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Bacterial infection of human hematopoietic stem cells induces monocytic differentiation
FEMS Immunology and Medical Microbiology 40 (2004) 147^153
www.fems-microbiology.org
Bacterial infection of human hematopoietic stem cells induces monocytic di¡erentiation Annette Kolb-Ma«urer a
a;
, Florian Weissinger b , Oliver Kurzai c , Mathias Ma«urer d , Martin Wilhelm b , Werner Goebel e
Department of Dermatology, University of Wu«rzburg, Josef-Schneider-Str. 2, D-97080 Wu«rzburg, Germany b Department of Internal Medicine, University of Wu«rzburg, Wu«rzburg, Germany c Institute for Hygiene and Microbiology, Wu«rzburg, Germany d Department of Neurology, University of Wu«rzburg, Wu«rzburg, Germany e Institute for Microbiology, Theodor-Boveri-Institute, University of Wu«rzburg, Wu«rzburg, Germany Received 1 September 2003; received in revised form 14 October 2003 ; accepted 22 October 2003 First published online 21 November 2003
Abstract Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-K were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34þ hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Hematopoietic stem cell; Bacterial infection ; Cytokine; Cell di¡erentiation
1. Introduction The availability of blood cells is sustained by pluripotent hematopoietic progenitors. These hematopoietic stem cells (HSCs), which have in common the expression of CD34 surface molecules, are rare pluripotent cells in the bone marrow with the capacity to di¡erentiate into erythrocytes, megakaryocytes, granulocytes, monocytes, and lymphocytes [1,2]. Hematopoiesis is a complex process in which multiple factors of the microenvironment are involved. The production of peripheral blood cells is regulated by hematopoietic
* Corresponding author. Tel. : +49 (931) 20126710; Fax : +49 (931) 20126700. E-mail address : [email protected] (A. Kolb-Ma«urer).
growth factors and related cytokines which promote the survival of stem cells and stimulate the proliferation and di¡erentiation of progenitors [3,4]. Disturbance of this complex network by infectious agents is critical, since infection can lead to dysregulation and destruction of these important progenitor cells [5]. Infection may augment the production of cytokines, and change proliferation and differentiation of stem cells. Concerning the interaction between infectious agents and HSCs the repertoire of cellular surface receptors on HSCs plays an important role in differential targeting of infectious agents. Therefore identi¢cation of surface phenotypes of HSCs is important for the understanding of basic steps in hematopoiesis. Recently, we have shown that human HSCs are fully resistant to infection with Listeria monocytogenes and Yersinia enterocolitica. However, when CD34þ HSCs were cultured in a cytokine mixture with stem cell factor, thrombopoietin, and £t-3 ligand to promote myeloid development [6,7],
0928-8244 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0928-8244(03)00305-5
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these partially di¡erentiated HSCs were able to take up pathogens [8]. In this study, we therefore characterize the developmental changes of HSC surface molecule expression and cytokine release after infection with Gram-negative Y. enterocolitica and Gram-positive L. monocytogenes.
2.3. Di¡erentiation of HSCs towards monocytes
2. Materials and methods
2.4. Bacteria
2.1. Isolation of human CD34+ progenitor cells derived from peripheral blood cells
Bacterial strains used in this study are: L. monocytogenes Sv 1/2a EGD and Y. enterocolitica. The bacteria were grown in brain heart infusion medium at 30‡C (Yersinia) or 37‡C (Listeria) until they reached the mid-exponential phase of growth (circa 1U109 bacteria ml31 ).
After informed consent, cells were collected by apheresis during hematopoietic recovery after chemotherapy and treatment with recombinant s.c. granulocyte colony-stimulating factor (G-CSF). 4 ml cell suspension from the pilot aliquot was used for positive selection of CD34þ cells. +
+
2.2. Positive selection of CD34 cells with the CD34 selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany) After washing, the cell pellet was resuspended in 300 Wl bu¡er (phosphate-bu¡ered saline supplemented with 0.5% bovine serum albumin, and 2 mM EDTA) per 5U108 cells. The cell suspension was incubated with Fc receptor-blocking reagent (human immunoglobulins) and subsequently with monoclonal hapten-conjugated anti-CD34 antibody (clone QBEND/10, mouse IgG1) for 15 min at 6‡C. Following an additional washing step, the stained cell suspension was incubated with colloidal super-paramagnetic MACS MicroBeads conjugated to an anti-hapten antibody in a ¢nal volume of 500 Wl per 5U108 cells for 15 min at 6‡C. For positive selection cells were again washed and resuspended in bu¡er (2U108 cells ml31 ). After passing through a ¢lter to remove clumps, cells were applied to the selection column (type VSþ0 ), inserted into the magnetic ¢eld of the VarioMACS0 system. Following rinsing three times, the VSþ column was removed from the magnetic ¢eld and the selected cells were eluted in bu¡er. To further enrich the CD34þ cells, eluted cells were applied to an additional positive selection column (RS+ column0 for 107 CD34þ cells) with an analogous procedure. The ¢nal CD34þ cell suspension was eluted in 1 ml bu¡er. The CD34þ cells were used for the experiments either directly or after storage in liquid nitrogen.
HSCs were di¡erentiated towards monocytes by incubation in long-term cell culture medium (Cell Systems, Vancouver, BC, Canada) supplemented with stem cell factor (100 ng ml31 ), thrombopoietin (75 ng ml31 ) and £t-3 ligand (15 ng ml31 ) (Pepro Tech, Rockyhill, SC, USA) [9].
