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Hck tyrosine kinase activity modulates tumor necrosis factor production by murine macrophages.

The hematopoietic cell kinase (hck) is a member of the src family of tyrosine kinases, and is primarily expressed in myeloid cells. Hck expression increases with terminal differentiation in both monocyte/macrophages and granulocytes and is further augmented during macrophage activation. Recent evidence has implicated src-related tyrosine kinases in critical signaling pathways in other hematopoietic lineages. Herein we demonstrate that manipulation of the level of hck expression in the murine macrophage cell line BAC1.2F5 alters the responsiveness of these cells to activation by bacterial lipopolysaccharide (LPS) but does not affect survival or proliferation. Overexpression of an activated mutant of hck in BAC1.2F5 cells augments tumor necrosis factor (TNF) production in response to LPS, whereas inhibition of endogenous hck expression, by antisense oligonucleotides, interferes with LPS-mediated TNF synthesis. Together, these observations suggest that hck is an important component of the signal transduction pathways in activated macrophages.


Summary
The hematopoietic cell kinase (hck) is a member of the src family of tyrosine kinases, and is primarily expressed in myeloid cells. Hck expression increases with terminal differentiation in both monocyte/macrophages and granulocytes and is further augmented during macrophage activation. Recent evidence has implicated src-related tyrosine kinases in critical signaling pathways in other hematopoietic lineages. Herein we demonstrate that manipulation of the level of hck expression in the murine macrophage cell line BAC1.2F5 alters the responsiveness of these cells to activation by bacterial lipopolysaccharide (LPS) but does not affect survival or proliferation. Overexpression of an activated mutant of hck in BAC1.2F5 cells augments tumor necrosis factor (TNF) production in response to LPS, whereas inhibition of endogenous hck expression, by antisense oligonucleotides, interferes with LPS-mediated TNF synthesis. Together, these observations suggest that hck is an important component of the signal transduction pathways in activated macrophages. E xposure of monocytes and macrophages to bacterial LPS triggers a series of biochemical and functional changes including the activation of protein kinase C (1), tyrosine phosphorylation of several proteins (2), production of reactive oxygen metabolites (3), and production and secretion of cytokines with critical roles in host defense (1,4,5). TNF is an important monokine with pleiotropic effects on the host inflammatory response, including the induction of fever, direct antiviral and antitumor activity, and augmentation of monocyte, granulocyte, and lymphocyte function (for a review see reference 5).
Expression of the hematopoietic cell kinase (hck) 1 tyrosine kinase is essentially limited to cells of monocyte/macrophage and granulocyte lineages (6)(7)(8)(9), increases with differentiation along either the macrophage or granulocyte pathways (7,10,11), and is further augmented by activation stimuli including LPS in mature monocytes and macrophages (9,10,12). This pattern of expression suggested a potential role for hck in the terminal differentiation of macrophages and/or in the activation pathways of mature monocytes and macrophages. Several lines of evidence support a role for other members of the src family of kinases in signal transduction pathways of lymphocytes, mast cells, and basophils (13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23).
To study the possible role of hck in macrophage activation 1 Abbreviations used in this paper: hck, hematopoietic cell kinase; LCM, L cell conditioned medium. and cytokine gene expression, we have manipulated the level of expression of hck in the murine macrophage cell line, BAC1.2F5. BAC1.2F5 cells require CSF-1 or GM-CSF for maintenance of viability and proliferation, grow as an adherent monolayer, and morphologically resemble normal macrophages (24)(25)(26). These cells express Ia antigen, possess Fc receptors, engage in Fc receptor-mediated phagocytosis, and produce cytokines in response to LPS (24)(25)(26). In this report, we show that BAC1.2F5 cells express hck mKNA, protein and kinase activity, and that hck expression and kinase activity are augmented by activation stimuli (e.g., LPS) and by growth factors (e.g., CSF-1 and GM-CSF). Furthermore, we demonstrate that manipulation of the level of hck kinase activity in BAC1.2F5 cells alters the response of these cells to LPS but does not affect viability or proliferation. Inhibition of endogenous hck expression, with antisense oligonucleotides, leads to diminished LPS-mediated TNF production in BAC1.2F5 cells, whereas constitutive expression of an activated mutant of hck (p59 hckrS~ in BAC1.2F5 cells augments TNF production in response to LPS.

Materials and Methods
Materials. DMEM, t-glutamine, penicillin, streptomycin, G418 (Geneticin), and herbimycin A were purchased from GIBCO (Grand Island, NY). FCS was obtained from HyClone Laboratories, Inc. (Logan, UT). LPS purified from Escherichia coli strain 0111:B4 was purchased from Sigma Chemical Co. (St. Louis, MO). L cell con-ditioned medium (LCM) was prepared as described by Stanley and Heard (27) and was used as the source of CSF-1 for routine growth and maintenance of the BAC1.2F5 cell line and subclones. Recombinant human CSF-1 was obtained from the Genetics Institute (Cambridge, MA) and recombinant murine GM-CSF was obtained from Amgen, Inc. (Thousand Oaks, CA).
