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IL-2 Signaling in Human Monocytes Involves the Phosphorylation and Activation of p59^{hck 1}

Maria C. Bosco^*, Rafael E. Curiel, Arnold H. Zea, Maria G. Malabarba^§, John R. Ortaldo^¶ and Igor Espinoza-Delgado^2,

^* Laboratory of Molecular Biology, Giannina Gaslini Institute, Genova Quarto, Italy; Departments of Medicine and Microbiology and Stanley S. Scott Cancer Center, Louisiana State University Medical Center, New Orleans, LA 70112; and Laboratories of ^§ Immunoregulation and ^¶ Experimental Immunology, Cytokines Molecular Mechanisms Section, National Cancer Institute-Frederick Cancer Research and Development Center, Frederick, MD 21702

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The activating properties of IL-2 and the structure of the IL-2R on human monocytes are well characterized. However, relatively little is known about the biochemical mechanisms involved in IL-2 signal transduction in these cells. We investigated the role of protein tyrosine kinases (PTKs) in the activation of monocytes by IL-2. Incubation of monocytes with the PTK inhibitor herbimycin A (HA) resulted in the dose-dependent suppression of IL-2-induced monocyte tumoricidal activity. This inhibition was rather potent, as a concentration of HA as low as 0.5 µM caused a complete abrogation of cytolytic activity. Furthermore, HA markedly suppressed the ability of IL-2 to induce IL-1ß, TNF-, IL-6, and IL-8 mRNA expression and protein secretion by monocytes. Anti-phosphotyrosine immunoblotting demonstrated that IL-2 induced a rapid and time-dependent increase in tyrosine phosphorylation of several cellular proteins of molecular masses ranging from 35 to 180 kDa. Interestingly, IL-2 caused a significant up-regulation of the constitutive levels of hck PTK mRNA and protein relative to medium-treated cells as well as an increase in p59^hck tyrosine phosphorylation. Finally, we demonstrated by in vitro kinase assay that the specific activity of p59^hck PTK was also induced by IL-2 in monocytes. Thus, these data show that the activation of PTKs is required for the triggering of monocyte effector and secretory functions by IL-2 and strongly suggest that p59^hck is a key participant in IL-2 signaling in human monocytes.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The IL-2R complex has been extensively characterized in T lymphocytes and is now known to comprise at least three distinct, noncovalently associated, and independently regulated membrane components, the -chain (IL-2R), the ß-chain (IL-2Rß), and the -chain (IL-2R). These components can be expressed in various combinations, resulting in receptors with different affinities for IL-2 as well as different functional attributes (1, 2). Previous studies have demonstrated that both the IL-2Rß and IL-2R subunits are required for the formation of functional IL-2Rs and for IL-2 intracellular signaling, whereas the IL-2R chain is endowed with IL-2 binding but is devoid of signal-transducing capabilities (3). Even though none of the IL-2R components possesses any intrinsic catalytic activity (1, 2), one of the earliest biochemical events observed after T lymphocyte stimulation by IL-2 is the increased tyrosine phosphorylation of several cellular proteins and the subsequent induction of nuclear proto-oncogenes critical for cellular proliferation (1), thus suggesting the activation of cytoplasmic IL-2R-coupled protein tyrosine kinases (PTKs).³ In this regard, evidence for the physical and functional association of PTK activity with the cytoplasmic domains of the ß- and -chains has been reported in lymphoid cells, and significant advances have recently been made in identifying the multiple signaling molecules that specifically interact with the IL-2R subunits (1, 3, 4). IL-2 binding to functional IL-2Rs on T cells leads to the recruitment and activation of distinct nonreceptor PTKs, such as p56^lck (5, 6) of the src family PTKs, Syk PTK of the Syk/ZAP-70 family (7), and JAK1 and JAK3 of the Janus kinase (JAK) family (8, 9, 10, 11). Specifically, p56^lck, Syk, and JAK1 couple with the cytoplasmic domain of the IL-2 ß-chain (5, 7, 8, 11, 12), whereas JAK3 associates with the cytoplasmic region of the IL-2R (8, 9, 10, 11).

Although originally identified as a T cell growth factor, IL-2 was later shown to exert a wide range of biological effects on several other cell types. Functional IL-2Rs have been found on B cells (13, 14, 15), NK cells (3), polymorphonuclear cells (16, 17, 18), and monocytes/macrophages (18, 19, 20, 21). We and others have previously shown that IL-2 is a powerful activator of human monocytes (19, 22). Monocyte stimulation with IL-2 leads to the secretion of several cytokines (23, 24, 25, 26, 27) and growth factors (28, 29, 30); to the expression of growth factor receptors (19, 21) and adhesion and costimulatory molecules (I. Espinoza-Delgado, S. Rottshafer, R. E. Curiel, and M. C. Bosco, manuscript in preparation); and to the enhanced production of hydrogen peroxide, superoxide, PGE₂, and thromboxane B₂ (20, 31). Furthermore, IL-2 can activate fresh human monocytes to exert microbicidal (20) and tumoricidal activities (19, 32) and can potentiate their Ag-presenting ability (I. Espinoza-Delgado et al., manuscript in preparation). Fresh peripheral blood monocytes constitutively express the IL-2Rß and IL-2R chains (19, 33, 34, 35), but not the IL-2R subunit, which is inducible by stimulation with IFN- (18, 20, 21, 36) or LPS (20, 37). IL-2, on the other hand, can up-regulate the expression of the ß-chain (21) and -chain (34), but is unable to induce the subunit (20, 21).

