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The Journal of Immunology, 2008, 180, 8048 -8056
Copyright © 2008 by The American Association of Immunologists, Inc.
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Essential Roles for the Tec Family Kinases Tec and Btk in M-CSF Receptor Signaling Pathways That Regulate Macrophage Survival1

Martin Melcher2,*, Bernd Unger2,{dagger}, Uwe Schmidt3,*, Iiro A. Rajantie{ddagger}, Kari Alitalo{ddagger} and Wilfried Ellmeier4,*

* Institute of Immunology, Center for Physiology, Pathophysiology and Immunology, Medical University of Vienna, Vienna, Austria; {dagger} Competence Center for Biomolecular Therapeutics, Vienna, Austria; and {ddagger} Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, University of Helsinki, Helsinki, Finland


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Tec family kinases have important roles in lymphocytes; however, little is known about their function in monocytes/macrophages. In this study we report that Tec family kinases are essential for M-CSF (M-CSF)-induced signaling pathways that regulate macrophage survival. Compared with wild-type bone marrow-derived macrophage (BMM) cultures, Tec–/–Btk–/– BMM cultures displayed increased cell death that correlated with a severe drop in macrophage numbers. In addition, macrophages deficient in either Tec or Btk showed expression and activation of caspase-11. Elucidation of M-CSF receptor (M-CSFR) signaling pathways revealed that the total tyrosine phosphorylation pattern upon M-CSF stimulation was altered in Tec–/–Btk–/– macrophages despite normal expression and phosphorylation of the M-CSFR. Further, Tec and Btk are required for proper expression of the GM-CSF receptor {alpha} (GM-CSFR{alpha}) chain in macrophages but not dendritic cells, implicating Tec family kinases in the lineage-specific regulation of GM-CSFR{alpha} expression. Taken together, our study shows that Tec and Btk regulate M-CSFR signaling-induced macrophage survival and provides a novel link between Tec family kinases and the regulation of caspase-11 and GM-CSFR{alpha} expression.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Macrophages are large phagocytic mononuclear cells that play important roles in innate and adaptive immunity. Their progenitors, the monocytes, enter the blood stream from the bone marrow (BM)5 and migrate to tissues where they mature into resident tissue macrophages (1). The differentiation, proliferation, and survival of macrophages are regulated by the M-CSF. In fact, M-CSF receptor (M-CSFR)-deficient mice or mice with an inactivating mutation of M-CSF have pleiotropic phenotypes including decreased macrophage numbers in vivo (2, 3). In mice, impaired M-CSF signaling has also been implicated in the pathogenesis of several disorders (for a detailed review see Ref. 4 and references therein). Thus, a better understanding of M-CSFR signaling may also be of medical relevance.

Members of the Tec kinase family (Bmx, Btk, Itk, Rlk, and Tec) constitute the second largest family of nonreceptor tyrosine kinases and are preferentially expressed in the hematopoietic system. A large number of studies have shown important roles for these kinases in the lymphoid system. Furthermore, mice with combinatorial deletions of Tec family kinases revealed both unique and redundant functions in B cells (Tec, Btk) and T cells (Rlk, Itk). Although the Tec family kinase members Tec, Btk, and Bmx are expressed in monocytes/macrophages (5, 6, 7), little is known about their function in this lineage. Several studies implicated Tec family kinases in the LPS-induced signaling in macrophages leading to the induction of TNF-{alpha} production. Btk-defective X-linked immunodeficient (xid) macrophages have impaired secretion of the proinflammatory cytokines TNF-{alpha} and IL-1β after stimulation with LPS (8) and are also incapable of producing efficient bursts of reactive oxygen intermediates (9). In line with this, xid macrophages show impaired p65 phosphorylation and transactivation upon LPS stimulation, whereas IkB{alpha}-degradation is normal (10). Meanwhile, another study could not find any differences in TNF-{alpha} expression between control and xid macrophages after LPS stimulation (5), which may reflect differences in the genetic backgrounds or different macrophage populations used in these studies. However, the importance of Tec family kinases for monocyte function has been confirmed through the analysis of Btk-deficient human monocytes. Blood monocytes isolated from X-linked agammaglobulinemia (XLA) patients who lack a functional Btk gene have impaired phagocytic functions and altered chemotactic responses (11) and are impaired in the production of TNF-{alpha} and IL-1β upon stimulation of TLR 2 or 4 (12), although another study reports that Btk is not essential for LPS/TLR4 signaling (13). Overexpression of Btk in wild-type human monocytes leads to the stabilization of TNF-{alpha} mRNA and therefore to an increase in TNF-{alpha} production (5, 12). Interestingly, incubation of XLA monocytes with M-CSF leads to an increase in the expression of Tec and restores their ability to produce TNF-{alpha} upon LPS stimulation (5). This suggests a compensatory role for Tec similar to the situation observed in murine B cells (14), which may also explain why XLA patients show normal innate immune responses (5).