2.5. In vitro bacterial infection of HSCs 5U105 cells ml31 were infected with bacteria. Bacteria were added at a multiplicity of infection (MOI) of 10. The cultures were incubated in RPMI 1640 medium supplemented with 10% pooled human AB serum at 37‡C for 1 h. For selective removal of extracellular bacteria, 100 Wg ml31 gentamicin (Gibco, Karlsruhe, Germany) was added. Twenty-four hours post infection (p.i.) cells were analyzed by £ow cytometry and the supernatant was collected and stored at 380‡C. 2.6. Uptake inhibition studies The uptake assay was performed as described above, except that the media contained 2 Wg ml31 of the inhibitor cytochalasin D throughout the duration of the experiment. 2.7. Flow cytometry The expression of surface molecules of uninfected and infected HSCs was evaluated by £ow cytometry after direct staining with PE- or FITC-conjugated anti-CD13, anti-CD14, anti-CD16 (FcQRIII), anti-CD33, anti-CD34, anti-CD54, anti-CD80, anti-CD83, anti-Toll-like receptor (TLR)-4, anti-CD86 antibodies, and B7-H1 (BioCarta, Hamburg, Germany; eBioscience, San Diego, CA, USA; Pharmingen, Hamburg, Germany). 2.8. Protein multiplex immunoassay kit (BioSource) Cytokine concentrations in the supernatants generated
6 Fig. 1. Surface marker analysis of human CD34þ HSCs. The x-axis of each histogram shows the log £uorescence intensity; the y-axis shows the relative cell number. a: Surface marker analysis of puri¢ed human CD34þ HSCs. Black histograms represent staining with matched-isotype antibodies, gray histograms represent d0 HSCs. b: d0 HSCs 24 h p.i. with L. monocytogenes or Y. enterocolitica : upregulation of CD54, CD86 and B7-H1. Black histograms represent staining with matched-isotype antibodies, thick black histograms represent uninfected HSCs, gray histograms represent HSCs after Y. enterocolitica infection, dashed histograms represent HSCs after L. monocytogenes infection. c: Expression of TLR-4 on monocytes and HSCs (d0, d6). Black histograms represent staining with matched-isotype antibodies, gray histograms represent TLR expression on HSCs or monocytes.
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in the infection experiments 24 h p.i. were measured with the Luminex technology for multiplex cytokine immunoassay (obtained from BioSource, Nivelles, Belgium) using beads for G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-K (TNFK), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-1L, IL12p40, IL-10, IL-13 and interferon-Q (IFN-Q). Supernatants were diluted 1:10 in sterile water ; 50 Wl of the dilution was used in the assay according to the detailed description supplied by the manufacturer. At least 100 beads per cytokine and sample were measured. Data were evaluated with the MasterPlex QT software (MiraiBio). Alternatively, standard enzyme-linked immunosorbent assay kits were used to determine TNF-K, IL-6, IL-8, IL-1L, IFN-Q (ImmunoKontact, AMS Biotech, Wiesbaden, Germany) and IL-10, IL-12p70 (Biocarta, Hamburg, Germany).
3. Results and discussion 3.1. CD34+ HSCs change surface marker expression after infection with L. monocytogenes and Y. enterocolitica The puri¢ed fraction of CD34þ HSCs from human blood consisted of s 95% CD34þ cells. Speci¢c markers for T-cells, B-cells, monocytes, and mature dendritic cells (CD3, CD19, CD14, or CD83) were found on 6 1% of the cells. CD34þ HSCs showed a slight expression of CD54 (ICAM-1), while other costimulatory signals such as B7.1 (CD80), B7.2 (CD86), and B7-H1 were almost not detectable (Fig. 1a). HSCs were incubated with the Gram-positive bacterium L. monocytogenes and the Gram-negative bacterium Y. enterocolitica. Four hours and 24 h p.i. the surface marker expression of CD13, CD14, CD16, CD33, CD34, CD54, CD80, CD83, CD86, and B7-H1 was evaluated. CD13 and CD33 are expressed on myeloid progenitors, monocytes, and mast cells. Four hours p.i. infected HSCs showed no altered surface molecule expression compared to uninfected HSCs. Twenty-four hours p.i. we observed a prominent upregulation of CD54 (ICAM-1). In addition to ICAM-1, the costimulatory molecules CD86 (B7.2) and B7-H1 [10] were upregulated, in particular after infection with Y. enterocolitica (Fig. 1b). The change in surface marker expression was unexpected, since recent studies have shown that undi¡erentiated HSCs are not able to perform macropinocytosis or receptor-mediated phagocytosis because essential receptors and internalization mechanisms for bacterial uptake have not yet been developed [8]. Therefore we examined whether these cells already express an important pathogen recognition receptor, namely TLR-4. Pathogen-recognition receptors include the family of TLRs, which recognize conserved motifs of pathogens [11]. We investigated TLR4, which is known to bind bacterial lipopolysaccharide
