect techniques (6). In some
experiments cell membranes were permeabilized by treatment with
ORTHOPermeafix (Ortho Diagnostic Systems, Roissy, France) before Ab
incubation for monitoring total (surface and cytoplasmic) Ag
expression. Unspecific staining was determined using identical
nonreactive isotype mAbs. Cells were analyzed on a FACScan (BD
Biosciences, Franklin Lakes, NJ) using CellQuest (BD Biosciences) and
WinMDI software programs (Scripps Research Institute, La Jolla,
CA).
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Multiple aliquots of cell-free supernatants were collected from
fibroblasts cultured alone or with hemopoietic
CD34+ cells, filtered, and frozen at -20°C.
Standard ELISA for IL-2, IL-15, SCF, Flt3L, TNF-
, and IFN-
were
performed using commercial kits from R&D Systems according to the
manufacturer’s instructions.
Confocal microscopic analysis
For double staining, cells were first permeabilized with
ORTHOPermeafix for 45 min at room temperature. Cells were then stained
with anti-perforin mAb 
G9 (BD PharMingen, Le Pont de
Claix, France), followed by incubation with Alexa Fluor 594-conjugated
goat anti-mouse Ab (Molecular Probes, Montluçon, France).
Cells were then labeled with biotinylated anti-granzyme B mAb
CLB-GB11 (Tebu, Le Perray en Yvelines, France), followed by
streptavidin-Alexa Fluor 488 conjugate (Molecular Probes).
Cytospin-stained cells were analyzed by laser scanning confocal
microscopy on a Leica TCS NT/SP interactive laser cytometer equipped
with confocal optics (Leica Microsystems, Wetzlar, Germany).
Cytotoxicity assay
The cytolytic activity of total nonadherent cultured cells was determined in a standard 4-h 51Cr release assay against the NK-sensitive K562 cell line, a gift from Dr. S. Chouaib (IGR, Villejuif, France). In some experiments cells were additionally incubated with 1000 U recombinant human IL-2 (Hoechst-Marion-Roussel, Romainville, France) for 48 h and then tested for cytotoxic activity.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The purified CD34+ cell populations isolated from adult nonmobilized peripheral blood cells showed no evidence of myeloid or NK and B lymphoid differentiated cells, with generally <1% of the population expressing CD14, CD15, CD56, and CD19 (data not shown). In contrast, cells expressing lymphoid CD2 (19.0 ± 9.6%; range, 3–28%) and megakaryocyte CD41 (46.4 ± 26.9%; range, 22–89%) lineage cell Ags were detected, indicating that CD34+ progenitors in resting blood were most likely committed cells (data not shown). Further phenotype characterization also indicated that significant (44–76%) proportions of CD34+ cells expressed a set of surface adhesion molecules necessary for blood cell interactions, including ICAM-1, VLA-4, and LFA-1. Cell surface expression of c-kit (23%) and Flt3 (12%) was detected in a fraction of cells (data not shown).
Differentiation of resting blood CD34+ progenitors cultured with human spleen-derived fibroblasts
When purified CD34+ cells were cultured with
splenic fibroblasts for 3 wk, a decline in nonadherent cell number was
generally observed by wk 1, followed by a low cell expansion (Table II). Phenotype characterization of
nonadherent surviving cells revealed a progressive lost of cells
positive for CD34, CD41, and CD2. In contrast, increasing percentages
of CD56+ and, to a much lesser extent,
CD15+ cells were detected. No cells positive for
CD3, CD19, and CD14 could be identified, indicating the lack of T or B
cell and monocyte development (data not shown). However, a fraction
(
40%) of the nonadherent cell population remained consistently
negative for each of the Ags tested, indicating that other lineages
might also be present.
