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(Journal of Leukocyte Biology. 2001;69:419-425.)
© 2001 by Society for Leukocyte Biology

A p74 common gamma receptor chain isoform facilitates IL-2 and IL-15 responses by the myelomonocytic cell line Tf-1ß2

Nancy L. Monson*, Timothy J. Fenske{dagger}, Shyng Wei#, Angela J. Okragly**, Jill L.O. de Jong*, Mary Haak-Frendscho**, John O’Shea||, Julie Djeu# and Paul M. Sondel*,#,**

Departments of
* Human Oncology,
{dagger} Molecular and Cellular Biology,
# Pediatrics, and
** Genetics, University of Wisconsin, and University of Wisconsin Comprehensive Cancer Center, Madison, Wisconsin;
{ddagger} H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida;
§ Promega Corp., Madison, Wisconsin; and
|| Arthritis and Rheumatoid Branch, National Institutes of Health, Bethesda, Maryland

Correspondence: Nancy L. Monson, Ph.D., Department of Neurology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9036. E-mail: nancy.monson{at}utsouthwestern.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Functional forms of the IL-2, IL-4, IL-7, IL-9, and IL-15 receptors require the {gamma}c receptor component. We have described previously a myeloid cell line called Tf-1ß, which binds IL-2 with intermediate-affinity and proliferates in response to IL-2. In this study, we characterize {gamma}c expression on Tf-1ß2 cells, a derivative of Tf-1ß cells stimulated exclusively with IL-2. Although Tf-1ß2 cells bind IL-2 with intermediate-affinity and proliferate in response to IL-2, this cell line does not express the p64 {gamma}c chain at the protein level. This result was surprising because prior studies suggest these cells should not be expected to proliferate in response to IL-2 or IL-15 in the absence of the p64 {gamma}c chain. A p74 protein was detected by western blot following immunoprecipitation with an anti-{gamma}c polyclonal antibody, and a p74 protein was identified consistently in complex with IL-2 and IL-15 on these cells. However, the {gamma}c gene in these Tf-1ß2 cells shows no evidence of mutation by sequence analysis. Furthermore, inhibition of glycosylation of these Tf-1ß2 cells by tunicamycin treatment yields a standard 39-kDa molecule recognized on western blot with anti-{gamma}c antibody, as seen for the standard 64-kDa isoform of {gamma}c. These results demonstrate that a 74-kDa {gamma}c receptor isoform was involved in the response of the Tf-1ß2 cells to cytokines which normally interact with the 64-kDa {gamma}c chain.

Key Words: monocytes • cytokines • cytokine receptors • signaling


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The responses induced by interleukin-2 (IL-2) on T-, B-, and natural killer (NK) cells and on monocytes are mediated by its binding to the IL-2 receptor (IL-2R). Three forms of the IL-2R exist, each with a distinct binding affinity for IL-2: the low-, intermediate-, and high-affinity forms composed through various combinations of three distinct receptor components (IL-2R{alpha}, -ß, and -{gamma}) [1 2 3 4 5 ]. The IL-2R{gamma} chain is also a component of several other cytokine receptor complexes (IL-4, IL-7, IL-9, and IL-15) [6 7 8 9 10 11 12 13 14 ] and has thus been renamed the common gamma chain ({gamma}c). Improper expression of {gamma}c causes alterations in the responses to all these cytokines and consequently defects in the immune repertoire. Indeed, defects in {gamma}c expression and function cause X-linked severe combined immunodeficiency (XSCID) in humans, which is manifested by the lack of lymphoid development [11 , 15 16 17 18 19 20 ]. However, myeloid development in patients with XSCID is apparently normal and suggests that normal {gamma}c expression is not essential for myeloid cell function.

