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Blood, 1 August 2006, Vol. 108, No. 3, pp. 965-973. Prepublished online as a Blood First Edition Paper on April 6, 2006; DOI 10.1182/blood-2005-12-5046.
IMMUNOBIOLOGY The Wingless homolog WNT5A and its receptor Frizzled-5 regulate inflammatory responses of human mononuclear cells induced by microbial stimulationFrom the Division of Molecular Infection Biology, Division of Clinical Infectious Diseases, Division of Clinical and Experimental Pathology, Division of Innate Immunity, and Division of Biological Chemistry, Research Center Borstel (RCB), Leibniz Center for Medicine and Biosciences, Borstel, Germany; Department of Mucosal Immunity, The German Research Center for Biotechnology (GBF), Braunschweig, Germany; and the Institute of Medical Microbiology, Hannover Medical School, Hannover, Germany.
Microarray - assisted gene - expression screens of human macrophages revealed WNT5A, a homolog of Wingless, a key regulator of Drosophila melanogaster embryonic segmentation and patterning, to be consistently up-regulated following stimulation with different mycobacterial species and conserved bacterial structures. The expression of WNT5A required Toll-like receptor signaling and NF-  B activation, which identifies a novel induction pathway for a Wingless homolog. We show that human peripheral-blood mononuclear cells express the WNT5A receptor Frizzled-5 (FZD5). Both WNT5A and FZD5 also were detected in granulomatous lesions in the lungs of Mycobacterium tuberculosis–infected patients. Functional studies showed that WNT5A and FZD5 regulate the microbially induced interleukin-12 response of antigen-presenting cells and interferon- production by mycobacterial antigenstimulated T cells. Our findings implicate the evolutionarily conserved WNT/Frizzled signaling system in bridging innate and adaptive immunity to infections.
Immunity to infections depends on the successful integration of innate and adaptive defense strategies.1 Cells of the innate immune system, such as macrophages and dendritic cells, recognize pathogen-associated molecular patterns shared by many microbes but not found in higher eukaryotes, via members of the Toll-like receptor (TLR) family.2,3 TLR-dependent signaling pathways can directly induce macrophage antimicrobial programs but also initiate inflammatory cell recruitment and help prime cells of the adaptive immune system to amplify bactericidal effector mechanisms. Experimental infections with microorganisms have been used successfully to uncover the intricacies governing the interplay between innate and adaptive immunity.4,5 For example, cell-wall components of Mycobacterium tuberculosis, the causative organism of tuberculosis, critically depend on TLR2 and TLR4 to induce secretion of the proinflammatory cytokines tumor necrosis factor alpha (TNF-  ) and interleukin (IL-12, necessary for differentiating T-helper 1 cells).6 Subsequently, mycobacteria-primed T cells secrete interferon gamma (IFN-) as a critical macrophage-activating agent. Eradication of mycobacteria is achieved only when both arms of the immune system are fine-tuned for full antimicrobial potency. Functionally, the Toll-mediated induction of antimicrobial effector systems is highly conserved between Drosophila melanogaster (D melanogaster) and Homo sapiens.7,8 Another example for human evolutionarily ancient effector mechanisms is granulysin (a granule-stored bactericidal molecule), which is homologous to the amoebapores of Entamoeba histolytica.9,10 Thus, there is ample precedence for evolutionarily conserved signatures governing individual facets of the immune response. To uncover novel regulatory pathways in innate responses to infection, we performed a microarray-based gene-expression screen with human macrophages infected with mycobacteria or conserved bacterial structures. We found mRNA for WNT5A, a homolog of Wingless in Drosophila species, to be consistently up-regulated in response to all stimuli. Wingless was originally characterized as a segment polarity gene in D melanogaster, which is essential in embryonic segmentation and patterning (reviewed in Klingensmith and Nusse11). Various homologs of the Wingless protein, termed WNT, are involved in embryonic development of nonvertebrates and vertebrates,12,13 where WNT signaling determines cell motility, differentiation, and apoptosis.14 In mammalian hematopoiesis, WNT signaling is essential for stem-cell homeostasis15 and lymphocyte differentiation.16,17 Most recently, one member of the WNT family of proteins, WntD, was shown to be involved in regulating antibacterial defenses in Drosophila.18 To date, however, WNT proteins have not been directly implicated in the human immune defense against infections.