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The production of CD56+ cells that was
reproducibly obtained in a series of experiments using fibroblasts and
blood CD34+ cells from different donors generally
reached maximal values of 33.5 ± 6.4% by wk 3 (Fig. 1a). Further analysis
demonstrated that these CD56+ cells displayed a
typical NK phenotype that closely resembled cells produced in
stroma-free medium supplemented with IL-15, Flt3L, and c-kit
ligand (c-kitL) (14, 15, 16). Thus, dual flow cytometry showed
that all CD56+ cells lacked CD3, and only a small
fraction of them expressed CD2 (<5%) or CD16 (10%; Fig. 1b). Furthermore, a high (>80%) percentage of
CD56+ cells coexpressed the C-type lectin
receptor CD94 and the NK receptors p46 (>70%), p44 (>60%), or p30
(50%). The surface expression levels of these NK Ags were
consistently high (mean fluorescent intensity between logs 2 and 3) in
a majority of cells (Fig. 1c). Other specific killer cell
IgG-like receptors, including p58-1, p58-2, p70, and leukocyte Ig-like
receptor-1, were lacking or expressed in only a small (<5%)
percentage of cells (data not shown). Further confocal microcopic
examination of NK cells demonstrated in a high proportion of
nonadherent cells (Fig. 2) an
intracytoplasmic colocalization of perforin and granzyme B, which are
thought to be lytic mediators for lymphocyte cytotoxicity
(17). Expression of Fas ligand, another mediator
responsible for the lytic NK cell function (18), was not
detected by flow cytometry (data not shown). When total nonadherent
cells were directly tested for their ability to lyse the classical
class I-negative target K562, a substantial (>35%) level of lytic
activity was detected at an E:T cell ratio of 5:1 (Fig. 1d).
Further stimulation by IL-2 did not increase cell cytotoxicity.
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Low amounts of the two ligands c-kitL/SCF and Flt3L, related to NK
progenitor amplification and differentiation (14, 15),
were detected in culture supernatants of spleen-derived fibroblasts as
early as day 4, with optimal values of 93.9 ± 24.3 and 19.0
± 5.2 pg/ml, respectively, by wk 1 (Fig. 3). In contrast, IL-15, the most potent
cytokine for NK cell differentiation (14), was barely
detectable, below or at the limit of ELISA detection and accuracy (3.9
pg/ml) during the first 4 days of culture and then reached an optimal
value of 17.7 ± 0.4 pg/ml by wk 1. Other cytokines, including
IL-2, TNF-, and IFN-, consistently remained undetectable (data
not shown). Except for SCF, no significant changes in secreted
cytokines were observed under coculture conditions.
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a). Unexpectedly, significant
surface IL-15 expression was also observed. Similarly to c-kitL and
Flt3L, high levels of IL-15 expression were detected after
permeabilization of the cell membrane, indicating that splenic
fibroblasts constitutively expressed IL-15 both on the cell surface and
intracellularly. Splenic fibroblasts express IL-15R -chain. To
determine whether the expression of IL-15 on the cell surface was
membrane-bound or secreted bound IL-15, cells were treated with
neutralizing anti-IL-15 from the start of culture to day 3, when
IL-15 was barely detectable in culture supernatants. No surface IL-15
expression was revealed in treated cells (data not shown), indicating
that the IL-15 expressed on the surface of fibroblasts was secreted,
rather than unsecreted, bound IL-15. After fixation, IL-15/IL-15R
interaction appeared stable because treatment with anti-IL-15R
mAb did not block bound IL-15 (Fig. 4b).
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To evaluate the importance of the three cytokines, IL-15, Flt3L,
and SCF, in the NK differentiation process stimulated by spleen-derived
fibroblasts, neutralizing mAbs directed at each cytokine were added to
the cultures for 3 wk, and cells with the NK phenotype were monitored
on the basis of CD56 expression. Under conditions using a combination
of the three neutralizing mAbs, no CD56+ cells
could be detected (Fig. 5a).
However, when used individually, neutralizing anti-IL-15 mAb was
found to block CD56+ cell development, whereas
incubation with anti-SCF or anti-Flt3L mAbs did not.
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b). Despite the lack of
CD34+ cells (data not shown), no
CD56+ cells were recovered from the inserts,
whereas a fraction (17%) of highly expressing
CD56+ cells was detected under cell-to-cell
contact standard culture conditions. Moreover, when exogenous rIL-15
was added to CD34+ cells at 50 ng/ml, a
concentration shown to elicit NK cell differentiation in stroma-free
cultures (14), CD56+ cells were
produced at levels comparable to those of cell-to-cell contact standard
culture conditions (data not shown). All these results indicated that
contact with fibroblasts and bound IL-15 interaction were necessary and
sufficient for CD34+ progenitors to differentiate
along the NK cell lineage. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Most fibroblast-induced NK cells showed CD56 and NK receptor expression closely related to circulating blood-derived NK subsets or cultured cells produced in stroma-free medium supplemented with cytokines (14, 15, 16). Thus, they highly expressed the CD56 NK lineage Ag, the C-type lectin CD94 receptor specific for MHC class I recognition, and other newly identified NK activator receptors, p46, p44, and p30, specific for non-MHC ligands (22). Additionally, expression of perforin and granzyme lytic granules was observed intracellularly. Thus, these NK cells displayed lytic potentialities, as suggested by the expression of killer cell-activating receptors and cytoplasmic lytic organelles. Indeed, these CD56+ cells were functionally activated cells, as evidenced by their ability to kill the classical class I-negative target K562 without additional cytokine activation.