(LPS) of Gram-negative bacteria and might therefore account for the upregulation of surface markers induced by Y. enterocolitica. While we could clearly show TLR-4 expression on monocytes, HSCs did not express TLR-4 (Fig. 1c). Therefore the altered surface marker expression pro¢le after infection is not mediated via TLR-4. HSCs were then di¡erentiated towards monocytes using a cytokine mixture consisting of stem cell factor, thrombopoietin, and £t-3 ligand (see Section 2). Incubation of HSCs in this di¡erentiation medium leads to the upregulation of the myeloid markers CD13 and CD33, and monocyte-speci¢c antigens such as CD14, while the stem cell marker CD34 was downregulated (Fig. 2a). CD54 (ICAM-1) was still detectable at low levels, but other costimulatory molecules were not expressed after incubation with the cytokine combination. Surface markers characteristic for T- and B-cells (CD3 and CD19), as well as for mature dendritic cells (CD83) were also undetectable. Infection studies were carried out after incubation of HSCs for 3 (d3 HSCs) or 6 days (d6 HSCs) in the cell culture medium. These time points were selected since monocytic di¡erentiation occurs within this period [8]. In comparison to the uninfected situation infection of d6 HSCs with Y. enterocolitica and L. monocytogenes led to an enhanced upregulation of CD13 and CD14 24 h p.i., which is expressed at high levels on monocytes (Fig. 2b). This e¡ect of altered cell surface expression was less pronounced after infection of d3 HSCs (data not shown). Our data indicate that Y. enterocolitica and to a lesser degree L. monocytogenes induced a more pronounced differentiation of HSCs towards monocytes. CD14 has been identi¢ed as a high-a⁄nity cell surface receptor for complexes of LPS and serum LPS-binding protein. Therefore, the more pronounced CD14 surface expression on HSCs could be a reaction to cope with further bacterial encounters. Although we noticed di¡erentiation towards monocytes, infected cells showed an upregulation of CD34. In addition HSCs express CD54 (ICAM-1) and B7-H1, and to a much lesser degree CD80 (B7.1), CD86 (B7.2), and CD83, a marker of mature dendritic cells after bacterial stimulation. In general the e¡ects were more pronounced after infection with Y. enterocolitica than with L. monocytogenes (Fig. 2b). To investigate if these changes in surface marker expression are related to bacterial uptake by partially di¡erentiated HSCs, we evaluated the surface marker expression after the addition of cytochalasin D, an inhibitor of bacterial uptake, to the culture medium. We observed no di¡erence between molecule expression in the presence or absence of the uptake inhibitor. These results underline that the uptake of bacteria was not essential for the surface molecule changes. In addition, these data indicate that reorganization of the cell membrane protein composition can occur independent of the actin cytoskeleton. Furthermore, we could show that changes in surface
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Fig. 2. a: Surface marker analysis of partially di¡erentiated d5 HSCs. FACS analysis was done after staining cells with FITC- (x-axis) and PE- (y-axis) labelled monoclonal antibodies. b: d6 HSCs 24 h p.i. with L. monocytogenes or Y. enterocolitica: upregulation of CD54, B7-H1, CD80, CD83, CD86, and CD34. Black histograms represent staining with matched-isotype antibodies, thick black histograms represent uninfected HSCs, gray histograms represent HSCs after Y. enterocolitica infection, dashed histograms represent HSCs after L. monocytogenes infection.
marker expression occur with both viable and heat-killed bacteria. 3.2. Cytokine pro¢le of HSCs after bacterial infection To investigate whether secreted factors are released from human HSCs after incubation with L. monocytogenes or Y. enterocolitica, we evaluated a panel of major cytokines and growth factors. We measured G-CSF, GM-CSF, IL-1L, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 (p40), IFN-Q, and TNF-K 24h p.i. in cell culture supernatant. Uninfected HSCs, either undi¡erentiated or grown for 6 days in the monocytic di¡erentiation cytokine combination, did not produce detectable levels of the majority of
the tested cytokines. Only a slight release of G-CSF (1 ng ml31 ), GM-CSF (500 pg ml31 ), and IL-1L (170 pg ml31 ) was observed in these cells. Subsequently, undi¡erentiated HSCs, d3 HSCs and d6 HSCs were incubated for 24 h with either Y. enterocolitica or L. monocytogenes and the production of cytokines was determined. In general, bacterial infection did not lead to an increased production of G-CSF, IFN-Q, IL-1L, IL-2, IL-4, IL-5, or IL-13. Undi¡erentiated HSCs produced a moderate level of GM-CSF (up to 3 ng ml31 ), while all other cytokines were hardly detectable. Infection of d3 and more pronouncedly of d6 HSCs resulted in IL-6 (Fig. 3a), IL-8, IL-10 (Fig. 3b), IL-12 (Fig. 3c), TNF-K (Fig. 3d), and
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Fig. 3. Cytokine production of uninfected and infected HSCs (d0 HSCs, d3 HSCs, d6 HSCs). Di¡erent cytokines in cell supernatant were measured 24 h after infection with L. monocytogenes or Y. enterocolitica (MOI 10). a: IL-6. b: IL-10. c: IL-12 p40. d: TNF-K. The results from a representative experiment are presented as mean values and standard deviations of triplicate determinations.