Our functional analyses demonstrate that both WNT5A and Frizzled-5 (FZD5) regulate mycobacteria-induced IL-12 production and IFN-
Bacteria, antibodies, and reagents M avium (strain SE01), M tuberculosis (strain H37Rv), synthetic lipopeptide (Pam3CSK4; kindly provided by Dr K. H. Wiesmüller, EMC Microcollections, Tübingen, Germany), lipopolysaccharide (LPS; Salmonella enterica, serovar friedenau H909; kindly provided by Prof H. Brade, Research Center Borstel, Germany), phorbol-12-myristate13-acetate (PMA), calcium ionophore A23187 [GenBank] (both Sigma Aldrich, Taufkirchen, Germany), and phytohemagglutinin (PHA) were used for stimulation. To rule out the presence of lipopolysaccharides (LPSs) in the assays, mycobacterial strains were tested in a limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD). The effective LPS concentration in the experiments when ratios of 10:1 were used was below 2 pg/mL.
The following antibodies, proteins, and inhibitors were used: anti–human TLR2 (kindly provided by Genentech, San Francisco, CA); anti–human TNF- Isolation, differentiation, and cultivation of human leukocyte subsets Mononuclear cells were isolated from peripheral blood (PBMC) of healthy volunteers by density gradient centrifugation. Lymphocytes and monocytes were separated (purity consistently > 97%) by counterflow elutriation. Macrophages were generated from highly purified monocytes as described.21 Stimulation was done in RPMI 1640 with 10% fetal calf serum (FCS) and 4 mM L-glutamine (all from Biochrom, Berlin, Germany). All experiments performed were approved by the Research Ethics Board of the University of Lübeck, Germany. Microarray analyses Microarray analyses were performed using Affymetrix GeneChips Human Genome U95Av2 consisting of 12 558 human genes and expressed sequence tags including all human WNT homologs. After 4 hours of stimulation total RNA from human macrophages was isolated by adding TriFastFL (PEQLAB, Erlangen, Germany), reverse transcribed, labeled, and hybridized according to the manufacturer's standard protocol. Primary image analysis of arrays and data analysis were performed using GeneChip Microarray Analysis Suite Version 5.0, MicroDB and Data Mining Tool (Affymetrix, Santa Clara, CA) as described previously.22 RT-PCR Total RNA was isolated from cell cultures (0.4 x 106 macrophages; 1 x 106 lymphocytes) (SV Total RNA Isolation System; Promega, Mannheim, Germany) and reverse transcribed using oligodT nucleotides. For reverse transcriptase–polymerase chain reaction (RT-PCR) the following gene-specific primer pairs were used: beta-2-microglobulin (B2M) forward 5'-GCTGTGCTCGCGCTACTCTC-3', reverse 5'-GCGGCATCTTCAAACCTCCAT-3'; WNT5A forward 5'-ACACCTCTTTCCAAACAGGCC-3', reverse 5'-GGATTGTTAAACTCAACTCTC-3'23; WNT5A (quantitative PCR) forward 5'-GTCTTGAGCTTGGGC-3', reverse 5'-AC GTCCATGTCTATAACGA-3', IL-2 forward 5'-TTACAAGAATCCCAAACTCACC-3'; reverse 5'-TAGCAAACCATACATTCAACAA-3', Fzd5 forward 5'-TGTCTGCTCTTCTCGGC-3', reverse 5'-CCGTCCAAAGATAAACTGCT-3'. For quantitative RT-PCR, LightCycler technology (Roche Diagnostics, Mannheim, Germany) was used. Gene expression is displayed as relative expression normalized to B2M. Specificity of amplified products was confirmed by sequence analyses. In situ hybridization and immunohistology Cytospins of human macrophages were prepared after 4 hours of cultivation and fixed overnight in HOPE (HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-glutamic acid buffer-mediated organic solvent protection effect).24 HOPE-fixed, paraffin-embedded sections of human lung tissue were deparaffinated. PCR product amplified with the WNT5A specific primer pair or nonspecific DNA (1356 bp of the bacterial vector pcDNA 3 cleaved by PstI) was labeled with the DIG-High-Prime system (Roche) according to the manufacturer's instructions. Hybridization (2 ng/µL freshly denatured probe, 0.1% sodium dodecyl sulfate (SDS), 50% formamide, and 250 µg/mL yeast