In this study, we aimed to further dissect the role of Tec family kinases in monocytes/macrophages. We used a genetic approach to study macrophages lacking various members of the Tec kinase family and generated combinatorial Tec family kinase knockout mice. We could show that Tec and Btk regulate the survival of BM-derived macrophages (BMM) by controlling M-CSFR signaling. A severe drop in cell numbers in Tec–/–Btk–/– macrophage cultures was observed, and this correlated with increased cell death of macrophages. Despite normal expression and M-CSF-induced autophosphorylation of the M-CSFR, M-CSF stimulation of Tec–/–Btk–/– macrophages resulted in an altered tyrosine phosphorylation pattern. Because Btk was activated upon M-CSF stimulation of primary BMMs, our study thus shows that Tec family kinases play an important role in M-CSFR signaling pathways that lead to macrophage survival. Interestingly, Tec–/– or Btk–/– macrophages showed constitutive expression of caspase-11, an inducible member of the caspase family (15). Finally, we found that Tec and Btk are required for proper expression of the GM-CSF receptor {alpha}-chain (GM-CSFR{alpha}) in macrophages but not dendritic cells, therefore implicating Tec kinases also in the lineage-specific regulation of GM-CSFR{alpha} expression.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Tec-deficient mice (14), Btk-deficient mice (16) (purchased from The Jackson Laboratory), and Bmx-deficient mice (17) were intercrossed and maintained in the animal facility of the Medical University of Vienna, Vienna, Austria. The mice used in this study were of mixed 129/Sv x C57BL/6 background. C57BL/6 Ly 5.1 mice were obtained from the European Mouse Mutant Archive (Strasbourg, France). All animal experiments were performed according to protocols approved by the Federal Austrian Ministry for Education, Science and Art (Vienna, Austria).

Flow cytometry and Abs

The spleen was removed from euthanized mice and placed into 60-mm tissue culture dishes containing staining buffer (PBS, 2% FCS, and 0.1% sodium azide). Peritoneal cells were obtained by lavage of the peritoneum with 10 ml of PBS. Single-cell suspensions were made by passing the tissue through a 70-µm nylon cell strainer. BM cells were harvested from reconstituted mice by flushing femur and tibiae with PBS containing 2% FCS. After hypotonic lysis of RBCs with ACK lysis buffer (0.15 M NH4Cl, 1.0 mM KHCO3, and 0.1 mM Na2EDTA (pH 7.2)), 1–5 x 105 cells were incubated on ice for 5 min with Fc block (BD Pharmingen) and subsequently stained with respective Abs for 30 min on ice in staining buffer. Afterward, the cells were washed once with staining buffer and analyzed. The following mAbs were used: FITC-anti-mCD11b, PE-anti-Gr1, and allophycocyanin-anti-B220 from Caltag Laboratories. Flow cytometric analysis was performed on a FACSCalibur device (BD Biosciences) and data were analyzed with CellQuest Pro software.

Generation of BMMs

BMMs were generated as described (18). Briefly, after RBC lysis, 107 BM cells were seeded onto 10-m bacterial dishes in 10 ml of DMEM (Sigma-Aldrich) supplemented with 10% FCS (Invitrogen), 100 U/ml penicillin, 10 µg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES (Sigma-Aldrich), and 50 µM 2-ME (Invitrogen) in the presence of 20% L929 cell conditioned medium (LCM). The culture medium was changed on days 3 and 6 unless indicated otherwise. Cells were analyzed by flow cytometry and microscopy (Nikon Eclipse TS100). LCM was produced as described (18). For determining the effects of M-CSF and GM-CSF and those of wild-type and Tec–/–Btk–/– supernatant exchanges on macrophage numbers, culture medium changes were done on days 2 and 5. For the M-CSF and GM-CSF rescue experiments, BMMs were removed on day 6 from the plate with citric saline (0.135 M potassium chloride and 15 mM sodium citrate) and reseeded in 6-well plates at 0.8 x 106 cells per well. Medium (total of 2 ml) containing M-CSF (10 and 30 ng/ml), GM-CSF (500 and 1500 U/ml), or LCM was exchanged daily until the end of the culture. For the supernatant exchange experiments, 107 wild-type and Tec–/–Btk–/– cells were reseeded onto 10-cm dishes on day 6, and the corresponding supernatants were added daily to the culture. For the M-CSF titration experiments, cells were reseeded in 6-well plates at 0.8 x 106 cells per well on day 5 and different concentrations of M-CSF (15, 30, 60, and 90 ng/ml) were added.

Generation of BM-derived dendritic cells (BMDCs)

BM cells were isolated and RBCs were lysed as described above for the generation of BMM. To generate BMDCs, 2 x 106 BM cells were cultured on 10-cm dishes in RPMI 1640 medium with 10% FCS, 100 U/ml penicillin, 10 µg/ml streptomycin, 2 mM L-glutamine, 10 mM HEPES (Sigma-Aldrich), 50 µM 2-ME (Invitrogen) and 700 U/ml recombinant murine GM-CSF (Peprotech) for 10 days (19).

RT-PCR

Total RNA from the various cell types was isolated with TRIzol reagent (Sigma-Aldrich), treated with DNase I (Boehringer Mannheim), and converted into cDNA by reverse transcription with oligo(dT) and random primer according to the manufacturer’s protocol (SuperScript II first-strand synthesis for RT-PCR; Invitrogen). The following primers were used for expression analysis: Tec, 5'-TAACCATGGTGACTCGTGGCCA-3' (forward) and 5'-GGTATACATGGCTGGCACTCA-3' (reverse): Btk, 5'-GAGTAACATTCTAGATGTGATGG-3' (forward) and 5'-CAGTCTGTTAGGAGTCTTGAA-3' (reverse); Bmx, 5'-gcagccctatgacttatatgat-3' (forward) and 5'-CAGATAAACAGCACATAGACC-3' (reverse); Hprt, 5'-GATACAGGCCAGACTTTGGTTG-3' (forward) and 5'-GGTAGGCTGGCCTATAGGCT-3' (reverse); and Csf2Ra, 5'-CCCCCACGGAGGTCACAAGGTCAA-3' and 5'-CAGGGCAACAGGGGTCCAGTCACA-3' (reverse).