Although a large body of information is available on the biological effects of IL-2 and the expression and regulation of the IL-2R components on human monocytes, the biochemical mechanisms involved in IL-2 signal transduction in these cells have not yet been elucidated. There is increasing evidence that tyrosine phosphorylation is important in monocyte functions (38), and different members of the src family PTKs, such as hck, fgr, and lyn, have been shown to be critical components of the signal transduction pathway of several monocyte/macrophage-activating factors (39, 40, 41, 42, 43), thus raising the possibility that PTK activation could also be involved in mediating monocyte responses to IL-2. However, because the expression of Lck is restricted to T lymphocytes and NK cells (38) and because cells of the monocytic lineage do not express JAK3 constitutively (39, 44), early IL-2 signaling in monocytes should occur via PTKs distinct from those required for regulating T cell functions. The present study was designed to explore the role of PTKs in the activation of human monocytes by IL-2 and to investigate whether a monocyte/macrophage-specific PTK is coupled to the IL-2 signaling pathway in human monocytes. We report that PTK activation is required for the induction of monocyte effector and secretory functions by IL-2, and that IL-2 can specifically affect the expression, phosphorylation, and activation of p59^hck kinase in human monocytes.

	Materials and Methods

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Monocyte isolation and culture conditions

Peripheral blood leukocytes were obtained from normal healthy volunteers by leukapheresis using a Fenwell CS-3000 blood cell separator (Fenwell, Deerfield, IL). Mononuclear cells were separated by density gradient centrifugation on lymphocyte separation medium (Organum Teknika, Durham, NC), and then purified in suspension from the unfractionated mononuclear leukocyte preparation by countercurrent centrifugal elutriation in a Beckman JE-6 elutriation chamber and rotor system (Beckman, Palo Alto, CA) as described previously (45). The purity of monocyte preparations were 94 ± 3%, as assessed by morphology on Giemsa-stained cytocentrifuge slide preparations and by flow cytometry using the monocyte-specific mAb Leu M3 (Becton Dickinson, Mountain View, CA). Other cells present in the monocyte preparations were as follows: 2–5% basophils, 1–2% lymphocytes, 1% neutrophils, and <1% large granular lymphocytes. Viability, as determined by trypan blue exclusion test, was >99%. Monocytes were cultured in RPMI 1640 (BioWhittaker, Walkersville, MD), supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, 2 mM glutamine, 20 mM HEPES (Life Technologies, Grand Island, NY), and 10% heat-inactivated FBS (HyClone, Logan, UT).

Cytokines and reagents

Highly purified rIL-2 from Escherichia coli (sp. act., 18 x 10⁶ IU/mg; 1 Chiron unit corresponds to 6 IU; LPS content, <0.6 pg/ml) was provided by Chiron (Emeryville, CA). Human rIFN- (sp. act., 2.02 x 10⁷ IU/mg) was provided by Dr. Michael Shepard (Genentech, San Francisco, CA). The PTK inhibitor herbimycin A (HA) was purchased from Life Technologies and/or was a gift from Dr. Satoshi Omura (Kitaato Mimato-Ku, Tokyo, Japan); it was prepared as a 1.75-mM stock solution in DMSO (Fisher Scientific, Pittsburgh, PA). ATP disodium salt was purchased from Sigma (St. Louis, MO). Special care was taken to ensure endotoxin-free conditions in all the experiments, and all reagents were demonstrated to be endotoxin free by the Limulus amebocyte lysate test (M. A. Bioproducts, Walkersville, MD; sensitivity, 0.06 IU/ml).

Northern blot analysis

Monocytes were cultured for the indicated time points in 15-cm Lux plates (Miles Scientific, Wapersville, CA) at 2 x 10⁶ cells/ml in the presence of IL-2 or HA, alone or in combination. Cells were then lysed in Trizol (Life Technologies), and total RNA was purified according to the manufacturer’s instructions. Twenty micrograms of total RNA from each sample was electrophoresed under denaturing conditions on a 1.2% agarose gel containing 2.2 M formaldehyde, blotted onto Nytran membranes (Schleicher & Schuell, Keene, NH), and cross-linked by UV irradiation. Membranes were prehybridized at 42°C in Hybrisol solution (Oncor, Gaithersburg, MD) and hybridized overnight with 2 x 10⁶ cpm/ml of an -³²P-labeled probe. Membranes were then washed three times at room temperature for 10 min each time in 2x SSC-0.1% SDS, and twice at 60°C for 15 min each time in 0.2x SSC-0.1% SDS before being autoradiographed using Kodak XAR-5 films (Eastman Kodak, Rochester, NY) and intensifying screens at -80°C. Probes were labeled by random priming reaction using a commercial kit (Roche, Indianapolis, IN) and [-³²P]dCTP (3000 Ci/mmol; Amersham, Arlington Heights, IL). The sp. act. was always >10⁹ cpm/µg. The following cDNAs were used as probes and were provided by each of the respective researchers listed below: human IL-8 full-length cDNA by Dr. K. Matsushima (Kanazawa University Cancer Institute, Kanazawa, Japan), human IL-1ß full-length cDNA by Dr. D. Carter (Upjohn Pharmacia, Kalamazoo, MI), the 900-bp PstI fragment of the human IL-6 cDNA (46), human TNF- cDNA by Dr. S. A. Nedospasov (Institute of Molecular Biology, Academy of Sciences of Russia, Moscow, Russia), and hck cDNA by Dr. Zack Howard (Laboratory of Molecular Immunoregulation, Division of Basic Sciences, National Cancer Institute, National Institutes of Health). The human GAPDH probe was purchased from Clontech (Palo Alto, CA).

Detection of cytokine release

Monocytes were cultured in 15-cm Lux plates at 2 x 10⁶ cells/ml and were stimulated for 18 h with the indicated factors. At the end of the incubation period, cell-free supernatants were harvested and assayed for IL-1ß, TNF-, IL-6, and IL-8 activity, using an IL-6-specific ELISA from BioSource (Camarillo, CA) and IL-1ß-, TNF--, and IL-8-specific ELISAs from R&D System (Minneapolis, MN), according to the manufacturer’s instructions.