tRNA [Roche]) with both probes was carried out overnight in moist chambers at 46°C, and detection was performed using an antidigoxigenin antibody conjugated with alkaline phosphatase (anti–DIG-AP, Roche) as previously described in detail.25 For immunohistochemistry, anti-FZD5 antibody (Upstate) was used. Detection was performed by a horseradish peroxidase–labeled streptavidine-biotin technique (LSAB2, Dakocytomation, Glostrup, Denmark) using aminoethylcarbazole as chromogenic substrate. Stained cytospins and tissue sections were analyzed with a Leica DMLB microscope (Leica Microsystems, Wetzlar, Germany) equipped with the following C Plan objective lenses: x20/0.4 NA; x40/0.65 NA; and x10/0.22 NA. Images were acquired as JPEG files using the Nikon Digital Sight DS-5M imaging system and software (Nikon, Düsseldorf, Germany). Minor adjustments of brightness, contrast, and color adjustment solely for the purpose of legibility were made with Adobe Photoshop 8.0 for Windows (Adobe Systems, San Jose, CA). Generation of the anti–Fzd5 antiserum Rabbits were immunized with a 21–amino acid peptide (CPILKESHPLYNKVRTGQVPN) derived from the cysteine-rich extracellular domain of human FZD5 that has previously been used for the successful generation of a functionally active anti-FZD5 antiserum.26 Immunogenicity of the peptide was enhanced by conjugation to keyhole limpet hemocyanine. Prior to immunization preimmune serum was obtained as a control. Peptide specificity of the obtained antiserum was monitored by competitive solid phase enzyme-linked immunosorbent assay (ELISA). An irrelevant peptide of the same size (amino acids 27-48 of human IL-8 receptor II; E. Brandt, Research Center Borstel) was used as an internal control in ELISA. Cytokine detection
Supernatants were assayed by ELISA according to the manufacturer: IL-12p40, IL-2 (Duoset, R&D Systems), IFN- Statistical analysis For statistical analysis, raw data were log10 transformed prior to analysis. Data obtained from independent experiments were compared using a t test for paired comparisons. mRNA levels or cytokine concentrations under different culture conditions were correlated to control cultures (normalized to 100%) and are represented as mean ± standard deviation (SD).
Differential expression of genes of the WNT/FZD signaling pathway in human macrophages
A microarray-based gene-expression screen was performed on human primary macrophages stimulated with mycobacteria (M tuberculosis strain H37Rv, M avium strain SE01) or conserved bacterial structures (LPS, synthetic lipopeptide [Pam3CSK4]). In addition to a number of cytokines (eg, TNF- WNT5A mRNA expression is restricted to professional antigen-presenting cells A similar induction of WNT5A mRNA expression in response to microbial stimuli also was observed in primary human monocytes and dendritic cells (data not shown). Under all conditions analyzed, up-regulation of WNT5A mRNA expression was apparent 2 hours after stimulation and persisted for 24 to 72 hours (Figure 1B). WNT5A mRNA expression by human macrophages was independently confirmed by in situ hybridization25 with a WNT5A-specific probe in human monocyte-derived macrophages and alveolar macrophages in uninvolved (tumor-free) human lung tissue derived from tumor patients (Figure 2). Next we investigated WNT5A expression in human lymphocytes. To this end, the lymphocyte fraction of peripheral-blood mononuclear cells (PBMCs), which comprise T- and B-lymphocytes as well as NK cells, was analyzed by RT-PCR. Both in the absence or presence of polyclonal stimuli (eg, crosslinking CD3 and CD28, addition of phytohemagglutinin or phorbol ester/calcium ionophore), there was no detectable WNT5A mRNA expression in the lymphocyte fraction, whereas IL-2 mRNA expression was readily induced (Figure 1C). These data indicate that activation-induced WNT5A expression in human blood–derived leukocytes is restricted to cells of the myeloid lineage.