BrdU incorporation

BM cells were differentiated into macrophages as described above. On day 5, cells were reseeded at 0.8 x 106 cells per well of a 6-well plate. At day 6, BMM cultures were incubated with 0.1 mM BrdU (Sigma-Aldrich) for a 1.5-h period. Cells were removed from the plate with citric saline as described above, resuspended in 500 ml 0.15 M NaCl. Ninety-five percent EtOH (–20°C) was added dropwise. After 30 min on ice, the cells were washed with PBS and resuspended in 1 ml of 1% paraformaldehyde with 0.01% Tween 20 in PBS and incubated at 4°C overnight. The cells were incubated in 1 ml DNase I solution (50 Kunitz units/ml DNase I, 0.15 M NaCl, 4.2 mM MgCl2, and 10 mM HCl; Sigma-Aldrich) at 37°C for 30 min. The samples were washed with PBS, stained with FITC anti-BrdU or isotype control Ab (BD Pharmingen), and analyzed by FACScan (BD Biosciences).

Propidium iodide (PI) stainings and cell death analysis

Macrophages were harvested with citric saline as described above and resuspended in PBS. PI (4 µg/ml in PBS) was added and the percentage of PI-positive cells was determined by flow cytometry (FACSCalibur; BD Biosciences).

Bone marrow competition

BM cells from wild-type and Tec–/–Btk–/–Bmx–/– as well as from Ly 5.1-positive wild-type mice were isolated and RBCs were lysed with ACK (ammonium chloride-potassium carbonate) buffer. The cells were washed three times with PBS and counted. Ly 5.1 BM cells were mixed in the ratio of 1:1 with either wild-type BM or knockout BM. Mixed BM cells (1 x 106) were injected into the tail vein of lethally irradiated Ly 5.1 mice (2 x 3500 mGy; Hille TH-150). Mice were treated with 25 µg/ml neomycin (Invitrogen) and 25,000 U/ml polymyxin B sulfate (Sigma-Aldrich) in the acidified drinking water for 1 wk. After 6–8 wk the reconstituted mice were sacrificed and analyzed by flow cytometry (FACSCalibur; BD Biosciences).

RNase protection assay

RNA from wild-type and Tec–/–Btk–/– BMMs was isolated with TRIzol reagent (Sigma-Aldrich) at day 10. The multiprobe RNase protection assay was performed according the manufacturer’s protocol (BD Biosciences). The hybridization products were separated on a 4.75% denaturating polyacrylamide gel. The gel was dried and exposed to autoradiography films (Kodak) overnight at –80°C. Probes for the housekeeping gene probes L32 and GAPDH were used as normalization controls.

Preparation of BMM lysates and immunoblot analysis

Cell lysates were prepared by washing the macrophages on the tissue culture dish with ice-cold PBS followed by their lysis in 120 µl (for 107 cells) lysis buffer (1% Nonidet P-40, 20 mM Tris-HCl (pH 8.0), 138 mM NaCl, 10 mM EDTA, and 10% glycerol) supplemented with 1 mM orthovanadate and complete protease inhibitor mix (Roche). Cell lysates were cleared by centrifugation and protein concentrations were determined using the Bradford method (Bio-Rad). The cell lysates were analyzed by standard Western blotting techniques using the following Abs: anti-phospho-Tyr (PY99; catalog no. sc-7020, Santa Cruz Biotechnology), rabbit-anti-phospho-Tyr223-Btk (catalog no.3531, Cell Signaling Technology), rabbit anti-Btk (catalog no. 556365, BD Pharmingen), rabbit anti-M-CSFR (catalog no. sc-692, Santa Cruz Biotechnology), rabbit anti-phospho-M-CSFR (catalog no.3155, Cell Signaling Technology), rat anti-caspase-11 (catalog no.C1354, Sigma-Aldrich), rabbit anti-actin (catalog no. A2066, Sigma-Aldrich), hypoxanthine phosphoribosyltransferase (HPRT)-coupled anti-rabbit Ig (Jackson ImmunoResearch Laboratories), HPRT-coupled anti-goat Ig (Jackson ImmunoResearch), HPRT-coupled anti-rat Ig (catalog no. P0450, DakoCytomation) and rabbit anti-Tec (gift from Prof. H. Mano, Jichi Medical University, Tochigi, Japan). Immunoblot protein bands were visualized by ECL (Amersham Biosciences).

Stimulation of BMM

BMMs were incubated overnight in 10 ml of medium without LCM at a density of 107 cells per 10-cm dish. The following day the adherent cells were stimulated directly on the dish with 100 ng/ml M-CSF (Peprotech) in a total volume of 4 ml for the indicated time periods at 37°C. To terminate the stimulation, the plates with the adherent cells were put on ice and the cells were washed with ice-cold PBS. Protein lysates were harvested as described above.

Cell surface M-CSFR immunoprecipitation

The protocol for immunoprecipitation of cell surface M-CSFR was adapted from Lee et al. (20). In brief, day 8 BMMs were reseeded at 107 cells per 10-cm dish and incubated overnight with medium without LCM. The next day, the adherent macrophages were stimulated with M-CSF as described above, washed three times with ice-cold PBS, and incubated with 3 ml of PBS containing 6 µg/ml sheep anti-M-CSFR Ab (catalog no. AF3818, R&D Systems) specific for the extracellular domain of the M-CSFR for 15 min at 4°C. Unbound Ab was removed by washing the cells five times with ice-cold PBS. Macrophages were lysed as described above and protein lysate (~300 µg of protein) was incubated with protein G-agarose beads (Roche) for 1 h at 4°C. The beads were pelleted by centrifugation and washed five times with lysis buffer. Proteins were removed from the beads by boiling in Laemmli buffer and the surface fraction of M-CSFR was determined by immunoblotting. Internal M-CSFR levels were measured in cell lysate aliquots taken after the incubation with protein G-agarose beads. Total M-CSFR levels were determined in cell lysate aliquots after the stimulation with M-CSF.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Normal myeloid cell development in Tec–/–Btk–/–Bmx–/– mice