Cytotoxicity assay

The cytotoxicity assay was performed as previously described (19). Briefly, monocytes were cultured for 18 h in 96-well round-bottom plates (Dynatech, Alexandria, VA) at 2 x 10⁵ cells/well in medium alone or in medium containing optimal concentrations of IL-2, IFN-, or various doses of HA, alone or in combination. The plates were then extensively washed before the addition of labeled tumor target cells. Cytolytic activity was measured in a 48-h ¹¹¹In release assay against the human colon carcinoma cell line HT29 (American Type Culture Collection, Manassas, VA). Target cells were labeled by incubating 5 x 10⁶ tumor cells with 40 µCi of ¹¹¹In (Amersham) for 20 min at room temperature. Effector cells were incubated with 5 x 10^{3 111}In-labeled target cells at an E:T cell ratio of 20:1 at 37°C for 48 h. Plates were then centrifuged at 350 x g, 75 µl of the supernatant was harvested, and the radioactivity was measured. The results are expressed as the percentage of ¹¹¹In released, calculated from the mean counts per minute of triplicate determinations as [(experimental counts per minute - spontaneous counts per minute)/(total counts per minute - spontaneous counts per minute)] x 100. The SEMs were consistently <10% of the means. The spontaneous release of ¹¹¹In from target cells cultured alone was between 8 and 10% of the total radioactivity incorporated.

Western blot analysis

For analysis of hck protein levels, monocytes were cultured in 15-cm Lux plates at 2 x 10⁶ cells/ml with IL-2 for the indicated times, washed in ice-cold PBS, and solubilized in lysis buffer (10 mmol/L Tris, 50 mmol/L NaCl, 5 mmol/L EDTA, and 1% Triton X-100, pH 7.6) containing 10 µg/ml of the protease inhibitors aprotinin, leupeptin, water-soluble PMSF, and pepstatin A (Roche, Mannheim, Germany) by end-over-end rotation for 20 min at 4°C. Insoluble material was removed by centrifugation, and the protein content was determined using a protein assay kit (Bio-Rad, Richmond, CA). Equal amounts of protein from each sample were denatured by boiling for 5 min after the addition of 1 vol of 2x sample buffer (125 mmol/L Tris-HCl (pH 6.8), 4% SDS, 10% 2-ME, and 20% glycerol), electrophoresed under reducing conditions on 10% SDS-PAGE, and transferred to Immobilon nitrocellulose membranes (Millipore, Bedford, MA) using a semidry transfer apparatus (Pharmacia LKB, Piscataway, NJ). Membranes were blocked for 2 h at room temperature in blocking buffer (5% dry milk, 0.1% Tween 20, and 1x PBS) and subsequently probed in blocking buffer for 1 h with affinity-purified rabbit polyclonal Ab specific for human p59^hck, Lyn, c-Fgr, c-Yes, fyn, JAK3 (Santa Cruz Biotechnology, Santa Cruz, CA) or anti-JAK1 mAb (Transduction Laboratories, Lexington, KY). The blots were then washed three times for 10 min each time in wash buffer (2.5% powdered milk, 0.1% Tween 20, and 1x PBS), incubated for 30 min in blocking buffer containing 200 µg/ml of HRP-linked affinity purified goat anti-rabbit antiserum (Kirkegaard & Perry Laboratories, Gaithersburg, MD), and extensively washed. Bound Ab was detected by the enhanced chemiluminescence Western blotting detection kit (Amersham, Aylesbury, U.K.), and the membranes were autoradiographed using Kodak XAR-5 films.

Immunoprecipitation and tyrosine phosphorylation analysis

Monocytes were stimulated in 50-ml conical polypropylene tubes (Falcon, Becton Dickinson Labware, Lincoln Park, NJ) at 5 x 10⁶ cells/ml of warm RPMI with 1000 U/ml of IL-2 for brief periods of time, washed in ice-cold PBS, and solubilized in lysis buffer containing protease and phosphatase inhibitors (10 mM sodium tetrapyrophosphate, 50 mM sodium fluoride, and 5 mM sodium orthovanadate). Depending on the experiment, clarified cell lysates were incubated rotating end-over-end overnight at 4°C with 3 µg/ml of anti-phosphotyrosine mAb 4G10 (Upstate Biotechnology, Lake Placid, NY), anti-JAK3 rabbit polyclonal antiserum, anti-JAK1 mAb, anti-hck rabbit polyclonal antiserum, or normal rabbit serum that had been prebound to protein A/G Plus-agarose beads (Santa Cruz Biotechnology). The beads were extensively washed with buffer containing 0.1% Triton X-100, and precipitated material was eluted by boiling in SDS sample buffer for 5 min, run on 10% SDS-PAGE, and transferred to Immobilon membranes. For immunoblotting, anti-phosphotyrosine and anti-hck Abs were used at a concentration of 1 µg/ml in blocking buffer, and Western blot analysis was performed as described above.

Tyrosine kinase assay

hck immune complex tyrosine kinase assays were conducted by incubating the immunoprecipitated hck tyrosine kinase from lysates of unstimulated and IL-2-stimulated cells in the presence or the absence of ATP and visualizing incorporated phosphate on tyrosines by immunoblotting. Immobilized proteins were washed three times with lysis buffer followed by a single wash with kinase buffer containing 25 mM HEPES (pH 7.3), 0.1% Triton X-100, 100 mM NaCl, 10 mM MgCl₂, 3 mM MnCl₂, and 200 µM sodium orthovanadate. Isotope-free tyrosine kinase reactions were initiated by the addition of 15 µM unlabeled ATP and allowed to incubate at 37°C for 15 min. The reactions were quenched by washing the protein A/G Plus-agarose beads with lysis buffer and eluting bound material by boiling in SDS-sample buffer for 4 min. The material in each lane represents immunoprecipitates from 1 x 10⁸ cells.