WNT5A expression in macrophages is mediated by Toll-like receptors and the NF- Receptors of the innate immune system that recognize conserved pathogen-associated molecular patterns (PAMPs) are called pattern recognition receptors (PRR). One family of PRR, the family of TLRs, mediates cell activation induced by defined conserved microbial ligands.1 Intact mycobacteria and lipoproteins require TLR2 signaling to activate cells; activation by LPS is mediated by the TLR4/MD2 complex (reviewed in Medzhitov1). Our analyses of TLR signaling in human macrophages showed that inhibitory anti-TLR2 antibodies selectively blocked mycobacteria-induced induction of WNT5A mRNA expression (Figure 3A). Analogously, the addition of the synthetic tetra-acyl LPS precursor Ia (compound 406), an antagonist of LPS-mediated activation in human monocytes,27 dose-dependently inhibited LPS-induced WNT5A expression (Figure 3C). We infer that WNT5A expression induced in macrophages by intact mycobacteria or purified microbial structures critically depends on TLR-mediated signaling. This TLR-mediated induction of WNT5A in human macrophages represents a hitherto undescribed pathway of WNT induction.
We further analyzed whether cellular activation by TLR ligation might be sufficient to induce WNT5A expression. To this end, human embryonic kidney cells (HEK293) transfected with the human TLR2 (HEK293-TLR2) were stimulated with mycobacteria or synthetic lipopeptide and functionally compared to control cells. HEK293 cells lacking TLR2 expression responded to TNF- , but not to bacterial stimulation, by secreting IL-8. TLR2-positive HEK293 cells additionally released IL-8 in response to mycobacteria and synthetic lipopeptide but not to LPS (Figure S1, available on the Blood website; see the Supplemental Figures link at the top of the online article). However, WNT5A expression in TLR2-transfected HEK293 cells was not further enhanced by stimulation with TNF- or TLR2 agonists (Figure S1). This suggests that the presence of TLR2 alone may not be sufficient to regulate WNT5A expression and that the cellular background or differentiation status also plays an important role. This is further supported by our findings that WNT5A expression cannot be induced by bacterial stimuli in human myeloid premonocytic cell lines such as HL-60, U937, and MonoMac6 (data not shown).
TLR-mediated activation of antigen-presenting cells involves various signaling cascades, the NF-
Early WNT5A expression in macrophages is not due to secondary effects induced by TNF-
Macrophages react to bacterial infection by rapidly releasing proinflammatory cytokines, such as TNF- Expression of the WNT5A receptor FZD5 by human monocytes/macrophages and lymphocytes WNT proteins bind to 7-pass transmembrane receptors of the FZD family and initiate at least 3 different intracellular signaling pathways, resulting in the regulation of gene expression and/or changes in cell behavior.30 FZD5 has been shown to be a functional receptor for WNT5A.31 We therefore analyzed the presence of FZD5 in human macrophages and lymphocytes (Figure 4A,B). Quantitative RT-PCR showed a basal expression of FZD5 mRNA in human macrophages, which is up-regulated in response to mycobacteria (5-fold) and LPS (50-fold) 4 hours after stimulation. A substantial up-regulation (10-fold) also was observed in lymphocytes following stimulation by anti-CD3 or anti-CD3/anti-CD28 antibodies. These data are the first to demonstrate that stimulation of human myeloid cells by Toll-like receptor agonists or T-cell stimulation induce transcription of the FZD5 gene.