Tec family kinases are broadly expressed in the hematopoietic system, and Tec, Btk, and Bmx have been detected in the murine and human monocyte/macrophage lineage (5, 6, 7). RT-PCR analysis showed that peritoneal macrophages expressed Btk, Tec, and Bmx (Fig. 1A), while BMMs expressed Btk and Tec (Fig. 1, A and B). Therefore, we focused our further studies on the analysis of Tec–/–Btk–/– BMMs and Tec–/–Btk–/–Bmx–/– mice (Btk and Bmx map to the X chromosome and thus the genotype of male knockout mice is Y/–; however, for simplicity we refer to Btk-deficient or Bmx-deficient mice as Btk–/– or Bmx–/– mice, respectively, throughout the article regardless of whether they were male of female).


Figure 1
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  		FIGURE 1. Myeloid cell development in Tec–/–Btk–/–Bmx–/– mice. A, RT-PCR analysis of RNA isolated from peritoneal macrophages (PM), BMM, and BM showing expression of Btk, Bmx, and Tec. Expression data are representative of two independent experiments. B, Immunoblot analysis showing expression of Tec and Btk in wild-type (wt), Tec–/–, and Btk–/– BMM. Expression data are representative of two independent experiments. C, Histograms showing Ly5.1 expression in macrophages (CD11bhigh/Gr1medium) or B cells (B220high) isolated from the spleen of irradiated mice that were reconstituted with a 1:1 mixture of either Ly5.1+ wild-type (wt) and Ly5.2+ wild-type (upper panels) or Ly5.1+ wild-type and Ly5.2+ (TBB; lower panels) BM cells. Numbers in the histograms indicate the percentage of cells in the indicated regions. One representative mouse from a total of three mice reconstituted with two different batches of BM cells is shown.

 
FACS analysis of various organs indicated that myeloid cell subsets are present in Tec–/–Btk–/–Bmx–/– mice under homeostatic conditions. However, because the lack of Tec and Btk leads to reduced peripheral B cell numbers (14), the relative percentage of the various subpopulations was different compared with wild-type mice (data not shown). To determine whether Tec–/–Btk–/– Bmx–/– myeloid cells display the same developmental kinetic, competitive BM reconstitution experiments were performed. Wild-type (Ly5.1+) BM cells were mixed in a 1:1 ratio with either Tec–/–Btk–/–Bmx–/– (Ly5.2+) or wild-type (Ly5.2+) BM cells and transplanted into irradiated Ly 5.1+ wild-type recipients. After 6–8 wk of reconstitution, the ratio of Ly 5.1+ and Ly 5.2+ population of the various cell lineages in the BM chimeras was determined. There was equal reconstitution of Ly5.2+ to Ly5.1+ macrophages (defined as CD11bhighGr1medium) (Fig. 1C, left panels). As expected, Tec–/–Btk–/–Bmx–/– BM cells were not able to reconstitute the B cell lineage (Fig. 1C, right panels) due to a severe block of B cell development in the combined absence of Tec and Btk (14).

Impaired survival of BM-derived macrophages in the absence of Tec and Btk

To generate BMMs, BM cells of the various genotypes were isolated and differentiated using LCM, an established source of M-CSF (21). Wild-type, Tec–/–, and Btk–/– BM cell cultures resulted in a similarly confluent layer of macrophages after 10 days in culture (Fig. 2A). In contrast, Tec–/–Btk–/– macrophage cultures were less dense (Fig. 2A) and showed dramatically reduced cell numbers already at day 8 of culture (Fig. 2B). The drop in cell numbers occurred after day 6, because until this time point cell numbers were equal in the various macrophage cultures. Tec–/–Btk–/–Bmx–/– BMM cultures showed a similar reduction in cell numbers as Tec–/–Btk–/– cultures (data not shown). Although cell numbers were reduced in the absence of Tec and Btk, the differentiation kinetics of macrophages according to the expression of F4/80 and CD11b were similar in all genotypes analyzed (Fig. 2C).


Figure 2
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  		FIGURE 2. Impaired survival of Tec–/–Btk–/– BM-derived macrophages. A, Pictures depicting day 10 BMM cultures. The data are representative of 10 independent experiments. Original magnification was x100. wt, Wild type. B, Diagram showing BMM numbers in day 6 and day 8 cultures. The summary of five independent experiments is shown. Error bars show SD. The p values were calculated using an unpaired Student’s t test. The p values shown are: *, 0.0104; **, 0.0011; and ***, <0.0001. wt, Wild type. C, Diagram showing the appearance of F4/80+CD11b+ macrophages (as determined by flow cytometry) in BM cultures. Numbers at the y-axis indicate the percentage of the F4/80+CD11b+ population. Data are representative of three independent experiments. wt, Wild type.

 
The reduced cell numbers could be caused by increased cell death or reduced proliferation. BrdU labeling experiments revealed that there was no difference in the proliferation of Tec–/–Btk–/– macrophages on day 6 (Fig. 3A), whereas on days 8 and 10 there was no detectable proliferation of wild-type and Tec–/–Btk–/– macrophages (data not shown). To determine whether increased cell death caused the reduction in cell numbers, the percentage of PI-positive Tec–/–Btk–/– and PI-positive wild-type macrophages was determined on days 6, 8, and 10 in BMM cultures. Although there was no difference in the percentage of PI-positive cells on days 6 and 10, there was an ~60% increase in the percentage of PI-positive Tec–/–Btk–/– macrophages compared with PI-positive wild-type macrophages on day 8 (Fig. 3B).