We have established an in vitro model for evaluating myeloid cell responses to IL-2. The cell line Tf-1ß was derived from the myeloid leukemia Tf-1 cell line [21 ] by introducing the IL-2Rß chain into these cells. This rendered them responsive to IL-2, as assessed by proliferation. Tf-1ß cells respond to IL-2 through intermediate-affinity IL-2 receptors and express similar numbers of intermediate-affinity IL-2 receptors as do the NK-like leukemic YT cells. However, the p64 {gamma}c chain could not be detected on Tf-1ß cells by surface iodination or flow cytometry. The goal of this study was to further assess the expression of the p64 {gamma}c chain on this myeloid cell line. The data summarized here indicate that the p64 {gamma}c chain is not expressed on these cells even though {gamma}c chain mRNA was detectable in these cells and was unaltered as evidenced by direct sequencing analysis. These results indicate that a p64 {gamma}c is not required in the generation of responses to IL-2 and IL-15 on these cells. In contrast, a p74 molecule was complexed with IL-15 and IL-2 and demonstrated a glycosylation pattern that suggested the p74 molecule is a functional {gamma}c isoform surrogate for p64 {gamma}c on these cells.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cytokines
Human recombinant IL-2 was provided by Hoffmann-La Roche, Inc. (Nutley, NJ) and has a specific activity of 15 x 106 U/mg protein so that 1 U/mL = 4.4 pM. Granulocyte-macrophage colony-stimulating factor (GM-CSF) was provided by Immunex Corporation (Seattle, WA). IL-4 was purchased from Genzyme Diagnostics (Cambridge, MA). Recombinant simian and human IL-15 were generously provided by Dr. M. Widmer of Immunex.

Cell lines
The Tf-1ß cell line was a variant of the cell line Tf-1 [22 , 23 ], infected with the recombinant retrovirus encoding the gene for human IL-2Rß and maintained in GM-CSF and G418 as described previously [21 ]. Tf-1 cells were maintained in 5 ng/mL GM-CSF. The Tf-1ß2 cell line was maintained in 100 U/mL IL-2 for at least 2 months in the absence of GM-CSF and G418. The Tf-1L cell line used as a negative control was a variant of the Tf-1 cell line infected with the recombinant retrovirus without the IL-2Rß gene insertion. Tf-1L cells and Tf-1ß cells were also maintained in 0.5 ng/mL G418 for vector selection. YT cells (provided by J. Yodoi of Kyoto University, Japan) were established from a patient with thymic lymphoma and express IL-2Rß and {gamma}c chains but not the IL-2R{alpha} chain [24 ]. The THP-1 cell line was a human monocyte cell line known to be negative for {gamma}c chain mRNA expression [25 ]. Phytohemagglutinin (PHA) blasts were generated by incubating Ficoll-Hypaque-generated, normal donor peripheral blood lymphocytes in RPMI complete medium containing PHA at a concentration of 1 µg/mL for 3 days [26 ].

Isolation of RNA, generation of cDNA, and amplification of the {gamma}c gene from Tf-1ß cells
This procedure was performed as described previously [21 ].

Sequence analysis
This procedure was performed as described previously [27 ] using primers designed to obtain the entire {gamma}c sequence with significant overlaps, which was then compared with the human {gamma}c sequence (Genbank Accession Number D11086). Sequencing analysis was performed at the University of Wisconsin Biotechnology Center (Madison, WI) by Dr. C. Nicolet.

Antibodies
M111 (IgG1) was an inhibitory antibody against human IL-15 and was provided by Genzyme Diagnostics. Monoclonal antibodies (mAbs) 341 [immunoglobulin G (IgG)1] and 561 (IgG2a) were noninhibitory antibodies against the human IL-2Rß chain [28 ]. Anti-JAK3 rabbit serum was used at a 1:2000 dilution for probing western blots [29 ]. The anti-{gamma}c rabbit serum used as the probing reagent in the western blots and as the immunoprecipitating reagent for the affinity-labeling experiments was a kind gift from W. Farrar (National Cancer Institute, Frederick MD) [30 ]. The immunoprecipitating antibody used for the surface iodinations and in the western blots was generated in a Leghorn hen following repeated immunization using a peptide generated from the C-terminus of {gamma}c (YTLKPET) [31 ]. The resultant polyclonal chicken antibody (pAb) was affinity-purified against the immunizing peptide and designated IgY{gamma}0 (Promega Corp., Madison, WI).