RNA Isolation and Northern Blot Analysis. Total cellular RNA was isolated from BAC1.2F5 cells by the guanidinium isothiocyanate/cesium chloride method and quantitated spectrophotometricall), as described (31). For blots, 20 #g RNA was electrophoresed on 2.2 M formaldehyde, 1% agarose gds, transferred to Nytran membranes, UV irradiated, and baked, as described (32). Blots were hybridized overnight with 106 dpm ml-t of 32p-labeled murine or human hck cDNA probes or a routine TNF cDNA probe, washed twice with 3x SSC, 0,1% SDS, and twice with 0.2x SSC, 0.1% SDS at 42~ then autoradiographed at -70~ in the presence of two enhancing screens.
In Vitro Kinase Assays. For in vitro kinase assays, PBS-washed cells were lysed in 2% NP-40 and TEN (50 mM Tris-HC1, pH 7.4, 1 mM EDTA, 0.15 M NaC1). Lysates were incubated with the rabbit polyclonal anti-hck antiserum and immunocomplexes were collected with protein A-agarose. Immunoprecipitated proteins were suspended in 10 #1 kinase buffer (containing 50 ram Pipes, pH 7.0, 1 mM MnC1, 1 mM Na3VO4, and 10 #Ci of'y-[32p]ATP) and incubated at room temperature for 20 rain as described (33). In some experiments, denatured rabbit muscle enolase was added as an exogenous substrate, as described (34). Products of the kinase reactions were analyzed by SDS-PAGE.
Determination of TNF Concentrations. TNF protein levels were measured with a solid-phase sandwich ELISA specific for murine TNF-c~, as specified by the manufacturer (Genzyme Corp., Cambridge, MA).

Results and Discussion
BAC1.2F5 cells express hck mKNA ( Fig. 1 a), immunoreactive p59 hc~ protein (Fig. 2 a), and hck kinase activity (Fig.  2, b and c). Hck m R N A levels increase three-to sixfold within 1-4 h of exposure of quiescent BAC1.2F5 cells to growth factors (CSF-1 and GM=CSF) and activating stimuli including LPS (Fig. 1 a); hck protein expression parallels the accumulation of hck m K N A (Fig. 2 a). The upregulation of hck expression by growth factors and LPS in BAC1.2F5 cells is similar to that reported in human monocyte-derived macrophages (9) and murine bone marrow-derived monocytes and macrophages (10,12).
Exposure of quiescent BAC1.2F5 cells to CSF-1, LPS (Fig.  2, b and c), and GM-CSF (data not shown) also promptly augments hck kinase activity, as measured both by autophosphorylation kinase reactions (Fig. 2 b) and trans-phosphorylation reactions with denatured rabbit muscle enolase serving as a substrate (Fig. 2 c). At optimal concentrations of CSF-1 and LPS, the kinetics of induction of augmented hck kinase activity by these stimuli are very similar (Fig. 3), with some increase in kinase activity observed as early as 5 min after stimulation and maximal kinase activity detected by 10-20    cells. BAC1.2F5 cells were incubated overnight in the absence of growth factors, then stimulated for 10 min with LPS 1 ~g ml -1. Cells were lysed, lysates subjected to immunoprecipitation with a rabbit polyclonal antiserum specific for tick, immunoprecipitates collected with protein A-agarose, and a t~ansphosphorylation in vitro kinase assay was performed in the presence of exogenous denatured rabbit muscle enolase, as described in Materials and Methods. The band corresponding to rabbit muscle enolase ('~45 kD) was cut out of the gel, solubilized, reacted with trypsin, and analyzed by thin-layer chromatography to determine the content ofphosphotyrosine (Y), phosphoserine (S), and phosphothreonine (T), as described in Materials and Methods. min after stimulation. Phosphoamino acid analysis confirmed that this in vitro kinase activity resulted in phosphorylation of enolase exclusively on tyrosine residues (Fig. 4). LPS and IFN-3' have also been reported to provoke increased hck kinase activity in murine bone marrow-derived macrophages (12), but the effect was noted only after more than 6 h of stimulation and may have primarily reflected increased steadystate levels of p59 hck protein, rather than augmented kinase activity per se.
Unstimulated BAC1.2F5 cells contain little or no T N F m K N A (Fig. 5 a) and do not secrete detectable amounts of T N F protein (data not shown). In response to LPS (but not CSF-1 or GM-CSF; data not shown), BAC1.2F5 cells accumulate large amounts of T N F ml~NA and secrete T N F protein (Fig. 5).
To determine if p59 h~ was an important signaling molecule in activated macrophages, we employed complementary strategies to modulate hck kinase activity in BAC1.2F5 cells.
Second, we used antisense oligonucleotides specific for mufine hck to inhibit endogenous p59 ~'~ expression in BAC1.2F5 cells. We also examined the effect of an inhibitor of tyrosine kinase activity, herbimycin A, on the activation requirements of these cells.