Densitometry analysis

The intensities of the bands were quantitated from the autoradiographs generated from every experiment using an Imager 2000 (Innotech, San Leandro, CA). Whenever applicable, the results were normalized to the housekeeping gene GAPDH.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Effects of HA on IL-2-induced monocyte tumoricidal activity

To study the requirement for PTKs in human monocyte activation by IL-2, experiments were performed to analyze the effects of the PTK inhibitor HA on IL-2-induced monocyte tumoricidal activity. Previous studies have indicated that this inhibitor is effective against most tyrosine kinases, but does not significantly affect PKC, phosphorylase kinase, phospholipase C, or cyclic nucleotide-dependent protein kinases (47, 48). Monocytes were stimulated with an optimal dose of IL-2 for 18 h in the presence or the absence of increasing concentrations of HA and then assayed for cytotoxic activity against the human colon carcinoma cell line HT-29. Fig. 1 shows the results of one representative experiment of three performed with monocytes from different donors. IL-2-treated monocytes exerted high levels of tumoricidal activity (49%), which was markedly decreased in a dose-dependent manner by treatment with HA. Interestingly, this function of monocytes was exquisitely sensitive to the drug, because a concentration of HA as low as 0.01 µM was sufficient to cause a 54% reduction and a concentration of 0.5 µM was able to completely suppress IL-2-induced monocyte-mediated cytotoxicity. As depicted in Fig. 1 and previously reported (19), a similar activation of human monocytes to a tumoricidal stage can be achieved by cell stimulation with IFN-; however, IFN--dependent monocyte cytotoxicity was relatively more resistant to the effects of HA at all doses tested than IL-2-induced monocyte cytotoxicity. In fact, concentrations of HA that inhibited the monocyte response to IL-2 (0.01–0.1 µM) did not affect IFN--induced effects, and a concentration as high as 1 µM was required for a 51% reduction of IFN--mediated cytolysis (Fig. 1). Similar results were obtained using the PTK inhibitor genistein, which at a dose of 20 µM inhibited IL-2-induced monocyte cytotoxicity by 48%, but only decreased IFN--induced monocyte cytotoxicity by 6% (data not shown). At the concentrations used, HA did not affect monocyte viability, as determined by the trypan blue dye exclusion test. The vehicle for HA, DMSO, alone at the equivalent concentrations used did not inhibit IL-2-induced monocyte cytotoxicity. These results demonstrate that the activation of monocyte effector functions by IL-2 is extremely sensitive to PTK inhibition, suggesting a role for PTK in monocyte response to IL-2.

View larger version (20K): [in this window] [in a new window]   FIGURE 1. Dose-dependent inhibition of IL-2-induced monocyte tumoricidal activity by HA. Monocytes were cultured for 18 h in medium alone or in medium supplemented with IL-2 (1000 U/ml) or IFN- (500 U/ml), in the presence or the absence of increasing concentrations of HA and then assayed for tumoricidal activity as described in Materials and Methods. Results from one representative experiment are plotted as the means of triplicate determinations (SEM, <10% of the mean).

Human monocytes can be stimulated by IL-2 to release several proinflammatory cytokines (49). If signaling via IL-2R is mediated by PTK activation, responses of monocytes to IL-2 other than cytotoxicity would also be susceptible to inhibition by HA. To investigate this possibility, HA was tested for its effects on IL-2-induced monokine production. Supernatants from monocytes stimulated for 18 h with IL-2, alone or in combination with HA (0.1 µM), were assayed for the presence of IL-1ß, TNF-, IL-6, and IL-8 (Fig. 2). Secretion of all four cytokines was induced by IL-2, as previously reported (22, 33, 50), although the absolute levels detected varied somewhat from donor to donor (33) (data not shown). HA almost completely abrogated the effects of IL-2 on TNF-, IL-1ß, and IL-6 secretion and decreased monocyte release of IL-8 relative to that in IL-2-treated cells in all the experiments performed. The specificity of this inhibition was demonstrated by the inability of HA to block in a meaningful manner IL-1ß induction of IL-6 and IL-8. The HA diluent DMSO had no effect on IL-2-induced monokine protein expression (Fig. 2). To investigate whether the effects of HA were exerted at the level of gene expression, total RNA was extracted from monocytes stimulated for 6 h with optimal doses of IL-2 or 0.1 µM HA, alone or in combination, and Northern blot analysis was performed (Fig. 3A). This time point was shown to be optimal for mRNA induction by IL-2 (22). As depicted in Fig. 3A, control monocytes expressed undetectable or very low constitutive levels of IL-6, IL-8, IL-1ß, and TNF- mRNAs that were markedly up-regulated by stimulation with IL-2. Addition of HA at the onset of the culture potently suppressed IL-1ß, IL-6, and IL-8 and completely inhibited TNF- mRNA expression in IL-2-stimulated monocytes. HA alone did not induce expression of the message for any of the cytokines tested and did not affect the mRNA levels of the housekeeping gene GAPDH.

View larger version (44K): [in this window] [in a new window]   FIGURE 2. HA suppresses monocyte secretion of proinflammatory cytokines. Monocytes were cultured in medium alone or in medium supplemented with 1000 U/ml of IL-2 or 10 ng/ml of IL-1ß, alone or in combination with 0.1 µM HA. Supernatants were harvested after 18 h and assayed for the indicated cytokines by specific ELISA. Results from one representative experiment of three performed, expressed as picograms per milliliter per 2 x 106 cells/ml, are shown.