In contrast to other human and murine stroma cultures composed of heterogeneous stromal cell populations and using multiple exogenous cytokines (23, 24), spleen-derived fibroblasts were capable, by themselves, of stimulating the development of activated NK cells from blood CD34+ progenitors, indicating that they provided the appropriate microenvironment for NK cell genesis. In this context, IL-15 is now recognized as one of the major factors triggering NK cell differentiation, whereas c-kitL and Flt3L can promote IL-15-mediated expansion and differentiation of NK progenitor cells in stroma-free cultures (25) In human spleen-derived fibroblasts, we detected the three cytokines both exposed on the cell surface and produced extracellularly.
The constitutive expression of IL-15 on the surface of human
spleen-derived fibroblasts was quite striking because stromal
fibroblasts isolated from other tissue sources did not show such
expression unless stimulated by inflammatory cytokines or derived from
pathologic lesions (3, 26). In contrast, blood-derived
cells, including monocytes, monoblastoid cells, and leukemic
progenitors, have been reported to constitutively express
membrane-associated IL-15 without any IL-15 secretion
(27, 28, 29). This membrane form has been proposed to be the
bioactive form of IL-15 (27) and the result of IL-15
mobilization from intracellular stores to the membrane
(30). Unlike resting and stimulated cells, secreted rather
than unsecreted bound IL-15 was detected on the surface of splenic
fibroblasts. Even though expressed at low levels, the membrane-bound
IL-15 appeared to play a predominant role over the surface expression
of the other two ligands and extracellular cytokine production in the
process of NK cell development reported here. First, NK cell production
was blocked only when blood CD34+ progenitors and
splenic fibroblasts were cocultured in the presence of neutralizing
IL-15, but not c-kitL and Flt3L, mAbs. Second, culture conditions that
left the cytokine microenvironment intact but prevented direct contacts
between the two cell populations totally abrogated NK cell production.
In stroma-free cultures it has been shown that nanomolar concentrations
of exogenous IL-15 were necessary to activate NK cell proliferation and
differentiation, whereas picomolar amounts were effective at
maintaining NK cell survival. Indeed, we found that 50 ng/ml exogenous
rIL-15 induced NK cell differentiation of resting blood
CD34+ cells as efficiently as surface IL-15
expressed on splenic fibroblasts. Moreover, elevated concentrations of
c-kitL in synergy with Flt3L have been found to promote NK progenitor
expansion. Therefore, the picogram amounts of all three cytokines that
were produced extracellularly by human spleen-derived fibroblasts were
most likely efficient at ensuring cell survival, but not proliferation,
and might explain why the yields of NK cells resulting from splenic
fibroblasts were reduced compared with those from stroma-free cultures
(16). It should be emphasized that IL-2, the cytokine
initially used for NK cell development (23), and the two
classical cytokines, TNF- and IFN-, known to elicit lymphocyte
activation, consistently remained undetectable in culture supernatants
under both standard and coculture conditions.
In conclusion, our data provide the first evidence that human spleen-derived fibroblasts directly contribute to NK cell development via exposure of bioactive cell surface IL-15, strengthening the importance of stromal fibroblasts as sentinel cells in the immune system (31). It is reasonable to speculate that IL-15 exposed on the surface of fibroblasts serves as a physiologic regulator in maintaining a small number of functionally activated NK cells within the spleen.
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Acknowledgments |
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mAb M162,
and the staff of Centre Transfusion Sanguine, Hôpital Paul
Brousse (Villejuif, France), and of Coordination Hospitalière des
Activités de Prélèvements, Hôpital
Kremlin-Bicêtre, for supplying us with normal human blood and
spleen tissue samples. We also thank Corine Pottin-Clémenceau for
her skillful technical assistance and Julien Giron-Michel for his
guidance in the confocal analysis. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Danièle Brouty-Boyé, Institut National de la Santé et de la Recherche Médical, Unité 268, Hôpital Paul Brousse, 14 Avenue Paul Vaillant Couturier, 94807 Villejuif Cedex, France. E-mail address: dbroutyb{at}infobiogen.fr