In vitro proliferative assay
This procedure was performed as described previously [21 , 32 ].

Two-dimensional gel analysis of immunoprecipitates from 125I-labeled YT and Tf-1ß cells
This procedure was performed as described previously [21 ].

Western blot analysis
This procedure was performed as described previously [21 ] using 108 cells/sample. In the JAK3 phosphorylation studies, human IL-2, IL-4, and IL-15 were used at 10 nM final concentration. Cell lysates were incubated with the immunoprecipitating antibody for 1–2 h at 4°C, rotating end-over-end, and then allowed to conjugate to Gammabind G Sepharose beads (Pharmacia Biotech, Uppsala, Sweden) for an additional 30 min under the same conditions. The anti-phosphotyrosine (anti-PY) antibody, 4G10 (Upstate Biotechnology, Lake Placid, NY), used as the primary probe, was added to the blot at a 1:2000 dilution, and the anti-JAK3 rabbit serum was added to the stripped blot at a 1:1000 dilution.

Stripping JAK3 phosphorylation western blots for reprobing
After chemiluminescent detection of the blot with the first probe, the blot was placed into TBS-Tween (1x TBS consisting of 0.02 M Tris, pH 7.5, 0.137 M NaCl, 0.05% Tween 20) for 5–10 min with constant shaking on a gyrorotator, followed by an additional incubation at 60°C for 30–45 min in 15 mL stripping solution [2% sodium dodecyl sulfate (SDS), 62.5 mM Tris, pH 6.8, 100 mM ß-mercaptoethanol]. The blot was then washed in TBS-Tween for 45 min, changing the buffer every 10 min, and blocked overnight at 4°C in blocking buffer [2% bovine serum albumin (BSA) in TBS-Tween]. The usual probing steps were carried out the following day.

Tunicamycin treatment
This procedure was performed as described previously [33 ]. The cells were treated with or without 10 or 20 µg/ml tunicamycin for 24 h. The cell lysates were prepared using lysis buffer [1% Nonidet P-40 (NP-40), 10 mM Tris-HCl, 140 mM NaCL, 10 mM iodoacetamide, 1 mM orthovanadate, 0.02% sodium azide, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 ug/ml each aprotinin, leupeptin, and antipain]. Lysates were immunoprecipitited with the IgY{gamma}0 antibody or an IgG control serum. The immunoprecipitates were separated on 10% SDS gel and western blotted with the rabbit anti-{gamma}c antibody.

Affinity-labeling of YT and Tf-1ß2 with 125I-IL-2 and 125I-IL-15
This procedure was performed as described previously [32 ].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tf-1ß2 cells do not express detectable p64 {gamma}c protein
In our previous study, the {gamma}c chain could not be detected on Tf-1ß cells by flow cytometry or surface iodination [21 ]. However, the surface iodination approach relied on IL-2 acting as an effective bridge to hold the entire IL-2Rß/IL-2/{gamma}c complex together and on the surface expression of the {gamma}c chain on Tf-1ß cells grown in GM-CSF. These factors may have prevented detection of the {gamma}c chain on the surface of the Tf-1ß cells. To circumvent reliance on IL-2 sustaining the integrity of the IL-2R complex for the detection of {gamma}c, anti-{gamma}c antibodies were generated and used for the immunoprecipitations. To ensure that all molecules necessary for an IL-2 response would be expressed, Tf-1ß2, a derivative of Tf-1ß [34 ], was generated by continuous culture in IL-2. Cell lysates from 125I-labeled YT cells and 125I-labeled Tf-1ß2 cells were prepared in parallel, and immunoprecipitates of IL-2 receptors were obtained using the anti-IL-2Rß antibody 561 or the anti-{gamma}c antibody IgY{gamma}0, followed by two-dimensional isoelectric focusing (IEF)/SDS-polyacrylamide gel electrophoresis (PAGE) analyses. Figure 1 is representative of five separate experiments of this design. The IL-2Rß/{gamma}c complexes on YT cells are evident at a 1:1 ratio in anti-IL-2Rß immunoprecipitates of IL-2-equilibrated cells (Fig. 1A) . However, only IL-2Rß is detected in the Tf-1ß2 cells prepared in the same manner (Fig. 1B) . The p64 {gamma}c chain is evident in the anti-{gamma}c immunoprecipitates of YT cells (Fig. 1C) but not the Tf-1ß2 cells (Fig. 1D) , Tf-1, or Tf-1L cells (unpublished results). No other specific iodinated molecular component was detected in the anti-IL-2Rß or anti-{gamma}c immunoprecipitations of surface-iodinated Tf-1, Tf-1L, Tf-1ß, or Tf-1ß2 cells, regardless of IL-2 equilibration.