To detect FZD5 at the protein level, a specific anti-FZD5 antibody was used.20 A single band at 62 kDa was detected by Western blot in lysates of unstimulated human monocytes and lymphocytes (Figure 4C). A recombinant FZD5/Fc fusion protein was added as a positive control and was detected at around 55 kDa as described by the manufacturer. In macrophages stimulated with mycobacteria as well as with LPS, no detectable changes in FZD5 protein level were observed (data not shown). These data demonstrate that FZD5 is expressed by human monocytes/macrophages as well as lymphocytes. The presence of FZD5 protein in human mononuclear cells and its transcriptional regulation in response to stimulation suggest that Wnt proteins may exert direct functional activities on human immune cells. WNT5A and FZD5 are present in granulomatous lesions from M tuberculosis–infected patients We wished to determine whether WNT5A and FZD5 would be present at the site of macrophage–T-cell interaction in an in vivo infection scenario. The granuloma is the structural correlate of the host's strategy to successfully fight mycobacterial infection and is a site of ongoing activation and interaction between macrophages and lymphocytes.32,33 We therefore analyzed the presence of WNT5A mRNA and FZD5 protein expression in granulomatous lesions of patients freshly diagnosed with pulmonary tuberculosis. Figure 5 depicts that FZD5 protein is detectable in various cells with mononuclear morphology using the antibody described in Figure 4. The arrows indicate that FZD5-positive cells are found predominantly in areas rich in mononuclear-cell infiltration associated with the granuloma. The parallel analysis of WNT5A mRNA expression by in situ hybridization showed mostly cells with a larger cytoplasm, indicative of macrophage morphology, to be positive for WNT5A. These data demonstrate that WNT5A- and FZD5-expressing cells are present in mononuclear infiltrates at the site of infection in M tuberculosis–infected patients. The immunohistochemical analyses show that not all macrophages and lymphocytes are positive for FZD5 protein WNT5A mRNA. The specific characteristics of the FZD5- as well as the WNT5A-expressing subsets are currently unknown.
Selective inhibition of IL-12 and IFN-
IFN-
The induction of IFN- The same anti-WNT5A antibody was used to specifically detect WNT5A in lysates and supernatants of WNT5A overexpressing NIH-3T3 fibroblasts37 (Figure S2). Attempts to demonstrate the presence of WNT5A at the protein level by Western blot in cultured primary human mononuclear cells were not successful, possibly due to the limited sensitivity of the assay or a low level of overall WNT5A protein expression. It bears mentioning that the literature contains numerous studies confirming WNT protein expression in overexpressing systems; however, detection of endogenous WNT proteins in primary cells appears to be very difficult.12,13
The FZD5-specific antibody20 used to demonstrate the protein expression of FZD5 (Figures 4C, 5) is directed against an intracellular proportion of FZD5 and can therefore not be used as an inhibitory agent to interfere with ligand receptor interactions. To investigate a potential functional role of FZD5 in the regulation of cytokine release, we generated an antiserum against the extracellular N-terminal cysteine-rich domain of FZD5, which is essential for WNT-ligand binding.38 The specificity of the antiserum was measured in a competition ELISA (Figure S3). The PPD-induced IFN- release of PBMCs was dose-dependently and significantly decreased with increasing concentrations of this anti-FZD5 anti-serum, when compared with the effect of the corresponding preimmune serum (Figure 6C,D). In contrast, the PPD-induced IL-2 and TNF- release, as well as the proliferative response of PBMCs, was not influenced by the anti-FZD5 antiserum (Figure 6C, and data not shown). Further experiments addressing the effect of the anti-FZD5 antiserum on mycobacteria-induced IL-12 release by macrophages showed a significant inhibition of IL-12 production compared with cultures treated with the preimmune serum (Figure 6D). From our data we conclude that WNT5A and its receptor, FZD5, play an important role in the regulation of key cytokines of the antimicrobial defense.