3 Abbreviations used in this paper: SCF, stem cell factor; c-kitL, c-kit ligand; Flt3L, Flt3 ligand.
Received for publication September 6, 2001. Accepted for publication February 26, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. J. Invest. Dermatol. 116:102.[Medline]
-upregulated interleukin 15. Blood 93:3531.This article has been cited by other articles:
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  M. P. Correia, E. M. Cardoso, C. F. Pereira, R. Neves, M. Uhrberg, and F. A. Arosa Hepatocytes and IL-15: A Favorable Microenvironment for T Cell Survival and CD8+ T Cell Differentiation J. Immunol., May 15, 2009; 182(10): 6149 - 6159. [Abstract] [Full Text] [PDF]
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 J.-J. Lataillade, O. Pierre-Louis, H. C. Hasselbalch, G. Uzan, C. Jasmin, M.-C. Martyre, M.-C. Le Bousse-Kerdiles, and on behalf of the French INSERM and the European EU Does primary myelofibrosis involve a defective stem cell niche? From concept to evidence Blood, October 15, 2008; 112(8): 3026 - 3035. [Abstract] [Full Text] [PDF]
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A. Chan, D.-L. Hong, A. Atzberger, S. Kollnberger, A. D. Filer, C. D. Buckley, A. McMichael, T. Enver, and P. Bowness CD56bright Human NK Cells Differentiate into CD56dim Cells: Role of Contact with Peripheral Fibroblasts J. Immunol., July 1, 2007; 179(1): 89 - 94. [Abstract] [Full Text] [PDF]
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B. Grzywacz, N. Kataria, M. Sikora, R. A. Oostendorp, E. A. Dzierzak, B. R. Blazar, J. S. Miller, and M. R. Verneris Coordinated acquisition of inhibitory and activating receptors and functional properties by developing human natural killer cells Blood, December 1, 2006; 108(12): 3824 - 3833. [Abstract] [Full Text] [PDF]
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J. Giron-Michel, M. Giuliani, M. Fogli, D. Brouty-Boye, S. Ferrini, F. Baychelier, P. Eid, C. Lebousse-Kerdiles, D. Durali, R. Biassoni, et al. Membrane-bound and soluble IL-15/IL-15R{alpha} complexes display differential signaling and functions on human hematopoietic progenitors Blood, October 1, 2005; 106(7): 2302 - 2310. [Abstract] [Full Text] [PDF]
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C.-S. Park, S.-O. Yoon, R. J. Armitage, and Y. S. Choi Follicular Dendritic Cells Produce IL-15 That Enhances Germinal Center B Cell Proliferation in Membrane-Bound Form J. Immunol., December 1, 2004; 173(11): 6676 - 6683. [Abstract] [Full Text] [PDF]
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A. Ozawa, H. Tada, Y. Sugawara, A. Uehara, T. Sasano, H. Shimauchi, H. Takada, and S. Sugawara Endogenous IL-15 Sustains Recruitment of IL-2R{beta} and Common {gamma} and IL-2-Mediated Chemokine Production in Normal and Inflamed Human Gingival Fibroblasts J. Immunol., October 15, 2004; 173(8): 5180 - 5188. [Abstract] [Full Text] [PDF]
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G. G. Neely, S. Epelman, L. L. Ma, P. Colarusso, C. J. Howlett, E. K. Amankwah, A. C. McIntyre, S. M. Robbins, and C. H. Mody Monocyte Surface-Bound IL-15 Can Function as an Activating Receptor and Participate in Reverse Signaling J. Immunol., April 1, 2004; 172(7): 4225 - 4234. [Abstract] [Full Text] [PDF]
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J. Barlic, J. M. Sechler, and P. M. Murphy IL-15 and IL-2 oppositely regulate expression of the chemokine receptor CX3CR1 Blood, November 15, 2003; 102(10): 3494 - 3503. [Abstract] [Full Text] [PDF]
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I. Barao, D. Hudig, and J. L. Ascensao IL-15-Mediated Induction of LFA-1 Is a Late Step Required for Cytotoxic Differentiation of Human NK Cells from CD34+Lin- Bone Marrow Cells J. Immunol., July 15, 2003; 171(2): 683 - 690. [Abstract] [Full Text] [PDF]
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J. Giron-Michel, A. Caignard, M. Fogli, D. Brouty-Boye, D. Briard, M. van Dijk, R. Meazza, S. Ferrini, C. Lebousse-Kerdiles, D. Clay, et al. Differential STAT3, STAT5, and NF-{kappa}B activation in human hematopoietic progenitors by endogenous interleukin-15: implications in the expression of functional molecules Blood, July 1, 2003; 102(1): 109 - 117. [Abstract] [Full Text] [PDF]
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) and IL-2-stimulated (
) nonadherent cells at a 5:1
E:T cell ratio. For b–d, results are representative of
at least three separate experiments