GM-CSF upregulation. In comparison to L. monocytogenes infection, infection with Y. enterocolitica resulted in particular in a stronger upregulation of IL-10, TNFK, and IL-12 p40 (Fig. 3). These di¡erences may be due to an alternative pathway of signal transduction induced by Gram-negative and Gram-positive bacteria and LPS and lipoteichoic acid (LTA). It was shown recently that human dendritic cells require a higher concentration of listerial LTA in comparison to LPS to become stimulated and to release cytokines [12]. Investigations with viable and heat-killed bacteria showed only one major di¡erence concerning the production of cytokines. Infection of d3 and d6 HSCs with heatkilled bacteria led to an approximately 10-fold increased release of IL-8 in comparison to viable bacteria. This could be explained by the fact that heat killing of the bacteria will lead to a release of bacterial components, namely bacterial CpG DNA which could be responsible for the increased IL-8 production. This is underlined by observations of Akhtar et al., who showed that bacterial DNA evokes epithelial IL-8 production in a time- and dose-dependent manner by a mitogen-activated protein kinase-dependent pathway, while other cytokines are dependent on the activation of nuclear factor UB [13]. This study clearly demonstrates that expression of sur-
face antigens and cytokine release of HSCs can be in£uenced by pathogens. Infection with bacteria promotes differentiation towards a monocytic phenotype. This e¡ect is more pronounced when HSCs have already started to differentiate as shown by infection experiments with undi¡erentiated HSCs cultured in a di¡erentiation medium. These cells express characteristic monocyte surface markers and adopt a monocytic cytokine secretion pro¢le. Interestingly, infection also results in secretion of GMCSF, even when undi¡erentiated HSCs encounter bacteria. This upregulation of GM-CSF might result in an increased amount of progenitor cells. Indeed, when we counted the number of viable cells 24 h p.i. and compared the results with the number of viable cells in uninfected controls we found no signi¢cant di¡erence, although L. monocytogenes and Y. enterocolitica are known to kill host cells upon encounter. In line with this observation, we could show that bacterial infection results in an elevation of the CD34þ cell fraction. This enhanced reconstitution of the myeloid progenitor pool might o¡er protection against challenge with bacterial pathogens [14] and is one possible explanation for leukocytosis during bacterial infections. Proliferative kinetics of the progenitor cells, in particular survival and expansion, are controlled by interacting
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cytokines. An e¡ect was described for IL-3, IL-4, IL-6, GM-CSF, G-CSF, IL-11, IL-12, leukemia inhibitory factor, and stem cell factor [15,16]. In addition the expression of receptors for IL-3, IL-6, and GM-CSF appears to be very low on the most immature subsets of CD34þ cells, but increases progressively during successive stages, in particular during myelo-monocytic di¡erentiation [17]. Therefore, the cytokine release of hematopoietic progenitor cells after bacterial infection is likely to in£uence proliferation and di¡erentiation of HSCs. However, it is so far not clear if the changes in the surface molecule receptors result from cytokine release or are mediated by direct signaling via pathogen recognition receptors. Since infection of bone marrow progenitors may contribute to hematologic manifestation of diseases, further studies are needed to elucidate this important interaction between pathogens and stem cells.
Acknowledgements This study was supported by a fellowship from the Bayerisches Staatsministerium fu«r Wissenschaft, Forschung und Kunst to A.K.-M. The authors thank R. Ottohal for excellent technical assistance. We are grateful to Prof. J. Heesemann for donating the Yersinia enterocolitica strain and F. Hartmann for help with the Luminex technology.
References [1] Weissman, I.L., Anderson, D.J. and Gage, F. (2001) Stem and progenitor cells: origins, phenotypes, lineage commitments and transdifferentiations. Annu. Rev. Cell Dev. Biol. 17, 387^403. [2] Ogawa, M. (1993) Di¡erentiation and proliferation of hematopoietic stem cells. Blood 81, 2844^2853. [3] Messner, H.A. (1995) Assessment and characterization of hemopoietic stem cells. Stem Cells 3, 13^18. [4] Ku, H., Yonemura, Y., Kaushansky, K. and Ogawa, M. (1996) Thrombopoietin, the ligand for the Mpl receptor, synergizes with steel factor and other early acting cytokines in supporting proliferation of primitive hematopoietic progenitors of mice. Blood 87, 4544^ 4551.
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[5] Klein, M.B., Miller, J.S., Nelson, C.M. and Goodman, J.L. (1997) Primary bone marrow progenitors of both granulocytic and monocytic lineages are susceptible to infection with the agent of human granulocytic ehrlichiosis. J. Infect. Dis. 176, 1405^1409. [6] Audet, J., Zandstra, P.W., Eaves, C.J. and Piret, J.M. (1998) Advances in hematopoietic stem cell culture. Curr. Opin. Biotechnol. 9, 146^151. [7] Ziegler, B., Testa, U., Condorelli, G., Vitelli, L., Valtieri, M. and Peschle, C. (1998) Unilineage hematopoietic di¡erentiation in bulk and single cell culture. Stem Cells 16 (Suppl. 1), 51^73. [8] Kolb-Ma«urer, A., Wilhlem, M., Weissinger, F., Bro«cker, E.-B. and Goebel, W. (2002) Interaction of human hematopoietic stem cells with bacterial pathogens. Blood 100, 3703^3709. [9] Weissinger, F., Schertlin, T., Buchhofer, S., Ottohal, R. and Wilhelm, M. (1999) Hematopoietic stem cells of CML patients lack monocytic di¡erentiation [abstract]. Blood 94, 350b. [10] Freeman, G.J., Long, A.J., Iwai, Y., Bourque, K., Chernova, T., Nishimura, H., Fitz, L.J., Malenkovich, N., Okazaki, T., Byrne, M.C., Horton, H.F., Fouser, L., Carter, L., Ling, V., Bowman, M.R., Carreno, B.M., Collins, M., Wood, C.R. and Honjo, T. (2000) Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation. J. Exp. Med. 192, 1027^1034. [11] Ulevitch, R.J. (1999) Toll gates for pathogen selection. Nature 401, 755^756. [12] Kolb-Ma«urer, A., Ka«mmerer, U., Ma«urer, M., Gentschev, I., Bro«cker, E.