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  		Figure 1. Direct immunoprecipitation of {gamma}c present on surface-iodinated Tf-1ß2 and YT cells. YT cells (A and C) and Tf-1ß cells (B and D), which had been stripped of surface-bound cytokine, surface-iodinated, and treated with saturating concentrations of IL-2, were detergent-solubilized. The resulting lysates were immunoprecipitated with the anti-IL-2Rß antibody 561 (A and B) or the anti-{gamma}c antibody IgY{gamma}0 (C and D). The immunoprecipitates were subjected to two-dimensional IEF/SDS-PAGE and placed on phosphorimager plates for 7 days at which time they were read and analyzed using ImageQuant 3.3 software. IEF tube gels are oriented acidic-to-basic (left to right). The IL-2Rß and {gamma}c spots are labeled within the panels. The molecular weight markers are indicated on the left.

 
Western blotting reveals p74 and p120 bands rather than a p64 molecule on Tf-1ß2
Although we and others have been unable to detect p64 {gamma}c protein on Tf-1 cells previously [35 , 36 ], others have shown that it is detectable in cells of the myeloid lineage by western blot analysis [33 , 37 38 39 ]. Because we now had access to a derivative of Tf-1ß that was cultured continuously in IL-2 and an antibody that could immunoprecipitate {gamma}c directly, western blots were performed, again attempting to detect {gamma}c protein on Tf-1ß cells using the anti-{gamma}c antibody, IgY{gamma}0. Lysates from YT, Tf-1, Tf-1L, and Tf-1ß2 cells were immunoprecipitated with IgY{gamma}0, resolved by one-dimensional SDS-PAGE, and probed for {gamma}c using the rabbit anti-{gamma}c antibody. Figure 2 represents one of four separate experiments of this design. As indicated in lane 1, the p64 {gamma}c chain was detected by western blot from YT cells, which express similar numbers of intermediate-affinity IL-2 receptors as Tf-1ß cells [21 ]. No p64 {gamma}c chain was detected from lysates of Tf-1, Tf-1L, and Tf-1ß2 cells (Fig. 2 , lanes 2–4). No bands were detected in YT, Tf-1, Tf-1L, or Tf-1ß2 cell lysates immunoprecipitated with IgY, the isotype control for IgY{gamma}0 (unpublished results). However, at least two prominent bands, at 74 and 120 kDa, were detected by western blot in the Tf-1, Tf-1L, and Tf-1ß2 cell lysates (Fig. 2 , lanes 2–4) that were not evident in the YT cell lysate sample (Fig. 2 , lane 1).



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  		Figure 2. Western blot analysis of total {gamma}c in Tf-1, Tf-1L, and Tf-1ß2 cells compared with YT cells. Samples of YT, Tf-1, Tf-1L, and Tf-1ß2 cells (108 cells/sample) were lysed, and immunoprecipitates were generated using the anti-{gamma}c antibody IgY{gamma}0 and resolved on 8% SDS-PAGE gels. After transferring to membrane, the blot was probed for {gamma}c expression using the anti-{gamma}c rabbit sera raised against the C-terminus of {gamma}c. The p64 {gamma}c chain is labeled "{gamma}c"; other potential {gamma}c isoforms are indicated with arrows. The molecular weight markers are indicated on the left. The p74 and p120 bands are not precipitated by the IgY isotype-control mAb in related experiments (unpublished results).