View larger version (12K): [in this window] [in a new window]   FIGURE 3. HA inhibits cytokine mRNA expression in IL-2-treated monocytes. A, Total RNA was extracted from monocytes stimulated for 6 h with IL-2 (1000 U/ml) or HA (0.1 µM), alone or in combination, and analyzed by Northern blotting. B, Monocytes were cultured for 6 h with IL-2 (1000 U/ml) in the presence or the absence of increasing concentrations of HA, and Northern blot analysis was performed on total RNA. The blots were sequentially hybridized with the cDNAs for the indicated cytokines. GAPDH levels were determined to ensure that equal amounts of RNA were loaded in each lane.

IL-2 induces protein tyrosine phosphorylation in human monocytes

Activation of the IL-2R in T lymphocytes rapidly induces tyrosine phosphorylation of a variety of substrates (51). Because tyrosine kinase activation appeared to be involved in regulating the responses of monocytes to IL-2, we next examined the effects of IL-2 on the phosphorylation of cellular proteins on tyrosine residues. Fresh human monocytes were stimulated with 1000 U/ml of IL-2 for the indicated times and subjected to detergent lysis and immunoprecipitation with anti-phosphotyrosine mAb. The protein samples were separated by SDS-PAGE and analyzed further by immunoblotting with a chemiluminescence detection system. As shown in Fig. 4, upper panel, IL-2 induced a marked increase in the phosphorylation on tyrosine residues of several cellular proteins, ranging from 46–180 kDa (indicated by the arrows), within the first 5 min of incubation. IL-2-dependent tyrosine phosphorylation was further augmented after 15 min of stimulation, reached a plateau at 30 min, and remained steady for at least 60 min during continuous incubation with IL-2. Among the cellular substrates that underwent phosphorylation in response to IL-2 were proteins migrating with apparent molecular masses from 35 kDa (after 30 min of treatment) to 46 kDa (after 5 min of treatment), which were detected upon overexposure of the film (Fig. 4, lower panel). This experiment as well as two other similar experiments gave comparable results, strongly suggesting that IL-2 is able to directly stimulate phosphorylation of proteins on tyrosine residues and provides further evidence that PTK activation is induced in response to treatment with IL-2 in fresh human monocytes.

View larger version (35K): [in this window] [in a new window]   FIGURE 4. Kinetics of IL-2-dependent induction of protein tyrosine phosphorylation in human monocytes. Monocytes were stimulated with IL-2 (1000 U/ml) for the indicated time points. Cell lysates were immunoprecipitated with 4G10 anti-phosphotyrosine mAb prebound to protein A/G Plus-agarose, run on a 10% SDS-polyacrylamide gel, and immunoblotted with the 4G10 mAb. Two different autoradiograms of the same blot from one of three separate experiments are shown. Molecular mass standards (kilodaltons) and Ig heavy chains (IgG) are indicated. The arrows denote those proteins whose level of tyrosine phosphorylation is increased by IL-2.

Several members of the src family PTKs are constitutively expressed in mononuclear phagocytes and have been shown to be important components of the signal transduction pathway of several monocyte/macrophage-activating factors (39). To gain insights into the biochemical mechanisms leading to monocyte activation by IL-2, experiments were designed to investigate whether IL-2 specifically affected the expression and activation of a specific member of the src family PTKs in human monocytes. Cell lysates from monocytes cultured for 6 and 12 h with IL-2 were subjected to Western blot analysis using specific Abs against the various src PTKs. In agreement with previous reports (38, 52), we detected constitutive levels of expression of fyn, Lyn, c-Fgr, and yes that were not up-regulated in response to IL-2 (Fig. 5). Interestingly, of the constitutively expressed src PTKs, only the steady-state levels of hck mRNA dramatically increased in a time-dependent manner upon exposure of resting monocytes to IL-2. As shown by Northern blot analysis (Fig. 6A), increased amounts of the 2.2-kb hck transcripts were observed as early as 3 h after IL-2 stimulation, while maximal mRNA accumulation (as determined by densitometric analysis of the intensities of the bands and normalization with GAPDH) occurred within 6 h and remained stable until 18 h after the onset of the culture. The accumulation of mRNA was paralleled by enhanced expression of p59^hck that reached a plateau after 12 h of culture, as assessed by Western blot (Fig. 6B). Both the 56- and 59-kDa isoforms of hck were equally up-regulated in IL-2-treated monocytes. Three independent experiments yielded comparable results, although slight fluctuations in the degree of induction were detectable due to donor variability. To determine whether the increased expression of hck in response to IL-2 was the general consequence of the activated phenotype on human monocytes, cells were treated with IFN-, a well-known monocyte activator. As shown in Fig. 7, IL-2 induced a 2.5-fold increase in hck expression over that in medium-treated cells. On the other hand, IFN- failed to affect the expression of hck in a meaningful way. These results provide the first evidence that the expression of hck PTK can be markedly induced by IL-2 in human monocytes.

View larger version (40K): [in this window] [in a new window]   FIGURE 5. The src PTKs are constitutively expressed in human monocytes. Monocytes were incubated in medium alone or in medium supplemented with 1000 U/ml of IL-2 for the indicated times. The cells were then lysed, and the amount of protein was equalized in each sample. The proteins were separated on a 10% SDS-PAGE and analyzed by immunoblotting as described in Materials and Methods using the indicated Abs. An anti-GAPDH immunoblot was performed to control that comparable amounts of protein were loaded into each lane.