-B., Rieckmann, P. and Ka«mpgen, E. (2003) Synthesis of Th1-stimulating cytokines IL-12 and IL-18 in human dendritic cells upon infection by Listeria monocytogenes. FEMS Immunol. Med. Microbiol. 35, 255^262. [13] Akhtar, M., Watson, J.L., Nazli, A. and McKay, D.M. (2003) Bacterial DNA evokes epithelial IL-8 production by a MAPK-dependent, NF-UB-independent pathway. FASEB J. 17, 1319^1321. [14] BitMansour, A., Burns, S.M., Traver, D., Akashi, K., Contag, C.H., Weissman, I.L. and Brown, J.M. (2002) Myeloid progenitors protect against invasive aspergillosis and Pseudomonas aeruginosa infection following hematopoietic stem cell transplantation. Blood 100, 4660^ 4667. [15] Ogawa, M. and Matsunaga, T. (1999) Humoral regulation of hematopoietic stem cells. Ann. NY Acad. Sci. 872, 17^23. [16] Ku, H., Hirayama, F., Kato, T., Miyazaki, H., Aritomi, M., Ota, Y., D’Andrea, A.D., Lyman, S.D. and Ogawa, M. (1996) Soluble thrombopoietin receptor (Mpl) and granulocyte colony-stimulating factor receptor directly stimulate proliferation of primitive hematopoietic progenitors of mice in synergy with steel factor or the ligand for Flt3/Flk2. Blood 88, 4124^4131. [17] Wognum, A.W., de Jong, M-O. and Wagemaker, G. (1996) Di¡erential expression of receptors for hemopoietic growth factors on subsets of CD34þ hematopoietic cells. Leukoc. Lymphoma 24, 11^ 25.
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Bacterial infection of human hematopoietic stem cells induces monocytic di¡erentiation Annette Kolb-Ma«urer a
a;
, Florian Weissinger b , Oliver Kurzai c , Mathias Ma«urer d , Martin Wilhelm b , Werner Goebel e
Department of Dermatology, University of Wu«rzburg, Josef-Schneider-Str. 2, D-97080 Wu«rzburg, Germany b Department of Internal Medicine, University of Wu«rzburg, Wu«rzburg, Germany c Institute for Hygiene and Microbiology, Wu«rzburg, Germany d Department of Neurology, University of Wu«rzburg, Wu«rzburg, Germany e Institute for Microbiology, Theodor-Boveri-Institute, University of Wu«rzburg, Wu«rzburg, Germany Received 1 September 2003; received in revised form 14 October 2003 ; accepted 22 October 2003 First published online 21 November 2003
Abstract Differentiation of hematopoietic stem cells (HSCs) can be influenced by different stimuli, including cytotoxic agents, certain cytokines, and contact with pathogens. Infection may result in dysregulation of these important progenitor cells and therefore interfere with the availability of blood cells. In this study we analyzed the effect of bacterial infection on HSCs concerning surface marker expression and cytokine release. Listeria monocytogenes and Yersinia enterocolitica accelerated maturation of hematopoietic progenitor cells along the myeloid lineage, as demonstrated by the upregulation of CD13, CD14, and costimulatory signals. By screening cytokine secretion, granulocyte-macrophage colony-stimulating factor, interleukin (IL)-6, IL-8, IL-10, IL-12, and tumor necrosis factor-K were found to be induced by bacterial infection. These data indicate that infection of HSCs with L. monocytogenes and Y. enterocolitica affects the differentiation of CD34þ hematopoietic progenitors in vitro and may lead to secretion of cytokines that can influence the HSC differentiation capacity and immune response. 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. Keywords : Hematopoietic stem cell; Bacterial infection ; Cytokine; Cell di¡erentiation
1. Introduction The availability of blood cells is sustained by pluripotent hematopoietic progenitors. These hematopoietic stem cells (HSCs), which have in common the expression of CD34 surface molecules, are rare pluripotent cells in the bone marrow with the capacity to di¡erentiate into erythrocytes, megakaryocytes, granulocytes, monocytes, and lymphocytes [1,2]. Hematopoiesis is a complex process in which multiple factors of the microenvironment are involved. The production of peripheral blood cells is regulated by hematopoietic
* Corresponding author. Tel. : +49 (931) 20126710; Fax : +49 (931) 20126700. E-mail address : [email protected] (A. Kolb-Ma«urer).
growth factors and related cytokines which promote the survival of stem cells and stimulate the proliferation and di¡erentiation of progenitors [3,4]. Disturbance of this complex network by infectious agents is critical, since infection can lead to dysregulation and destruction of these important progenitor cells [5]. Infection may augment the production of cytokines, and change proliferation and differentiation of stem cells. Concerning the interaction between infectious agents and HSCs the repertoire of cellular surface receptors on HSCs plays an important role in differential targeting of infectious agents. Therefore identi¢cation of surface phenotypes of HSCs is important for the understanding of basic steps in hematopoiesis. Recently, we have shown that human HSCs are fully resistant to infection with Listeria monocytogenes and Yersinia enterocolitica. However, when CD34þ HSCs were cultured in a cytokine mixture with stem cell factor, thrombopoietin, and £t-3 ligand to promote myeloid development [6,7],
0928-8244 / 03 / $22.00 9 2003 Federation of European Microbiological Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/S0928-8244(03)00305-5
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these partially di¡erentiated HSCs were able to take up pathogens [8]. In this study, we therefore characterize the developmental changes of HSC surface molecule expression and cytokine release after infection with Gram-negative Y. enterocolitica and Gram-positive L. monocytogenes.