 
In our previous study, Tf-1ß cells equilibrated with IL-2 were immunoprecipitated with anti-IL-2Rß antibody and evaluated by western blot. A very faint band of 60–75 kDa was noted (see figure 8 in [21 ]). To better characterize the bands identified in Figure 2 and compare these results with our previous western blot (see figure 8 in [21 ]), YT and Tf-1ß2 cells were equilibrated with or without IL-2, immunoprecipitated with the anti-IL-2Rß or anti-{gamma}c antibody, and probed with the anti-{gamma}c rabbit antisera. Figure 3 represents six separate experiments of this design. In the absence of IL-2, some p64 {gamma}c chain was detected in association with IL-2Rß on YT cells (Fig. 3 , lane 1), which in our experience, can occur when large numbers of YT cells are used. The addition of exogenous IL-2 clearly increases the amount of p64 {gamma}c chain detected in association with IL-2Rß on YT cells (Fig. 3 , lane 2). This same p64 band is detected in YT cell lysates immunoprecipitated directly with IgY{gamma}0 (Fig. 3 , lane 5) and represents a more precise estimate of size for the p64 {gamma}c chain.



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  		Figure 3. Detection of p64 {gamma}c in YT but not in Tf-1ß2 cells by anti-IL-2Rß and anti-{gamma}c immunoprecipitations. YT and Tf-1ß2 cells were stripped of bound cytokine and equilibrated with or without 100 nM IL-2. Cell lysates (108 cells/sample) were generated, and immunoprecipitations were performed using anti-IL-2Rß or anti-{gamma}c antibodies. The resultant immunoprecipitates were resolved by one-dimensional SDS-PAGE, transferred to immobilon, and probed for {gamma}c detection using the anti-{gamma}c rabbit sera. (A) An unmarked copy of the blot; (B) labeled for ease in visualization—the p64 bands and the novel 74- and 120-kDa bands are enclosed in boxes. The cell type, immunoprecipitating antibody, and presence or absence of IL-2 for each lane are indicated at the bottom. Molecular weight markers are as indicated at the left.

 
The p64 {gamma}c chain was not detected in the anti-IL-2Rß immunoprecipitation of Tf-1ß2 cells equilibrated with IL-2 (Fig. 3 , lane 3). However, the p74 and p120 molecules were detected in the anti-IL-2Rß immunoprecipitations and gained intensity with the addition of exogenous IL-2 (Fig. 3 , lane 4). Additional bands were detected as well (Fig. 3 , lane 3) but did not increase in intensity with the addition of IL-2 (Fig. 3 , lane 4). The p74 and p120 bands were also detected in the direct anti-{gamma}c immunoprecipitations of Tf-1ß2 and Tf-1 cells (Fig. 3 , lanes 6 and 7). As expected, THP-1 cells used as negative control cells do not express the p64 {gamma}c chain nor the p74 and p120 proteins (Fig. 3 , lane 8).

Tf-1ß2 cells respond to IL-15 by proliferation despite the lack of a p64 {gamma}c molecule
It was apparent that Tf-1ß2 cells were functionally responsive to IL-2 in the absence of detectable p64 {gamma}c chain protein expression. To determine if responsiveness by Tf-1ß2 cells lacking p64 {gamma}c expression was limited to IL-2, Tf-1ß2 cells were tested for responsiveness to IL-15 [14 , 32 , 40 41 42 ]. Proliferative assays were performed that included IL-2, IL-15, and the anti-IL-15 antibody M111, which neutralizes IL-15 responses by blocking binding sites on IL-15 itself. As indicated in Table 1 , Tf-1ß2 cells proliferated in response to IL-2 and IL-15. The M111 antibody was able to inhibit the response of Tf-1ß2 cells to IL-15 but not to IL-2.