View larger version (21K): [in this window] [in a new window]   FIGURE 6. IL-2 up-regulates the constitutive expression of hck mRNA and protein in human monocytes. Monocytes were cultured for the indicated time points in medium alone (-) or in medium supplemented with 1000 U/ml of IL-2 (+). A, Total RNA was isolated and analyzed by Northern blotting for hck mRNA expression (upper panel). The blot was then rehybridized with the GAPDH probe as a control for RNA loading (lower panel). B, Protein lysates were prepared and analyzed by Western blotting using a rabbit polyclonal anti-hck antiserum. The two 56- and 59-kDa isoforms of hck are indicated.

View larger version (15K): [in this window] [in a new window]   FIGURE 7. IL-2, but not IFN-, up-regulates the expression of p59hck. Cells were cultivated for the indicated lengths of time in the presence or the absence of 1000 U/ml of IL-2. Monocytes were then lysed, and the amount of protein was equalized in each sample. The proteins were separated on a 10% SDS-PAGE and analyzed by immunoblotting as described in Materials and Methods using rabbit anti-hck Ab. An anti-GAPDH immunoblot was performed to control that comparable amounts of protein were loaded into each lane.

Having established that IL-2-activated monocytes express increased levels of p59^hck, we asked whether this PTK could be involved in the IL-2 signal transduction pathway in monocytes. Monocytes were stimulated with 1000 U/ml of IL-2 for the indicated periods of time, and tyrosine phosphorylation was assessed on immunoprecipitated p59^hck by immunoblotting with an anti-phosphotyrosine Ab. As shown in Fig. 8, upper panel, a certain degree of basal tyrosine phosphorylation of p59^hck was present in unstimulated monocytes. IL-2 treatment resulted in a rapid, time-dependent augmentation of hck phosphorylation that started as early as 1 min after IL-2 addition, peaked at 5 min after stimulation, and declined thereafter. A second peak of phosphorylation was evident after 60 min of culture. Both the p56 and p59 isoforms of hck were equally tyrosine phosphorylated. Stripping of the blot followed by direct immunoblotting for hck demonstrated that at these early time points IL-2 treatment did not affect the amounts of hck immunoprecipitated (Fig. 8, bottom panel). Similar results were obtained in three independent experiments. These results demonstrate that the hck protein becomes tyrosine phosphorylated in response to IL-2 and suggest that p59^hck may be involved in IL-2 signal transduction in human monocytes.

View larger version (26K): [in this window] [in a new window]   FIGURE 8. Tyrosine phosphorylation of hck in response to IL-2. Monocytes were stimulated with 1000 U/ml of IL-2 for the indicated time points and then lysed. Cell lysates were incubated overnight with anti-hck polyclonal rabbit antiserum prebound to protein A/G Plus-agarose. Immunoprecipitated hck was run on a 10% SDS-PAGE and immunoblotted with 4G10 anti-phosphotyrosine mAb (-PY; top). An anti-hck immunoblot (bottom) was performed to control that comparable amounts of p59hck had been immunoprecipitated.

To better assess the importance of hck in regulation of the monocyte response to IL-2, we next examined the catalytic activity of hck PTK following IL-2 stimulation. Human monocytes were exposed to 1000 U/ml of IL-2 for 2 min, and cell lysates were subjected to immunoprecipitation by a specific anti-hck or a control antiserum. Immunoprecipitated proteins were then subjected to the in vitro tyrosine kinase assay. As shown in Fig. 9, although a certain degree of basal hck kinase activity was present in unstimulated monocytes (lane 2), a significant (11-fold) increase in phosphate incorporation on tyrosine residues was detectable when hck immunoprecipitates from IL-2-stimulated cells were incubated with ATP in vitro (lane 4). Both the 56- and 59-kDa isoforms of hck were equally phosphorylated upon cell exposure to IL-2. By contrast, cell lysates subjected to immunoprecipitation by normal rabbit serum (control) were negative (Fig. 9, lanes 5–8), and reprobing of the immunoblots with hck antiserum verified equal protein loading (data not shown). To further substantiate the potential role of hck in the activation process of monocytes by IL-2, experiments were performed to investigate whether HA inhibited IL-2-induced hck catalytic activity. Monocytes were preincubated with medium in the presence or the absence of increasing concentrations of HA. After 8 h of culture, 1000 U/ml of IL-2 was added for 2 min, lysates were prepared, and hck catalytic activity was determined as described above. As depicted in Fig. 10, HA decreased p59^hck catalytic activity in a dose-dependent manner. These results demonstrate that the specific activity of hck PTK is increased by IL-2 stimulation of monocytes and strongly suggest that hck is an integral component of the signaling pathway involved in IL-2-induced monocyte activation.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. IL-2-induced p59hck catalytic activity analyzed by in vitro tyrosine kinase assay. Monocytes were incubated with medium (-) or 1000 U/ml of IL-2 (+) for 2 min at 37°C. Lysates were prepared and subjected to immunoprecipitation with rabbit polyclonal anti-hck (-hck; lanes 1–4) or preimmune rabbit serum (CTRL; lanes 5–8). Immunoprecipitates were assayed for in vitro kinase activity in the absence (-) or the presence (+) of 15 µM unlabeled ATP. Reaction products were then resolved by SDS-PAGE, followed by anti-phosphotyrosine immunoblotting, as described in Materials and Methods.

View larger version (22K): [in this window] [in a new window]   FIGURE 10. HA inhibits IL-2-induced p59hck catalytic activity. Monocytes were incubated in medium alone or in medium supplemented with 0.05 µM HA (lanes 3 and 7) or 0.1 µM HA (lanes 4 and 8). After 8 h of incubation, 1000 U/ml of IL-2 was added for 2 min, lysates were prepared and subjected to immunoprecipitation as described in Fig. 9, and in vitro kinase activity was determined as described in Materials and Methods.