2.3. Di¡erentiation of HSCs towards monocytes
2. Materials and methods
2.4. Bacteria
2.1. Isolation of human CD34+ progenitor cells derived from peripheral blood cells
Bacterial strains used in this study are: L. monocytogenes Sv 1/2a EGD and Y. enterocolitica. The bacteria were grown in brain heart infusion medium at 30‡C (Yersinia) or 37‡C (Listeria) until they reached the mid-exponential phase of growth (circa 1U109 bacteria ml31 ).
After informed consent, cells were collected by apheresis during hematopoietic recovery after chemotherapy and treatment with recombinant s.c. granulocyte colony-stimulating factor (G-CSF). 4 ml cell suspension from the pilot aliquot was used for positive selection of CD34þ cells. +
+
2.2. Positive selection of CD34 cells with the CD34 selection kit (Miltenyi Biotec, Bergisch Gladbach, Germany) After washing, the cell pellet was resuspended in 300 Wl bu¡er (phosphate-bu¡ered saline supplemented with 0.5% bovine serum albumin, and 2 mM EDTA) per 5U108 cells. The cell suspension was incubated with Fc receptor-blocking reagent (human immunoglobulins) and subsequently with monoclonal hapten-conjugated anti-CD34 antibody (clone QBEND/10, mouse IgG1) for 15 min at 6‡C. Following an additional washing step, the stained cell suspension was incubated with colloidal super-paramagnetic MACS MicroBeads conjugated to an anti-hapten antibody in a ¢nal volume of 500 Wl per 5U108 cells for 15 min at 6‡C. For positive selection cells were again washed and resuspended in bu¡er (2U108 cells ml31 ). After passing through a ¢lter to remove clumps, cells were applied to the selection column (type VSþ0 ), inserted into the magnetic ¢eld of the VarioMACS0 system. Following rinsing three times, the VSþ column was removed from the magnetic ¢eld and the selected cells were eluted in bu¡er. To further enrich the CD34þ cells, eluted cells were applied to an additional positive selection column (RS+ column0 for 107 CD34þ cells) with an analogous procedure. The ¢nal CD34þ cell suspension was eluted in 1 ml bu¡er. The CD34þ cells were used for the experiments either directly or after storage in liquid nitrogen.
HSCs were di¡erentiated towards monocytes by incubation in long-term cell culture medium (Cell Systems, Vancouver, BC, Canada) supplemented with stem cell factor (100 ng ml31 ), thrombopoietin (75 ng ml31 ) and £t-3 ligand (15 ng ml31 ) (Pepro Tech, Rockyhill, SC, USA) [9].
2.5. In vitro bacterial infection of HSCs 5U105 cells ml31 were infected with bacteria. Bacteria were added at a multiplicity of infection (MOI) of 10. The cultures were incubated in RPMI 1640 medium supplemented with 10% pooled human AB serum at 37‡C for 1 h. For selective removal of extracellular bacteria, 100 Wg ml31 gentamicin (Gibco, Karlsruhe, Germany) was added. Twenty-four hours post infection (p.i.) cells were analyzed by £ow cytometry and the supernatant was collected and stored at 380‡C. 2.6. Uptake inhibition studies The uptake assay was performed as described above, except that the media contained 2 Wg ml31 of the inhibitor cytochalasin D throughout the duration of the experiment. 2.7. Flow cytometry The expression of surface molecules of uninfected and infected HSCs was evaluated by £ow cytometry after direct staining with PE- or FITC-conjugated anti-CD13, anti-CD14, anti-CD16 (FcQRIII), anti-CD33, anti-CD34, anti-CD54, anti-CD80, anti-CD83, anti-Toll-like receptor (TLR)-4, anti-CD86 antibodies, and B7-H1 (BioCarta, Hamburg, Germany; eBioscience, San Diego, CA, USA; Pharmingen, Hamburg, Germany). 2.8. Protein multiplex immunoassay kit (BioSource) Cytokine concentrations in the supernatants generated
6 Fig. 1. Surface marker analysis of human CD34þ HSCs. The x-axis of each histogram shows the log £uorescence intensity; the y-axis shows the relative cell number. a: Surface marker analysis of puri¢ed human CD34þ HSCs. Black histograms represent staining with matched-isotype antibodies, gray histograms represent d0 HSCs. b: d0 HSCs 24 h p.i. with L. monocytogenes or Y. enterocolitica : upregulation of CD54, CD86 and B7-H1. Black histograms represent staining with matched-isotype antibodies, thick black histograms represent uninfected HSCs, gray histograms represent HSCs after Y. enterocolitica infection, dashed histograms represent HSCs after L. monocytogenes infection. c: Expression of TLR-4 on monocytes and HSCs (d0, d6). Black histograms represent staining with matched-isotype antibodies, gray histograms represent TLR expression on HSCs or monocytes.
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in the infection experiments 24 h p.i. were measured with the Luminex technology for multiplex cytokine immunoassay (obtained from BioSource, Nivelles, Belgium) using beads for G-CSF, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor-K (TNFK), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-8, IL-1L, IL12p40, IL-10, IL-13 and interferon-Q (IFN-Q). Supernatants were diluted 1:10 in sterile water ; 50 Wl of the dilution was used in the assay according to the detailed description supplied by the manufacturer. At least 100 beads per cytokine and sample were measured. Data were evaluated with the MasterPlex QT software (MiraiBio). Alternatively, standard enzyme-linked immunosorbent assay kits were used to determine TNF-K, IL-6, IL-8, IL-1L, IFN-Q (ImmunoKontact, AMS Biotech, Wiesbaden, Germany) and IL-10, IL-12p70 (Biocarta, Hamburg, Germany).