Total RNA was isolated from the indicated cells and analyzed by Northern blotting with a murine TNF cDNA probe. (b) TNF secretion by BAC1.2F5 cells and subclones expressing vector alone (LNSL7), human kinase-negative p59 h' kE269, and human activated kinase, p59 hCkFs01. Cells were cultured in 6-well tissue culture plates in medium containing LCM as a source of CSF-1, and were exposed to LPS 1 #g ml-1 for 16 h. Supernatants were collected and stored at -70~ until analysis. TNF protein levels were determined by use of a solid-phase sandwich ELISA (Genzyme Corp.). Data represent means of three experiments _+SD.
expressing either human or murine p59 hckFs~ accumulated 5-20-fold more T N F m K N A (Fig. 5 a) and secreted twoto four-fold more T N F protein (Fig. 5 b) than did parental cells, clones expressing vector alone (LNSL7), or clones expressing human kinase-defective p59 ~'kE269. In the absence of LPS, BAC1.2F5 clones expressing p59 ~kFs~ accumulated appreciable T N F m R N A (Fig. 5 a) but produced little or no immunoreactive T N F (data not shown).
In parallel experiments, we employed antisense oligonucleotides to study the effects of inhibiting endogenous p59 hck expression in BAC1.2F5 cells. BAC1.2F5 cells were exposed to either sense or antisense hck oligonucleotides at concentrations varying from 1-30 # M for intervals ranging from 24 h to 4 wk. Exposure of BAC1.2F5 cells to hck antisense oligonucleotides for 72 h resulted in a significant reduction (two-to five-fold) in the expression of p59 hck protein (Fig.  6 a) and kinase activity, as measured by autophosphorylation (Fig. 6 b) and trans-phosphorylation (Fig. 6 c) kinase assays, whereas exposure of the cells to the sense control oligonucleotide had no effect.
BAC1.2F5 cells exposed to hck antisense oligonucleotides 72 h, then exposed to LPS 100 ng ml-1 for 10 min. Lysates were prepared and trans-phosphorylation kinase assays performed with denatured rabbit muscle enolase as a substrate, as described in Materials and Methods. (d) TNF secretion by Bacl.2F5 cells exposed to hck-specific oligonucleotides. Bacl.2F5 cells were cultured in 6-well plates for 72 h in the presence of medium alone (with LCM as a source of CSF-1) or in the presence of 10 #M concentrations of sense or antisense hck oligonucleotides, then exposed to LPS 250 ng ml-1 (solid bars) or LPS 1 #g ml-1 (open bars) for 16 h. Supernatants were collected and frozen at -70~ until analyzed. TNF concentrations were quantitated by use of a solid-phase antibody sandwich ELISA. Data represent means of three experiments _+SD. accumulated significantly less T N F m R N A (25-50% of control, data not shown) and produced significantly less T N F protein (30-35% of control, Fig. 6 d) in response to LPS than did untreated cells or cells exposed to an equimolar concentration of the sense oligonucleotide control. In parallel experiments, exposure of BAC1.2F5 cells to sense or antisense Herbimycin A inhibits TNF production by BAC1.2F5 cells and subclones expressing p59 h'kFs~ BAC1.2F5 cells and subdones expressing human p59 ~kFs~ were exposed to herbimycin A at the indicated concentrations for 4 h, then stimulated with LPS 250 ng ml-1 for 16 h. Supernatants were collected and stored at -70~ until analyzed. TNF concentrations were quantitated by use of a solid-phase antibody sandwich ELISA.
oligonudeotides corresponding to the analogous seven codons of murine lira kinase (33) had no effect on TNF production by BAC1.2F5 cells, which also express p56tr" (data not shown). Proliferation of BAC1.2F5 cells in response to CSF-1 was not affected by exposure to hck sense or antisense oligonucleotides (data not shown).
The effect of hck antisense oligonucleotides on TNF produc-tion by BAC1.2F5 cells was comparable with that observed with preincubation of these cells with the tyrosine kinase inhibitor herbimycin A. Pretreatment of BAC1.2F5 parental cells and subclones overexpressing p59 ~kFs~ with herbimycin A inhibited LPS-stimulated TNF production by these cells in a dose-dependent manner, leading to a maximal four-to five-fold reduction in TNF secretion at optimal concentrations (Fig. 7). Weinstein, et al. (2), have reported that herbimycin A blocks the LPS-stimulated release of arachidonic acid metabolites in the murine RAW 264.7 macrophage cell line, and herbimycin A also inhibits LPS-induced TNF production by human alveolar macrophages (Beatty, C., and C. B. Wilson, personal communication). These experiments provide the first direct evidence for a functional role for the hck tyrosine kinase and suggest that p59 hck is an integral component of the signaling pathways involved in macrophage activation and TNF production. Whereas other stimuli (e.g., CSF-1 and GM-CSF) also upregulate hcle expression and kinase activity, the results of our experimental manipulation of hck expression in BAC1.2F5 cells suggest that p59 ~ does not play a critical role in the mitogenic responses to those stimuli. An improved understanding of the role of p59 hc~ in macrophage signaling pathways will require identification of associated cell surface molecules and substrates of the hck kinase.