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  		Table 1. IL-15 Induced Proliferation of Tf-1ß2 Cells is Inhibited by the Anti-IL-15 Antibody, M111

 
IL-2 and IL-15 induce JAK3 phosphorylation of Tf-1ß2 cells despite the lack of a p64 {gamma}c molecule
Tf-1ß2 cells do not use the p64 {gamma}c chain, yet they proliferate in response to IL-2 or IL-15. Because this proliferative response by Tf-1ß2 cells does not involve the p64 {gamma}c chain, we wanted to determine whether the same signaling cascade used by cells expressing a p64 {gamma}c chain is triggered in Tf-1ß2 cells in response to IL-2 and IL-15. JAK3 phosphorylation was targeted for these studies because this kinase couples the p64 {gamma}c subunit to downstream signaling events directly in other IL-2 responsive cells [29 , 30 , 43 ] and those of the myeloid lineage [44 ]. Tf-1 and Tf-1ß2 cells were equilibrated with IL-2, IL-4, IL-15, GM-CSF, or no cytokine and lysates immunoprecipitated with the anti-JAK3 antibody. The immunoprecipitates were resolved by one-dimensional SDS-PAGE and probed with an anti-phosphotyrosine antibody to determine the extent of JAK3 phosphorylation in each sample (Fig. 4A ). The blots were stripped and reprobed with an anti-JAK3 antibody to ensure that JAK3 was immunoprecipitated in each sample (Fig. 4B) .



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  		Figure 4. Phosphorylation of JAK3 following IL-2 and IL-15 equilibration of Tf-1ß2. This assay was performed as outlined in Materials and Methods. Tf-1ß2 cells were equilibrated with 10 nM IL-2, 10 nM IL-15, GM-CSF, or no cytokine for 15 min at 37°C; lysed; and immunoprecipitated with the anti-JAK3 rabbit sera. Tf-1 cells were equilibrated with 10 nM IL-2, 10 nM IL-4, 10 nM IL-15, or no cytokine for 15 min at 37°C; lysed; and immunoprecipitated with the anti-JAK3 rabbit sera. (A) The samples were resolved by one-dimensional SDS-PAGE and probed with the anti-phosphotyrosine antibody (anti-PY). (B) The blot was stripped and reprobed with the anti-JAK3 rabbit sera. Molecular weight markers are indicated at the right. The cytokine stimulus is indicated at the top of each lane, and the JAK3 bands are marked with an arrow. The band marked "IgHC" is the Ig heavy chain that is cross-reactive with the secondary reagent used in the probing process.

 
As demonstrated in Figure 4B , JAK3 was precipitable from all the lysates, regardless of equilibration conditions, as expected. However, only Tf-1ß2 preparations, which received IL-2 or IL-15, activated (phosphorylated) JAK3 (Fig. 4A , lanes 6 and 7). Tf-1 cells did not activate JAK3 in response to IL-2 or IL-15 because they do not express the IL-2Rß chain (Fig. 4A , lanes 2 and 3). However, Tf-1 cells did activate JAK3, as expected in response to IL-4, demonstrating that they are capable of activating JAK3 under appropriate conditions (Fig. 4A , lane 4). As expected, Tf-1 and Tf-1ß2 cells stimulated with GM-CSF do not activate JAK3 (Fig. 4A , lane 8), because this signaling cascade is mediated by JAK2 [45 , 46 ].

The 74-kDa, IL-2 receptor component does not represent the product of an alternatively spliced or mutated {gamma}c mRNA in Tf-1ß2 cells
Direct sequencing of the {gamma}c gene from Tf-1ß cDNA did not indicate any alteration of the {gamma}c gene itself that might account for this lack of expression (unpublished results). Thus, the increased mass of this 74-kDa IL-2 receptor component is not the result of changes to the {gamma}c gene or mRNA in Tf-1ß2 cells.