To determine whether JAK1 and/or JAK3 are involved in the early stages of monocyte activation by IL-2, we investigated their expression on resting and activated monocytes. Northern blot analysis revealed very low basal levels of JAK1 mRNA in medium-treated cells (Fig. 11, upper panel). Incubation of monocytes with either 1000 U/ml of IL-2 or 500 U/ml of IFN- for up to 6 h did not affect JAK1 mRNA expression. Similar results were obtained in four independent experiments. To determine whether JAK1 protein paralleled the expression of JAK1 mRNA, Western blot analysis was performed. Monocytes cultured for 3 h in medium alone expressed very low levels of JAK1. The expression of JAK1 increased with culture, reaching a maximum at 9 h, the latest point tested. Neither IL-2 nor IFN- meaningfully affected JAK1 expression (Fig. 11, lower panel). The same lysates were then used to investigated the expression of JAK3 in monocytes. Western blot analysis revealed that JAK3 was almost undetectable in medium-treated monocytes. On the other hand, both IL-2 and IFN- induced the expression of JAK3 in a time-dependent manner (Fig. 12).

View larger version (36K): [in this window] [in a new window]   FIGURE 11. Expression of JAK1 in human monocytes. Upper panel, Monocytes were cultured for the indicated lengths of time in medium alone or in medium supplemented with either 1000 U/ml of IL-2 or 500 U/ml of IFN-. Total RNA was isolated and analyzed by Northern blot for JAK1 mRNA expression. The blot was then rehybridized with GAPDH probe as a control for RNA loading. Lower panel, Cells were cultivated for the indicated lengths of time in the presence or the absence of 1000 U/ml of IL-2. Monocytes were then lysed, and the amount of protein was equalized in each sample. The proteins were separated on a 10% SDS-PAGE and analyzed by immunoblotting as described in Materials and Methods using JAK1 mAb. An anti-GAPDH immunoblot was performed to control that comparable amounts of protein were loaded into each lane.

View larger version (28K): [in this window] [in a new window]   FIGURE 12. JAK3 expression is induced by either IL-2 or IFN-. Human monocytes were cultivated for the indicated lengths of time in the presence or the absence of 1000 U/ml of IL-2 or 500 U/ml of IFN-. Cells were then lysed, and the amount of protein was equalized in each sample. The proteins were separated on a 10% SDS-PAGE and analyzed by immunoblotting as described in Materials and Methods using rabbit anti-JAK1 Ab. An anti-GAPDH immunoblot was performed to control that comparable amounts of protein were loaded into each lane.

View larger version (52K): [in this window] [in a new window]   FIGURE 13. The tyrosine phosphorylation status of JAK3 and JAK1 in resting and preactivated monocytes in response to IL-2. Upper panel, Fresh resting monocytes were stimulated with 1000 U/ml of IL-2 for 5 min (lane 1) or with 500 U/ml of IFN- for 18 h followed by IL-2 for 5 and 10 min (lanes 2 and 3, respectively). YT cells were treated for 5 min with IL-2 (lane 4). Cells were then lysed and incubated overnight with anti-JAK3 polyclonal rabbit antiserum prebound to protein A/G Plus-agarose. Immunoprecipitated JAK3 was run on a 10% SDS-PAGE and immunoblotted with 4G10 anti-phosphotyrosine mAb (-PY; top). An anti-JAK3 immunoblot (bottom) was performed to control for immunoprecipitation. Lower panel, The same cellular lysates and experimental procedures used in the upper panel experiment were used to determine JAK1 tyrosine phosphorylation status.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