Figure 3
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  		FIGURE 3. Normal proliferation but increased cell death in Tec–/–Btk–/– macrophage cultures. A, Diagram showing the percentage of BrdU-positive cells in wild-type (wt) and Tec–/–Btk–/– macrophages cultures (day 6) after 90 min of incubation with BrdU. Data are representative of four different experiments. Error bars show SD. B, Diagram showing the percentage of wild-type (Wt) and Tec–/–Btk–/– PI-positive macrophages in day (d) 6, 8, and 10 cultures. The summary of four (day 6 and 8) and three (day 10) independent experiments each performed in triplicates is shown. Error bars show SD. The p value was calculated using a paired Student’s t test. The p value (*) is 0.0299.

 
Constitutive expression of caspase-11 in the absence of Tec family kinases

The increase in the percentage of PI-positive macrophages indicated increased cell death in the absence of Tec and Btk. To test whether the expression of pro- or anti-apoptotic genes is altered in Tec–/–Btk–/– macrophages, RNase protection assays were performed. The expression of most of the genes analyzed was similar between wild-type and Tec–/–Btk–/– macrophages (Fig. 4A); however, we observed that caspase-11 expression was induced in Tec–/–Btk–/– macrophages. Two isoforms of 43 and 38 kDa exist that can be processed to an active form of 30 kDa (15). Expressed and activated forms of caspase-11 could be detected by immunoblot analysis not only in Tec–/–Btk–/– but also in Tec–/– and Btk–/– single knockout macrophages. This indicates a link between Tec family kinases and the regulation of caspase-11 expression (Fig. 4B).


Figure 4
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  		FIGURE 4. Expression of caspase-11 in the absence of Tec or Btk. A, RNase protection assay using RNA isolated from day 10 cultures of wild-type (wt) and Tec–/–Btk–/– macrophages. One representative of two independent experiments is shown. B, Immunoblot analysis of wild-type (wt), Tec–/–, Btk–/– and Tec–/–Btk–/– macrophages showing caspase-11 expression. Actin was used as loading control. Expression data are representative of two independent experiments. {alpha}-, Anti- (antibody).

 
Tec–/–Btk–/– BMM survival can be rescued by M-CSF

The reduced survival of Tec–/–Btk–/– BMM indicates that culture conditions that allow the survival of wild-type BMM are not sufficient for Tec–/–Btk–/– BMM. This could be the result of a cell-intrinsic alteration. However, it is also possible that Tec–/–Btk–/– BMMs secret a toxic factor, a survival factor is missing, or a survival factor is depleted faster compared with wild-type cells. To distinguish between these possibilities, medium exchange experiments were performed. Daily replacement of macrophage culture medium (supplemented with LCM) led to a dense layer of Tec–/–Btk–/– macrophages (Fig. 5A, left panels) with normal macrophage numbers (Fig. 5B), indicating that the survival defect of Tec–/–Btk–/– macrophages is not caused by a cell autonomous process. Daily exchange of Tec–/–Btk–/– macrophage culture supernatant to wild-type cultures did not affect the survival of wild-type macrophages (Fig. 5, A, middle panels, and B). This argues against a toxic factor that is produced by Tec–/–Btk–/– macrophages or a faster depletion of a survival factor. Adding wild-type macrophage culture supernatant (i.e., without additional LCM) to Tec–/–Btk–/– cultures did not rescue the cell numbers of Tec–/–Btk–/– macrophages (Fig. 5, A, middle panels, and B), indicating that wild-type cells do not produce a survival factor that is missing in Tec–/–Btk–/– macrophages. As expected, daily supernatant replacement with a culture medium not supplemented with LCM led to a severe reduction in cell numbers for both wild-type and Tec–/–Btk–/– macrophages (Fig. 5A, right panels).


Figure 5
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  		FIGURE 5. Effects of M-CSF and GM-CSF on macrophage survival. A, Pictures depicting day 10 wild-type (wt; upper panels) and Tec–/–Btk–/– (lower panels) BMM cultures. The culture medium was replaced daily with new LCM-containing medium (daily + LCM; left panels), supernatant (SN) from Tec–/–Btk–/– (daily Tec–/–Btk–/– SN; upper middle panel) or wild-type (daily wt SN; lower middle panel) cultures, or medium not supplemented with LCM (daily – LCM; right panels). Original magnification was x100. B, Diagram showing macrophage numbers in day 10 cultures after medium exchange experiments shown in A. Error bars show SD. Data are representative of two independent experiments. SN ex, SN exchange between wt and Tec–/–Btk–/– cultures; + LCM, culture conditions in which LCM was added on days 1, 3, and 6. C, Diagram showing macrophage numbers in day 10 cultures after daily addition (from day 6 on) of either LCM or M-CSF (10 and 30 ng/ml). Error bars show SD. Data shown are representative of two independent experiments. D, Diagram showing macrophage numbers in day 10 cultures after addition (day 5) of increasing amounts of M-CSF (0, 15, 30, 60, and 90 ng/ml). Error bars show SD. Data show summary of two independent batches each performed in triplicates. The p values shown are as follows: ***, <0.0001; **, 0.029 (for 60 ng/ml M-CSF).