3. Results and discussion 3.1. CD34+ HSCs change surface marker expression after infection with L. monocytogenes and Y. enterocolitica The puri¢ed fraction of CD34þ HSCs from human blood consisted of s 95% CD34þ cells. Speci¢c markers for T-cells, B-cells, monocytes, and mature dendritic cells (CD3, CD19, CD14, or CD83) were found on 6 1% of the cells. CD34þ HSCs showed a slight expression of CD54 (ICAM-1), while other costimulatory signals such as B7.1 (CD80), B7.2 (CD86), and B7-H1 were almost not detectable (Fig. 1a). HSCs were incubated with the Gram-positive bacterium L. monocytogenes and the Gram-negative bacterium Y. enterocolitica. Four hours and 24 h p.i. the surface marker expression of CD13, CD14, CD16, CD33, CD34, CD54, CD80, CD83, CD86, and B7-H1 was evaluated. CD13 and CD33 are expressed on myeloid progenitors, monocytes, and mast cells. Four hours p.i. infected HSCs showed no altered surface molecule expression compared to uninfected HSCs. Twenty-four hours p.i. we observed a prominent upregulation of CD54 (ICAM-1). In addition to ICAM-1, the costimulatory molecules CD86 (B7.2) and B7-H1 [10] were upregulated, in particular after infection with Y. enterocolitica (Fig. 1b). The change in surface marker expression was unexpected, since recent studies have shown that undi¡erentiated HSCs are not able to perform macropinocytosis or receptor-mediated phagocytosis because essential receptors and internalization mechanisms for bacterial uptake have not yet been developed [8]. Therefore we examined whether these cells already express an important pathogen recognition receptor, namely TLR-4. Pathogen-recognition receptors include the family of TLRs, which recognize conserved motifs of pathogens [11]. We investigated TLR4, which is known to bind bacterial lipopolysaccharide
(LPS) of Gram-negative bacteria and might therefore account for the upregulation of surface markers induced by Y. enterocolitica. While we could clearly show TLR-4 expression on monocytes, HSCs did not express TLR-4 (Fig. 1c). Therefore the altered surface marker expression pro¢le after infection is not mediated via TLR-4. HSCs were then di¡erentiated towards monocytes using a cytokine mixture consisting of stem cell factor, thrombopoietin, and £t-3 ligand (see Section 2). Incubation of HSCs in this di¡erentiation medium leads to the upregulation of the myeloid markers CD13 and CD33, and monocyte-speci¢c antigens such as CD14, while the stem cell marker CD34 was downregulated (Fig. 2a). CD54 (ICAM-1) was still detectable at low levels, but other costimulatory molecules were not expressed after incubation with the cytokine combination. Surface markers characteristic for T- and B-cells (CD3 and CD19), as well as for mature dendritic cells (CD83) were also undetectable. Infection studies were carried out after incubation of HSCs for 3 (d3 HSCs) or 6 days (d6 HSCs) in the cell culture medium. These time points were selected since monocytic di¡erentiation occurs within this period [8]. In comparison to the uninfected situation infection of d6 HSCs with Y. enterocolitica and L. monocytogenes led to an enhanced upregulation of CD13 and CD14 24 h p.i., which is expressed at high levels on monocytes (Fig. 2b). This e¡ect of altered cell surface expression was less pronounced after infection of d3 HSCs (data not shown). Our data indicate that Y. enterocolitica and to a lesser degree L. monocytogenes induced a more pronounced differentiation of HSCs towards monocytes. CD14 has been identi¢ed as a high-a⁄nity cell surface receptor for complexes of LPS and serum LPS-binding protein. Therefore, the more pronounced CD14 surface expression on HSCs could be a reaction to cope with further bacterial encounters. Although we noticed di¡erentiation towards monocytes, infected cells showed an upregulation of CD34. In addition HSCs express CD54 (ICAM-1) and B7-H1, and to a much lesser degree CD80 (B7.1), CD86 (B7.2), and CD83, a marker of mature dendritic cells after bacterial stimulation. In general the e¡ects were more pronounced after infection with Y. enterocolitica than with L. monocytogenes (Fig. 2b). To investigate if these changes in surface marker expression are related to bacterial uptake by partially di¡erentiated HSCs, we evaluated the surface marker expression after the addition of cytochalasin D, an inhibitor of bacterial uptake, to the culture medium. We observed no di¡erence between molecule expression in the presence or absence of the uptake inhibitor. These results underline that the uptake of bacteria was not essential for the surface molecule changes. In addition, these data indicate that reorganization of the cell membrane protein composition can occur independent of the actin cytoskeleton. Furthermore, we could show that changes in surface
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Fig. 2. a: Surface marker analysis of partially di¡erentiated d5 HSCs. FACS analysis was done after staining cells with FITC- (x-axis) and PE- (y-axis) labelled monoclonal antibodies. b: d6 HSCs 24 h p.i. with L. monocytogenes or Y. enterocolitica: upregulation of CD54, B7-H1, CD80, CD83, CD86, and CD34. Black histograms represent staining with matched-isotype antibodies, thick black histograms represent uninfected HSCs, gray histograms represent HSCs after Y. enterocolitica infection, dashed histograms represent HSCs after L. monocytogenes infection.