The ability of HA to suppress the monocyte response to IL-2 was not restricted to cytotoxicity, but was also evident on IL-2-induced expression of IL-1ß, TNF-, IL-8, and IL-6 mRNA, and accordingly, cytokine secretion was reduced to baseline levels in the presence of the drug. This inhibition was specific, because HA failed to inhibit IL-1ß-induced IL-6 and IL-8. Inhibition was dose dependent and occurred at the same doses as those that were effective in blocking cytotoxicity. No effect on cell viability or on GAPDH mRNA levels was observed upon treatment with the drug, demonstrating that the decrease in monocyte responses to IL-2 could not be accounted for by toxicity of the treatment and excluding the possibility of a general and nonspecific inhibition of mRNA synthesis. A causal relationship between inhibition of PTK activity and suppression of IL-2-induced monocyte functions was also supported by the observation that 20 µM genistein, another tyrosine kinase inhibitor structurally unrelated to HA and known to have different nonspecific activities (58), markedly decreased monocyte tumoricidal activation by IL-2 (data not shown). The simplest interpretation of our results is that IL-2R engagement can trigger the activation of one or more PTKs and protein tyrosine phosphorylation events critical for IL-2 intracellular signal transduction, and that HA (and genistein) inhibited PTK activation by IL-2, thereby preventing monocyte responses. Indeed, we demonstrated that IL-2, at a concentration optimal for inducing monocyte biological activities, was able in resting monocytes to directly stimulate rapid tyrosine phosphorylation of several proteins, ranging from 35 to 180 kDa. This increase was detectable within the first 5 min of treatment, reached a maximum by 30 min, and remained steady for at least 60 min during continuous incubation with IL-2. This time course of tyrosine phosphorylation is similar to that observed by Saltzman et al. (59) in the cytotoxic T cell line CTLL and peripheral blood T lymphocytes. The recruitment and activation of Lck, JAK3, JAK1, and Syk PTKs following IL-2 binding to its receptor on T cells are well documented (1, 3, 4, 51). However, the expression of Lck PTK is restricted to T lymphocytes and NK cells, and we demonstrated that resting monocytes express low levels of JAK1. Furthermore, JAK1 was not tyrosine phosphorylated on resting or preactivated monocytes despite the relatively high levels of JAK1 protein on the later cells. In contrast, Syk PTK, in agreement with previous observations (7), was constitutively expressed, but did not undergo phosphorylation in response to IL-2 (data not shown). In agreement with Musso et al. (39), we demonstrated that JAK3 was present in preactivated, but not in resting, monocytes. Furthermore, Villa et al. (44) recently reported that monocytes from a JAK3-SCID patient exhibited normal response to IL-2 in terms of cytokine production, thus demonstrating that the stimulatory activity of IL-2 on monocytes was unaffected by the lack of JAK3. These observations indicate that these kinases, although involved in T lymphocyte activation by IL-2, are not absolutely required for early IL-2 signaling in freshly isolated monocytes and suggest the participation of other PTKs in this pathway. Several members of the src family PTKs are constitutively expressed in mononuclear phagocytes and have been shown to be important components of the signal transduction pathway of different monocyte/macrophage-activating factors (39, 40, 41, 42, 43). Because HA at 0.1 µM has been reported to have a direct blocking effect on src PTKs (47), and consistent increases in the tyrosine phosphorylation of proteins in the 50–60 kDa range, which is the size of the src kinases (52), were observed in response to IL-2, we hypothesized that a myeloid-specific member of the src family PTK could be involved in the early signal transduction events activated by IL-2 in monocytes. Herein, we provide the first evidence that IL-2 induced increased expression, tyrosine phosphorylation, and rapid activation of p59^hck src family PTK. Monocyte treatment with IL-2 resulted in a significant up-regulation of the expression of hck mRNA relative to that in control cells; this was paralleled by p59^hck protein accumulation. hck mRNA induction occurred very rapidly, within 3 h of stimulation, suggesting a direct response to IL-2, and was likely to be IL-2 specific, because no changes in hck transcript levels were observed in IFN--treated monocytes (42) (data not shown). The finding that p59^hck underwent tyrosine phosphorylation within 5 min of stimulation with IL-2 and the fact that this phosphorylation is associated with an increase in p59^hck kinase activity strongly indicate that one of the earliest intracellular events triggered by IL-2 in monocytes is the activation of hck PTK. The functional relevance of hck in fresh monocytes is further suggested by the fact that HA inhibits IL-2-induced hck catalytic activity. These findings clearly suggest a critical role for p59^hck kinase in IL-2-induced protein tyrosine phosphorylation and the IL-2 signaling pathway in monocytes. The effects of IL-2 on hck were selective, because we did not detect any change in the expression of the other src family PTKs constitutively expressed in monocytes, such as fgr, lyn, fyn, and yes. However, we cannot completely rule out the involvement of others src family PTK in IL-2 signaling in freshly isolated monocytes. Studies are currently ongoing in our laboratory to investigate the effects of IL-2 on the catalytic activity of the others src PTK members. The effect of IL-2 on hck expression was specific and was not associated with the activated phenotype of monocytes, because IFN-, a potent activator of monocytes, failed to enhance the expression of hck.

Despite the close structural relationship among the src family members, it is likely that they probably subserve different functions in cells of the monocyte/macrophage lineage. Moreover, these results, together with earlier findings by others (60, 61) showing that IL-2 could specifically regulate the activities of fyn, lyn, and blk src family members in B cell lines not expressing p56^lck, indicate that some flexibility exists in the ability of different src-like PTKs to couple to the IL-2R and participate in IL-2 signal transduction. In addition, it raises the possibility that cell lineage-specific responses to IL-2 may be determined, at least in part, by the repertoire of src family PTKs expressed in the cell. Extensive studies in T cells have demonstrated the importance of the cooperation among distinct PTKs in IL-2 signaling (1, 3). It is likely that more than one class of signal transduction molecule contributes to the cascade of intracellular events that results in IL-2-induced monocyte activation. IL-2 has been shown to induce the expression of JAK3 mRNA and protein in IFN--pretreated monocytes and to trigger subsequent kinase phosphorylation, thus suggesting that JAK3 is a component of the IL-2 signal transduction pathway in activated monocytes (Ref. 39 and the present study). Our results indicate that in fresh resting monocytes the hck gene product is involved at an early step in IL-2 signal transduction cascade, and that JAK3 phosphorylation occurs at a later point from the src PTK, although additional experiments will be required to establish a causal role for the two kinases in IL-2 signaling. We propose a model in which IL-2 binding to its receptor in monocytic cells elicits the rapid activation of hck kinase and the triggering of downstream events resulting in monocyte responses, including up-regulation of IL-2R ß/-chains (21, 34, 35), increases in p59^hck expression (present study), and induction as well as latter activation of JAK3 (39), thereby augmenting the number of functional IL-2Rs on the cell surface and the expression of intracellular signaling molecules, leading to a more efficient cell response to IL-2. Studies are currently in progress to determine whether hck is constitutively associated with the IL-2R complex in freshly isolated monocytes or whether it is recruited upon IL-2 stimulation; furthermore, the studies will attempt to elucidate the contributions of other PTKs to signal transduction events activated by IL-2. In conclusion, the current observations provide important initial insights into IL-2 signal transduction processes in monocytic cells and emphasize a key role for specific tyrosine phosphorylation events.

	Acknowledgments

 
We thank Dr. Carmen S. Garcia and Dr. James M. Mwatibo for their critical review of this manuscript.

	Footnotes

 
¹ This work was supported in part by grants from the Italian Association for Cancer Research and from Telethon Italy (Project A 75).

² Address correspondence and reprint requests to Dr. Igor Espinoza-Delgado, Louisiana State University Medical Center, 1542 Tulane Avenue, Hematology-Oncology, Suite 604K, New Orleans, LA 70112.

³ Abbreviations used in this paper: PTK, protein tyrosine kinase; JAK, Janus kinase; HA, herbimycin A; LAK, lymphokine-activated killer cells.

Received for publication December 3, 1999. Accepted for publication February 24, 2000.

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