This study demonstrates that (1) WNT5A is expressed by human antigen-presenting cells in response to mycobacteria and conserved bacterial structures, (2) the expression of WNT5A is dependent on TLR signaling and the activation of the NF- B pathway, (3) WNT5A and its receptor FZD5 are present on human mononuclear cells in vitro and in vivo, and (4) WNT5A and FZD5 are functionally involved in the regulation of the human Th1 cytokine response, indicating their role in antimicrobial defenses. Wingless, a morphogen in Drosophila species, and its phylogenetically highly conserved homologs (termed WNT) are critical regulators of cell polarity, motility, differentiation, apoptosis, and carcinogenesis in both nonvertebrates and vertebrates. In mammals, members of the WNT family (currently comprising 19 human and murine homologs) are involved in early T-cell differentiation and B-cell proliferation (reviewed by Staal and Clevers39 and Reya et al17). Little is known about the expression and the potential function of WNT homologs under pathophysiologic conditions, such as in an inflammatory setting. In one report, transcripts for WNT1, 5A, 10B, 11, and 13 were detected in synovial fibroblasts from patients with rheumatoid arthritis.23 WNT2 and WNT5A were detected in tumor-associated macrophages of the Lamina propria in colorectal cancer patients,40 although the inducing stimulus was not determined. Recently, other investigators reported a differential expression of WNT5A in response to various microbial stimuli using microarrays for studying pathogen-induced gene-expression profiles of human macrophages and dendritic cells.41,42 However, these findings were not confirmed by an independent method. To date, the role of WNT5A during the immune response, particularly as it relates to the defense against infections, has not been further characterized.
We identified WNT5A as a differentially expressed gene in response to mycobacteria (M avium and M tuberculosis). Further experiments demonstrated the induction of WNT5A expression after contact with various conserved microbial structures such as lipopolysaccharide and lipopeptide, which indicates that this molecule is commonly induced in myeloid cells in infectious processes. The identification of TLR2 and TLR4 in human macrophages as 2 important pattern recognition receptors initiating Wnt5a expression and the fact that the NF-
In Drosophila species, wingless gene expression during embryonic development is induced by Hedgehog (Hh), a soluble factor, which binds to a 7-pass transmembrane receptor termed Patched (Ptc).43 Homologs of Hh also have been identified in vertebrates. Cellular activation by Hh leads to the stabilization of the transcription factor Cubitus interruptus (Ci). Three homologs of the ci-gene were identified in vertebrates, all of which encode for Gli-proteins (from glioblastoma; reviewed in Ingham and McMahon43). The expression of WNT homologs in vertebrates can be induced by Hh-induced signaling and Gli-proteins.44 Deletion of the murine Sonic hedgehog (Shh) led to a reduced Wnt5a expression during development of the genital organs.45 A further direct relation between Shh and the regulation of Wnt5a expression was found in the morphogenesis of hair follicles.46 Additionally, further Hh-independent mechanisms have been described to regulate Wnt5a expression. In a human epithelial cell line, a protein kinase C (PKC) and protein tyrosine kinase–dependent induction of WNT5A was observed.47 The results of our present study clearly demonstrate a TLR-mediated, NF-
TNF- Studies on the presence of FZD receptors as potential interaction partners for WNT homologs on human immune cells have been focused primarily on their role in early T-cell development (reviewed in Staal and Clevers39). A recent report by Murphy and Hughes50 described the expression of FZD5 mRNA by human T cells isolated from peripheral blood. The detection of FZD5 protein in human mononuclear cells and its increased mRNA expression in macrophages and T cells in response to stimulation in vitro, as shown in our study, strongly suggest that FZD5 may have a function in immunologic responses. The presence of FZD5 on human immune cells further implies that WNT proteins may exert a direct functional activity on human immune cells in an autocrine or paracrine manner. Indeed, both WNT5A and FZD5 were found to be expressed under pathophysiologic conditions in vivo, in alveolar macrophages adjacent to tumor-bearing tissue (Figure 2), and in tuberculous granulomas (Figure 6), indicating a direct involvement in antimicrobial responses. The presence of the ligand and its receptor in granulomatous lesions of tuberculosis patients prompted us to investigate in which way WNT/FZD signaling might be important in the host response to mycobacterial infections.