 
The supernatant exchange experiments indicated that daily addition of new culture medium that is supplemented with LCM rescues the survival defect of Tec–/–Btk–/– BMM. LCM is viewed mainly as a source of M-CSF. Therefore, we tested whether daily addition of M-CSF can rescue macrophage numbers in Tec–/–Btk–/– BM cultures. Macrophage numbers were restored in a dose-dependent manner to a similar extent in wild-type and Tec–/–Btk–/– macrophage cultures (Fig. 5C). Next, we tested whether a sufficiently high concentration of M-CSF can be identified that, if added at day 5, would yield similar numbers of wild-type and Tec–/–Btk–/– macrophages. Thus, increasing amounts of M-CSF were added at day 5 to wild-type and Tec–/–Btk–/– cultures. This led to a dose-dependent increase both in wild-type and Tec–/–Btk–/– macrophage numbers. Although at lower M-CSF concentrations the wild-type macrophage numbers were higher compared with Tec–/–Btk–/– cultures, at the highest M-CSF concentration the numbers were equal between wild-type and Tec–/–Btk–/– cultures (Fig. 5D). This indicated that similar numbers of Tec–/–Btk–/– macrophages compared with wild-type cells can be generated if the cells are generated under a sufficiently high concentration of M-CSF.

Impaired M-CSFR signaling in Tec–/–Btk–/– macrophages

Our data suggested that the M-CSFR signaling pathway is altered in Tec–/–Btk–/– macrophages. To test whether Tec family kinases are activated by M-CSFR stimulation, wild-type BMMs were stimulated with M-CSF for 1, 5, 10, and 30 min. M-CSF stimulation resulted in the activation of Btk, indicated by the phosphorylation of Y223 (Fig. 6A), a known autophosphorylation site of Btk (22).


Figure 6
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  		FIGURE 6. M-CSF receptor signaling in wild-type and Tec–/–Btk–/– macrophages. A, Immunoblot analysis of wild-type BMM (day 9) showing Btk activation upon M-CSF stimulation for various time points. pY223, Phosphotyrosine at position 223; {alpha}-, anti- (antibody). B, Immunoblot analysis showing the tyrosine phosphorylation of the M-CSFR (P-Y-M-CSFR) in wild-type (wt) and Tec–/–Btk–/– BMM (day 9) upon M-CSF stimulation (upper panel). Total M-CSFR levels are shown as control (lower panel). {alpha}-, Antibody. C, Immunoblot analysis showing surface (top row), internal (second row from top) and total (fourth row from top) expression levels of M-CSFR in wild-type (wt) and Tec–/–Btk–/– BMM (day 9) upon M-CSF stimulation. Actin was used as a loading control (third and fifth rows from top). {alpha}-, Anti- (antibody). D, Immunoblot analysis showing the tyrosine phosphorylation (P-Y) pattern of M-CSF stimulated wild-type (wt), Tec–/–, Btk–/–, Tec–/–Btk–/–, and Tec–/–Btk–/–Bmx–/– BMM (day 9). (A, B, and D) Actin was used as loading control. (A–D) The cell lysate equivalent of 2 x 106 cells was used for each immunoblot. Data are representative of two independent experiments. {alpha}, Anti- (antibody).

 
The M-CSFR showed normal tyrosine phosphorylation upon M-CSF stimulation in Tec–/–Btk–/– macrophages (Fig. 6B), and M-CSFR levels were similar in wild-type and the various knockout macrophages (Fig. 6C and data not shown). Daily addition of M-CSF rescued the cell numbers in Tec–/–Btk–/– BMM cultures (Fig. 5C). Thus, it is possible that the decrease in cell numbers in the Tec–/–Btk–/– BMM cultures was caused by lower levels of M-CSF due to a more rapid endocytosis of the M-CSFR and, therefore, an increased use of M-CSF in the absence of Tec and Btk. However, surface M-CSFR disappeared with a similar kinetic in wild-type and Tec–/–Btk–/– macrophages (Fig. 6C), indicating a similar turnover of the M-CSFR upon M-CSF stimulation. To investigate the activation of signaling pathways of Tec–/–, Btk–/–, Tec–/–Btk–/–, and Tec–/–Btk–/–Bmx–/– macrophages to M-CSF in more detail, the tyrosine phosphorylation pattern upon M-CSF stimulation was determined. Tec–/–Btk–/– and Tec–/–Btk–/–Bmx–/– macrophages displayed a changed tyrosine phosphorylation pattern as compared with wild-type, Tec–/– or Btk–/–cells (Fig. 6D). Tyrosine phosphorylation of proteins of ~110–130 kDa was reduced or absent in macrophages lacking both Btk and Tec.

Myeloid subset-specific regulation of GM-CSFR{alpha} expression by Tec and Btk

By performing RNase protection assays, we also noted that Csf2ra (the gene encoding GM-CSFR{alpha}) expression was reduced in Tec–/–Btk–/– macrophages as compared with wild-type cells (Fig. 7A). In contrast, Tec–/–Btk–/– and Tec–/–Btk–/–Bmx–/– BMDCs showed normal expression levels of GM-CSFR{alpha} as compared with wild-type cells (Fig. 7B), indicating Tec/Btk-dependent expression of the Csf2ra gene in BMMs but not in BMDCs. The reduced expression levels of Csf2ra were only observed in Tec–/–Btk–/– BMMs but not in Tec–/– or Btk–/– BMMs (data not shown). Unlike M-CSF, which upon addition restored cell numbers in Tec–/–Btk–/– BMM cultures to wild-type levels (Fig. 5D), the addition of GM-CSF restored cell numbers in Tec–/–Btk–/– BMM cultures only partially (Fig. 7C). The reduced expression of GM-CSFR{alpha} in the absence of Tec and Btk, however, may explain why the cell numbers upon GM-CSF addition were always lower in Tec–/–Btk–/– cultures compared with wild-type cultures (Fig. 7C). Culture of BM cells with GM-CSF instead of M-CSF leads to the generation of a confluent layer of adherent macrophages and the cogeneration of loosely attached CD11c+ BMDCs (23). The macrophage layer was observed in GM-CSF cultures (day 10) of wild-type but not Tec–/–Btk–/–Bmx–/– BM cells (Fig. 7D). However, the generation of BMDCs was not affected, because similar numbers of wild-type and Tec–/–Btk–/–Bmx–/– BMDCs developed in the presence of GM-CSF (Fig. 7E).