The p74 molecule is a heavily glycosylated isoform of the {gamma}c chain receptor
To determine whether the p74 molecule was a unique receptor component or an isoform of the {gamma}c receptor, the glycosylation pattern of the Tf-1 cell subsets were compared with that of YT using tunicamycin as a deglycosylating agent. Cell lysates were prepared from YT, Tf-1, Tf-1L, and Tf-1ß2 cells, which were treated with tunicamycin previously, as well as cell lysates from these same cells that had not been treated with tunicamycin. Lysates immunoprecipitated with the IgY{gamma}0 antibody were resolved by one-dimensional SDS-PAGE and probed for {gamma}c using the rabbit anti-{gamma}c antibody. As indicated in Figure 5 , the p64 {gamma}c receptor is evident in the YT cell lysate without tunicamycin treatment (Fig. 5 , lane 1). No p64 {gamma}c chain was detected from untreated lysates of Tf-1, Tf-1L, or Tf-1ß2, although the p74 molecule was observed in all three Tf-1 cell subsets. Upon treatment with tunicamycin, the nonglycosylated p64 {gamma}c migrates as a 39-kDa band as expected [33 , 39 ] in the YT cell lysates (Fig. 5 , lanes 2 and 3). This 39-kDa band is also apparent in the tunicamycin-treated Tf-1, Tf-1L, and Tf-1ß2 lysates, although several intermediate forms remain even when the tunicamycin concentration is increased significantly.



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  		Figure 5. Effect of glycosylation inhibition on expression of the p74 molecule. YT, Tf-1, Tf-1L, and Tf-1ß2 cells were treated with 0, 10, or 20 µg/mL tunicamycin, lysed, and immunoprecipitated with the anti-{gamma}c antibody. Samples were resolved by one-dimensional SDS-PAGE and probed with the anti-{gamma}c rabbit sera. Molecular weight markers are as indicated on the left. The p64 {gamma}c chain is labeled "p64 {gamma}c", and the p74 molecule is indicated with a thick arrow. The expected, fully nonglycosylated p64 {gamma}c migrates at 39 kDa and is enclosed in a box across all lanes of the figure.

 
The 74-kDa molecule on Tf-1ß2 cells binds to IL-2 and IL-15
To clarify whether this p74 molecule binds to IL-2 and IL-15, Tf-1ß2 and YT cells were equilibrated with 125I-IL-2 or 125I-IL-15, and the complexes cross-linked with the noncleavable chemical cross-linker bis[sulfosuccinimidyl]suberate (BS3). Anti-{gamma}c immunoprecipitates were then generated from the lysates. Figure 6 represents three separate experiments of this design. A band at 80 kDa was detected in YT cells equilibrated with radiolabeled IL-2 or IL-15. These 80-kDa bands represent the 64-kDa {gamma}c chain cross-linked to the 15-kDa IL-2 or IL-15 molecule. No band at 80 kDa (which would be composed of the 64-kDa {gamma}c chain cross-linked to the 15-kDa IL-2 or IL-15 molecule) was detected in complex with IL-2 or IL-15 on Tf-1ß2 cells, although a 90-kDa band (corresponding to the complex of the 74-kDa molecule with IL-2 or IL-15) was detected for IL-2 and IL-15. No band at 90 kDa was detected for YT cells. No other molecules complexed to IL-2 or IL-15 on Tf-1ß2 were detected by this technique.