The validation of a functional role for WNT5A and FZD5 in vivo, for example, in the well-established animal model of low-dose aerosol M tuberculosis infection in mice,51,52 is hampered by the fact that mice deficient in WNT5A and FZD5 are severely compromised in development53,54 and die in utero.55 Therefore, we focused on a human in vitro model to investigate if the activation of antigen-presenting cells (IL-12-, TNF-
Collectively, the data presented in our study demonstrate that both WNT5A and FZD5 are capable of regulating IL-12 and IFN-
Based on the results of our present study, we would like to propose the following model of WNT5A and FZD5 as modulators of the interplay between antigen-presenting cells and T lymphocytes (Figure 7): in the context of an infectious process, WNT5A produced by antigen-presenting cells regulates the IL-12 response in an autocrine manner and thereby primes specifically activated T lymphocytes for IFN-
The detailed molecular mechanism by which WNT5A and FZD5 regulate the cytokine release of primary human T cells and monocytes/macrophages are not yet clear: one scenario is the involvement of the so-called canonic signaling WNT signaling pathway, which leads to the stabilization of
A successful immune response to infection needs to be carefully choreographed. IL-12 and IFN- are critical mediators of cellular immunity to intracellular infections. We showed that WNT5A and FZD5, expressed by human mononuclear cells in response to microbial stimuli in a TLR- and NF-B–dependent manner, are necessary for the regulation of IL-12 and IFN- in response to infectious agents. Both Toll and WNT signaling pathways are evolutionarily highly conserved and have only recently been found to intersect in Drosophila.18 They may both have evolved to efficiently regulate the adaptive immune response in higher organisms. These findings, for the first time, implicate the phylogenetically conserved WNT/FZD system in bridging innate and adaptive immunity to infections.
The authors thank especially R. Kemler (Freiburg) for Wnt5a-, Wnt1-, and LacZ-transfected NIH-3T3 fibroblasts and critical reading of the manuscript. The authors thank J. M. Reichhart (Strassbourg), J. Gerdes, S. Uhlig, E. Vollmer, and E. Th. Rietschel (Borstel) for helpful discussions and suggestions. The authors gratefully acknowledge H. Kühl, E. Kaltenhäuser, R. Bergmann, and S. Kröger for expert technical assistance. This paper is dedicated to the memory of Jörg Lauber, who unexpectedly passed away at the age of 38 on May 11, 2003.
Submitted December 21, 2005; accepted March 21, 2006.
Prepublished online as Blood First Edition Paper, April 6, 2006; DOI 10.1182/blood-2005-12-5046.
The online version of this article contains a data supplement.
Supported in part by Deutsche Forschungsgemeinschaft grant SFB 415-C7 (N.R., S.E.) and the National Genome Research Network, grant NIES05-T22 (S.E.).
A.B. performed research, designed research, analyzed data, and wrote the paper; S.E. designed research, analyzed data, and wrote the paper; J.L. performed research, contributed vital new analytic tools, and analyzed data; J.B. contributed vital new analytic tools; C.L. contributed clinical samples; T.G. contributed vital new analytic tools; H.H. contributed vital new analytic tools; E.B. contributed vital new reagents; and N.R. designed research, performed research, analyzed data, and wrote the paper.
An Inside Blood analysis of this article appears at the front of this issue.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Norbert Reiling, Division of Molecular Infection Biology, Research Center Borstel (RCB), Leibniz Center for Medicine and Biosciences, 23845 Borstel, Germany; e-mail: nreiling{at}fz-borstel.de.
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Abstract
Introduction
), chemokines (eg, RANTES, MIP-1
and 
in the affected pathways.