Figure 7
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  		FIGURE 7. Reduced expression of GM-CSFR{alpha} in Tec–/–Btk–/– BMM. A, RNase protection assay using RNA isolated from day 10 cultures of wild-type (wt) and Tec–/–Btk–/– macrophages. One representative of two independent experiments is shown. B, Semiquantitative RT-PCR showing expression of Csf2Ra in BMDCs of the indicated genotype. Hprt expression was used as input control. Data are representative of two independent experiments. wt, Wild type. C, Diagram showing macrophage numbers in day 10 cultures after daily addition (from day 6 on) of GM-CSF (500 and 1500 U/ml). daily + LCM, Culture conditions in which LCM was added daily; –LCM, control cultures without LCM. Error bars show SD. Data shown are representative of two independent experiments. The p values shown are as follows: *, 0.0168; **, 0.0017. Wt, wild type. D, Pictures depicting adherent GM-CSF-generated wild-type (wt, upper panel) and Tec–/–Btk–/–Bmx–/– (lower panel) BMMs in day 10 GM-CSF cultures (after removal of nonadherent DC). E, Diagram showing the relative number of wild-type (wt) and Tec–/–Btk–/–Bmx–/– BMDCs. BM cells were differentiated with GM-CSF and nonadherant BMDCs were counted at day 10. The summary of three different experiments, each performed in duplicate, is shown. The percentages of Tec–/–Btk–/–Bmx–/– BMDC cell numbers compared with wild-type (wt) BMDC were 147, 108, and 75% for experiments 1, 2, and 3, respectively. The absolute cell numbers (x105) were 4.6 ± 1.2 (wild type) and 6.8 ± 0.9 (Tec–/–Btk–/–Bmx–/–, Tec–/–Btk–/–Bmx–/–) for experiment 1, 8.6 ± 1.0 (wild type) and 9.3 ± 2.6 (Tec–/–Btk–/–Bmx–/–) for experiment 2, and 6.3 ± 1.5 (wt) and 4.7 ± 0.9 (Tec–/–Btk–/–Bmx–/–) for experiment 3.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we performed a genetic approach to investigate in detail the role of Tec family kinases in murine macrophages. We observed reduced survival rates of Tec–/–Btk–/– bone marrow-derived macrophages. A severe drop in macrophage numbers correlating with increased numbers of dead cells occurred in the absence of Tec and Btk. Elucidation of M-CSFR signaling pathways revealed an impaired total tyrosine phosphorylation pattern in Tec–/–Btk–/– macrophages upon M-CSF stimulation despite normal expression and phosphorylation of the M-CSFR. Thus, our data provide a novel link between Tec family kinases and M-CSF receptor signaling pathways that regulate macrophage survival. Finally, Tec and Btk are required for the proper expression of GM-CSFR{alpha} in macrophages but not in dendritic cells, implicating Tec kinases in the lineage-specific regulation of GM-CSFR{alpha} expression.

The generation of BMMs as assessed by surface marker expression was not influenced by the absence of Tec and Btk. This was also reflected by similar macrophage numbers on day 6 in wild-type and Tec–/–Btk–/– cultures. Therefore, Tec and Btk are not required for the differentiation of precursor cells into macrophages. The drop in cell numbers starting after day 6 could either be caused by reduced proliferation or reduced survival of differentiated macrophages. Because BrdU incorporation was the same in wild-type and Tec–/–Btk–/– macrophages, it is likely that Tec and Btk regulate macrophage numbers by promoting macrophage survival. This is supported by the observation that the drop in cell numbers correlated with an increase in PI-positive cells and also by the occurrence of a sub-N2 population in Tec–/–Btk–/– macrophages as revealed by DNA content analysis (data not shown). Tec family kinases have already been implicated in regulating cell survival and/or apoptosis in other cell lineages (24, 25, 26) and in the apoptosis of macrophages after stimulation (9). Our findings indicate that Tec and Btk also regulate cell survival of BMM. Neither Tec nor Btk single-deficient BMM displayed a survival defect. Thus, our data also indicate redundant activities of Tec and Btk during macrophage generation, similar to the murine B cell lineage where Tec and Btk are required for proper B cell development (14).