marker expression occur with both viable and heat-killed bacteria. 3.2. Cytokine pro¢le of HSCs after bacterial infection To investigate whether secreted factors are released from human HSCs after incubation with L. monocytogenes or Y. enterocolitica, we evaluated a panel of major cytokines and growth factors. We measured G-CSF, GM-CSF, IL-1L, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 (p40), IFN-Q, and TNF-K 24h p.i. in cell culture supernatant. Uninfected HSCs, either undi¡erentiated or grown for 6 days in the monocytic di¡erentiation cytokine combination, did not produce detectable levels of the majority of
the tested cytokines. Only a slight release of G-CSF (1 ng ml31 ), GM-CSF (500 pg ml31 ), and IL-1L (170 pg ml31 ) was observed in these cells. Subsequently, undi¡erentiated HSCs, d3 HSCs and d6 HSCs were incubated for 24 h with either Y. enterocolitica or L. monocytogenes and the production of cytokines was determined. In general, bacterial infection did not lead to an increased production of G-CSF, IFN-Q, IL-1L, IL-2, IL-4, IL-5, or IL-13. Undi¡erentiated HSCs produced a moderate level of GM-CSF (up to 3 ng ml31 ), while all other cytokines were hardly detectable. Infection of d3 and more pronouncedly of d6 HSCs resulted in IL-6 (Fig. 3a), IL-8, IL-10 (Fig. 3b), IL-12 (Fig. 3c), TNF-K (Fig. 3d), and
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Fig. 3. Cytokine production of uninfected and infected HSCs (d0 HSCs, d3 HSCs, d6 HSCs). Di¡erent cytokines in cell supernatant were measured 24 h after infection with L. monocytogenes or Y. enterocolitica (MOI 10). a: IL-6. b: IL-10. c: IL-12 p40. d: TNF-K. The results from a representative experiment are presented as mean values and standard deviations of triplicate determinations.
GM-CSF upregulation. In comparison to L. monocytogenes infection, infection with Y. enterocolitica resulted in particular in a stronger upregulation of IL-10, TNFK, and IL-12 p40 (Fig. 3). These di¡erences may be due to an alternative pathway of signal transduction induced by Gram-negative and Gram-positive bacteria and LPS and lipoteichoic acid (LTA). It was shown recently that human dendritic cells require a higher concentration of listerial LTA in comparison to LPS to become stimulated and to release cytokines [12]. Investigations with viable and heat-killed bacteria showed only one major di¡erence concerning the production of cytokines. Infection of d3 and d6 HSCs with heatkilled bacteria led to an approximately 10-fold increased release of IL-8 in comparison to viable bacteria. This could be explained by the fact that heat killing of the bacteria will lead to a release of bacterial components, namely bacterial CpG DNA which could be responsible for the increased IL-8 production. This is underlined by observations of Akhtar et al., who showed that bacterial DNA evokes epithelial IL-8 production in a time- and dose-dependent manner by a mitogen-activated protein kinase-dependent pathway, while other cytokines are dependent on the activation of nuclear factor UB [13]. This study clearly demonstrates that expression of sur-
face antigens and cytokine release of HSCs can be in£uenced by pathogens. Infection with bacteria promotes differentiation towards a monocytic phenotype. This e¡ect is more pronounced when HSCs have already started to differentiate as shown by infection experiments with undi¡erentiated HSCs cultured in a di¡erentiation medium. These cells express characteristic monocyte surface markers and adopt a monocytic cytokine secretion pro¢le. Interestingly, infection also results in secretion of GMCSF, even when undi¡erentiated HSCs encounter bacteria. This upregulation of GM-CSF might result in an increased amount of progenitor cells. Indeed, when we counted the number of viable cells 24 h p.i. and compared the results with the number of viable cells in uninfected controls we found no signi¢cant di¡erence, although L. monocytogenes and Y. enterocolitica are known to kill host cells upon encounter. In line with this observation, we could show that bacterial infection results in an elevation of the CD34þ cell fraction. This enhanced reconstitution of the myeloid progenitor pool might o¡er protection against challenge with bacterial pathogens [14] and is one possible explanation for leukocytosis during bacterial infections. Proliferative kinetics of the progenitor cells, in particular survival and expansion, are controlled by interacting
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cytokines. An e¡ect was described for IL-3, IL-4, IL-6, GM-CSF, G-CSF, IL-11, IL-12, leukemia inhibitory factor, and stem cell factor [15,16]. In addition the expression of receptors for IL-3, IL-6, and GM-CSF appears to be very low on the most immature subsets of CD34þ cells, but increases progressively during successive stages, in particular during myelo-monocytic di¡erentiation [17]. Therefore, the cytokine release of hematopoietic progenitor cells after bacterial infection is likely to in£uence proliferation and di¡erentiation of HSCs. However, it is so far not clear if the changes in the surface molecule receptors result from cytokine release or are mediated by direct signaling via pathogen recognition receptors. Since infection of bone marrow progenitors may contribute to hematologic manifestation of diseases, further studies are needed to elucidate this important interaction between pathogens and stem cells.
Acknowledgements This study was supported by a fellowship from the Bayerisches Staatsministerium fu«r Wissenschaft, Forschung und Kunst to A.K.-M. The authors thank R. Ottohal for excellent technical assistance. We are grateful to Prof. J. Heesemann for donating the Yersinia enterocolitica strain and F. Hartmann for help with the Luminex technology.
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