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  		Figure 6. Detection of affinity-labeled {gamma}c on YT cells and affinity-labeled p74 on Tf-1ß2 cells. YT cells (left panel) and Tf-1ß2 cells (right panel) were equilibrated with 10 nM 125I-IL-2 (left lanes of each panel) or 125I-IL-15 (right lanes of each panel) for 1 h at 4°C and cross-linked using BS3, and lysates were immunoprecipitated with the anti-{gamma}c rabbit sera as outlined in Materials and Methods. The resultant samples were resolved by one-dimensional SDS-PAGE. Molecular weight markers are as indicated at the left. The p74 band is indicated with a thick, shaded arrow.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The experiments performed here have documented further that the p64 {gamma}c protein is not detected on Tf-1ß2 cells despite their ability to bind and respond to IL-2 and IL-15. Furthermore, IL-2 and IL-15 induce JAK3 phosphorylation on the Tf-1ß2 cells, a signal that involves the p64 {gamma}c chain on other cells. In contrast, immunoprecipitation with anti-{gamma}c antibody, followed by western blotting shows the expected 64-kDa molecule on YT cells but only the 74- and 120-kDa molecules on Tf-1ß2 cells. Moreover, affinity-labeling shows a 64-kDa molecule binding IL-2 and IL-15 on YT cells but only a 74-kDa molecule on Tf-1ß2 cells. These data indicate that there is a 74-kDa IL-2 receptor component on Tf-1ß2 cells, which binds IL-2 and IL-15 and is recognized by two separate anti-{gamma}c antisera. Direct sequencing of the {gamma}c mRNA in Tf-1ß2 cells demonstrated that these cells expressed the wild type {gamma}c message, unaltered at the DNA or RNA level. Therefore, this p74 molecule likely represented an alternative isoform of the {gamma}c molecule, possibly resulting from modified glycosylation [33 , 37 38 39 ]. In fact, experiments using tunicamycin as an agent to inhibit glycosylation indicated that this was the case and suggest that our inability to detect the {gamma}c by flow cytometry or surface iodination may be hindered by this heavy glycosylation pattern in the myeloid-derived p74 {gamma}c isoform. The purpose that this altered glycosylation pattern serves requires clarification, although it is clear that this p74 {gamma}c isoform is capable of facilitating the response to IL-2 and IL-15 by Tf-1ß cells in the absence of the p64 {gamma}c chain.

To date, no other cell line exhibiting a robust response to IL-2 and IL-15 in the absence of the p64 {gamma}c chain has been shown. Previous studies documented that Tf-1 cells expressed the {gamma}c chain on their surface by western blot [30 , 47 ] and affinity-labeling [48 ]. However, given the nature of these bands identified as the p64 {gamma}c chain, it is difficult, as it was in our experiments, to conclude that these bands are not a slightly different molecular weight than the p64 {gamma}c chain. Furthermore, although several {gamma}c isoforms are co-expressed with the p64 {gamma}c chain on fresh myeloid cells and neutrophils, only the p64 isoform has been shown to transmit a signal. Perhaps in the case of Tf-1 cells, the absence of the p64 {gamma}c chain allows one of these alternative isoforms to assume its functions. One of the nonfunctional {gamma}c isoforms found in fresh monocytes and neutrophils is larger than 64 kDa (~69 kDa) [33 , 37 38 39 ]. It is possible that the 74-kDa protein described here could be the 69-kDa {gamma}c chain isoform described by the teams of Djeu and Varesio [33 , 37 38 39 ] because subtle differences in molecular markers could account for the perceived differences in size.

Because Tf-1ß2 cells respond to IL-2 and IL-15 in the absence of the 64-kDa {gamma}c chain, these studies indicate that Tf-1ß2 cells circumvent this nonexpression by using a 74-kDa {gamma}c isoform as a surrogate for the p64 {gamma}c chain. Hence, the conclusion that the 64-kDa {gamma}c chain is the only functional {gamma}c form would not apply to Tf-1ß2 cells. Furthermore, these results also suggest further characterization is needed of the 120-kDa molecule expressed by Tf-1ß2 cells (which is recognized by the anti-{gamma}c antibody) to determine its exact molecular origin. It will be important to evaluate the presence of these 74- and 120-kDa molecules using a panel of monocyte lineage populations and cell lines to determine their potential involvement in IL-2 and IL-15 binding and activation in other cells of the myeloid lineage.


   ACKNOWLEDGEMENTS
 
This work was supported by grants from the American Cancer Society, IM-678, NIH CA-32685, p30-CA14520-2, CM-87290, and RR03186.

Received May 24, 1999; revised November 17, 2000; accepted November 21, 2000.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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