M-CSF is a crucial cytokine required for the differentiation, proliferation, and survival of macrophages and is often provided in cultures of BM-derived macrophages with LCM (18). M-CSF starvation in macrophage cultures induces apoptosis (27). The observation that high levels of M-CSF (by daily addition of new LCM) can rescue Tec–/–Btk–/– macrophage numbers suggested that there are differences in the use of M-CSF due to an increased internalization of the M-CSFR or an impairment of M-CSFR signaling or both. However, because the kinetic of the internalization of the M-CSFR upon M-CSF stimulation was similar in wild-type and Tec–/–Btk–/– macrophages, these data point toward a M-CSFR signaling defect in the absence of Tec and Btk. This is supported by the observation that the tyrosine phosphorylation pattern upon M-CSF stimulation was changed in Tec–/–Btk–/– macrophages despite normal expression and phosphorylation of the M-CSFR. These data indicate that Tec family kinases are required for the proper transmission of M-CSF signals in macrophages. Thus, suboptimal concentrations of M-CSF that still allow macrophage survival in wild-type cells do not induce a sufficient signal to allow survival of Tec–/–Btk–/– macrophages. However, higher M-CSF concentrations (due to daily replacement of M-CSF) presumable provide a stronger (i.e., above "threshold") signal to Tec–/–Btk–/– macrophages that rescues the survival defect. Thus, macrophages use Tec family kinases for proper "sensing" of M-CSF levels. In support of this hypothesis, we also observed a dose-dependent rescue of Tec–/–Btk–/– macrophage numbers that can reach wild-type cell numbers if sufficiently high amounts of M-CSF are added at day 5. Activation of Tec family kinases is a process involving their localization to the plasma membrane followed by a Src family kinase-mediated phosphorylation of a tyrosine residue in the activation loop of the kinase domain. Full activation of Tec family kinases is achieved after a subsequent autophosphorylation of a tyrosine residue in the Src homology 3 (SH3) domain of Tec kinases (28, 29). Membrane recruitment is mediated by the interaction of the pleckstrin homology domain of Tec family kinases with phosphatidylinositol 3, 4, 5-trisphosphate (PIP3), generated by PI3K activity (30). Interestingly, it has been shown that PI3K is required for macrophage survival (31), thus further supporting our finding of a novel link between Tec family kinases and M-CSFR signaling pathways that regulate macrophage survival. In a total phosphotyrosine blot, some tyrosine-phosphorylated bands of ~110–130 kDa are missing upon M-CSF stimulation in Tec–/–Btk–/– macrophages compared with wild-type cells. In preliminary experiments no difference could be observed in the phosphorylation status of several known signaling components of the M-CSFR pathway including SHIP1, Erk1,2, AKT, p38, JNK, and Erk5 (data not shown). Therefore, additional experiments including proteomic approaches are required to reveal the molecular nature of these factors that are not properly tyrosine phosphorylated in Tec–/–Btk–/– macrophages.

Another finding of our study was that caspase-11 is expressed in Tec family kinase-deficient BMM. Caspase-11 can act as an upstream caspase for caspase-1 in inflammation and for caspase-3 in apoptosis (32). In contrast to other caspases, caspase-11 is generally not expressed in cells and tissues under homeostatic conditions (15). However, expression can be induced by stimuli such as LPS, systemic inflammation, or ischemic brain injury (33). Expression of caspase-11 in macrophages depends on NF-{kappa}B and STAT1 (34), p38 MAPK (35), and the transcription factor CHOP, a C/EBP family transcription factor (36). CHOP is implicated in endoplasmatic reticulum stress-mediated apoptosis, providing a link between stress response and caspase-11 expression. However, we consider it unlikely that the up-regulation of caspase-11 expression in Tec family kinase-deficient macrophages is linked with the increase in cell death, because caspase-11 is expressed in Tec–/–Btk–/–macrophages on day 10 when there are no differences in the percentage of PI-positive cells between wild-type and Tec/Btk-deficient cells. Furthermore, the observation that Tec–/– or Btk–/– macrophages show caspase-11 expression also argues against a direct link between caspase-11 and the drop in macrophage numbers. However, caspase-11 expression might indicate a stress response in Tec- or Btk-deficient macrophages. Finally, we noted that Tec and Btk are required for the expression of GM-CSFR{alpha} in BMMs but not in BMDCs, implicating Tec family kinases in the lineage-specific regulation of GM-CSFR{alpha} expression. This may also explain why the addition of GM-CSF does not rescue macrophage numbers to the same extent in Tec–/–Btk–/– cultures as compared with wild-type cultures. However, it remains possible that Tec and Btk are, in addition, also required for proper GM-CSFR signaling.

Taken together, our analysis showed that Tec and Btk are crucially involved in macrophage survival by M-CSFR signaling. Our study provides a novel link between Tec family kinases and M-CSFR signaling as well as with the regulation of caspase-11 and GM-CSFR{alpha} expression. Future in vivo studies addressing the roles of Tec and Btk in inflammation and infection will be of interest because both caspase-11 and GM-CSF are already implicated in these processes.


   Acknowledgments
 
We thank Dr. Thomas Decker for providing L929 cells and Drs. Mathias Müller and Herbert Strobl for critical reading of the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by the START program (Grant Y-163) of the Fonds zur Förderung der Wissenschaftlichen Forschung and the Austrian Ministry of Education, Science and Culture (to W.E.), by the K-Plus Competence Center for Biomolecular Therapeutics (to W.E.), by the Sonderforschungsbereich project F2305-B13 of the Austrian Research Fund (to W.E.), and by a postdoctoral fellowship (to U.S.) from the Deutsche Forschungsgemeinschaft (Schm 2128/1-1). Back

2 M.M. and B.U. contributed equally to this work. Back

3 Current address: Nabriva Therapeutics, Brunnerstrasse 59, 1235 Vienna, Austria. Back

4 Address correspondence and reprint requests to Dr. Wilfried Ellmeier, Institute of Immunology, Medical University Vienna, Center for Physiology, Pathophysiology and Immunology, Lazarettgasse 19, A-1090 Vienna, Austria. E-mail address: wilfried.ellmeier{at}meduniwien.ac.at Back

5 Abbreviations used in this paper: BM, bone marrow; BMDC, BM-derived dendritic cell; BMM, BM-derived macrophage; HPRT, hypoxanthine phosphoribosyltransferase; LCM, L929 cell-conditioned medium; M-CSFR, M-CSF receptor; PI, propidium; xid, X-linked immunodeficient (gene); XLA, X-linked agammaglobulinemia. Back

Received for publication August 7, 2007. Accepted for publication April 10, 2008.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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