B-Lymphocyte Proliferation during Bovine Leukemia VirusInduced Persistent Lymphocytosis Is Enhanced by T-Lymphocyte-Derived Interleukin-2

Abstract

Bovine leukemia virus (BLV)-induced persistent lymphocytosis is characterized by a polyclonal expansion of CD5⁺ B lymphocytes. To examine the role of the cytokine microenvironment in this virus-induced B-lymphocyte expansion, the expression of interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-γ) mRNA, was measured in stimulated peripheral blood mononuclear cells from persistently lymphocytotic BLV-infected cows, nonlymphocytotic BLV-infected cows, and uninfected cows. IL-2 and IL-10 mRNA expression and IL-2 functional activity were significantly increased when peripheral blood mononuclear cells from persistently lymphocytotic cows were stimulated with concanavalin A (ConA). Additionally, during persistent lymphocytosis, peak IL-2 and IL-10 mRNA expression was delayed, and elevated expression was prolonged. To determine the potential biologic importance of increased IL-2 and IL-10 expression, the response of isolated B lymphocytes from persistently lymphocytotic cows to human recombinant cytokines and to cytokine-containing supernatants from isolated T lymphocytes was examined. While recombinant human IL-10 (rhIL-10) did not consistently induce detectable changes, rhIL-2 increased viral protein (p24) and IL-2 receptor expression in isolated B lymphocytes from persistently lymphocytotic cows. Additionally, rhIL-2 and supernatant from ConA-stimulated T lymphocytes enhanced B-lymphocyte proliferation. The stimulatory activity of the T-lymphocyte supernatant could be completely inhibited with a polyclonal anti-rhIL-2 antibody. Finally, polyclonal anti-rhIL-2 antibody, as well as anti-BLV antibody, inhibited spontaneous proliferation of peripheral blood mononuclear cells from persistently lymphocytotic cows, demonstrating that the spontaneous lymphoproliferation characteristic of BLV-induced persistent lymphocytosis is IL-2 dependent and antigen dependent. Collectively, these findings strongly suggest that increased T-lymphocyte expression of IL-2 in BLV-infected cows contributes to development and/or maintenance of persistent B lymphocytosis.

Bovine leukemia virus (BLV), the causative agent of enzootic bovine leukosis, is an exogenous oncogenic B-lymphotropic retrovirus in the human T-cell leukemia virus (HTLV) group (48). Like other members of the HTLV group, BLV induces preneoplastic lymphocyte dysregulation and lymphoid neoplasia (6, 57). However, BLV is unique among the HTLV-like viruses in predominantly targeting B lymphocytes (37).

Similar to other chronic retroviral infections, BLV infection results in a prolonged asymptomatic period with low viral load and low viral gene expression which persists for 1 to 8 years (24, 30, 37). Following the asymptomatic period, approximately 30% of BLV-infected cattle develop persistent lymphocytosis, characterized by a polyclonal expansion of B lymphocytes coexpressing CD5 and surface immunoglobulin M (sIgM) (10). During persistent lymphocytosis, the percentage of peripheral lymphocytes containing provirus is greatly increased and viral gene expression is enhanced (24, 37). Additionally, BLV gene transcription, which can be stimulated through normal cellular transcriptional pathways, is increased in activated B lymphocytes (25, 29). Therefore, activation and proliferation of B lymphocytes likely contributes to increased viral gene expression and disease progression to persistent lymphocytosis.

Activation, proliferation, and differentiation of normal B lymphocytes are largely mediated by T lymphocytes, both by direct cell-to-cell contact and by cytokine release (34). Interleukin-2 (IL-2), IL-4, IL-10, and gamma interferon (IFN-γ) act alone and synergistically to promote B-lymphocyte proliferation and differentiation (9, 12–14, 21, 46, 47). During BLV-induced persistent lymphocytosis, expression of IL-2 and IL-10 mRNA is increased in freshly isolated peripheral blood mononuclear cells (PBMC) (44), and IL-2 activity is increased in culture supernatants of concanavalin A (ConA)-stimulated PBMC (49). Importantly, alterations in cytokine expression are correlated with disease progression in other chronic retroviral infections, including HTLV type 1 (HTLV-1), human immunodeficiency virus (HIV), and murine leukemia virus (8, 11, 16, 33). Consequently, we hypothesize that cytokine dysregulation, either through the direct effects of viral regulatory genes or through a response to antigenic stimuli, results in B-lymphocyte stimulation during BLV-induced persistent lymphocytosis.

In this report, initial experiments measuring the expression of IL-2, IL-4, IL-10, and IFN-γ mRNAs indicated that IL-2 and IL-10 transcription was significantly increased in ConA-stimulated PBMC from cows with persistent lymphocytosis. Based on these results, the effects of IL-2 and IL-10 on B-lymphocyte proliferation and viral expression were investigated. The data indicate that during persistent lymphocytosis, increased IL-2 expression by T lymphocytes enhances B-lymphocyte proliferation, upregulates the IL-2 receptor (IL-2R) on B lymphocytes, and increases virus expression.

MATERIALS AND METHODS

Animals.

Adult Holstein cows from the University of Idaho Dairy herd were classified as persistently lymphocytotic, nonlymphocytotic BLV infected, or uninfected based on BLV serology, a complete blood count, a differential blood count, and phenotypic analysis of PBMC. Cows defined as persistently lymphocytotic were seropositive to BLV in an agar gel immunodiffusion test (Leukassay B; Pittman Moore, Mundelein, Ill.), had a lymphocyte count greater than 8,000 cells/μl which persisted for greater than 3 months, and had greater than 20% CD5⁺ B lymphocytes in their PBMC. Nonlymphocytotic cows were seropositive to BLV, had a lymphocyte count within the normal range (2,500 to 7,500 cells/μl), and had less than 20% CD5⁺ B lymphocytes in their PBMC. Uninfected cows were seronegative to BLV, had a lymphocyte count within the normal range, and had less than 20% CD5⁺ B lymphocytes in their PBMC. BLV-infected cows with normal lymphocyte counts but a high percentage of CD5⁺ B lymphocytes were excluded from the present studies (15, 32).

Antibodies.

Mouse monoclonal antibody (MAb) 4′G9, against BLV capsid protein p24, was a kind gift from Daniel Portetelle, Faculty of Agronomy, Gembloux, Belgium. MAbs specifically recognizing bovine leukocyte surface molecules were obtained from the Washington State University Monoclonal Antibody Center, Pullman. Profiles to characterize cell populations included markers for monocytes/macrophages (DH59B and CAM36A), B lymphocytes (BIG73A [sIgM], PIG45A [sIgM], and BAQ44A [B-B2]), bovine CD2 (boCD2) (MUC2A), boCD3 (MM1A), boCD4 (CACT138A and GC50A), boCD5 (CACT105A and B29A), boCD8 (CACT80C and BAQ111A), boCD21 (BAQ15A and GB25A), bovine γδ T-cell receptor (TCRγδ) δ chain (GB21A), and bovine IL-2Rα (CACT108A, CACT116A, and GB112A). MAbs COLIS52 (IgM), COLIS69 (IgG1), COLIS169 (IgG2b), and COLIS205 (IgG2a), which do not recognize bovine antigens, were used as isotype controls. All MAbs were isotyped, quantitated, and used at 15 μg/ml for flow cytometric analysis. MAb GB25A, which recognizes the B-lymphocyte marker boCD21, was used for positive selection of B lymphocytes (see below).

A neutralizing polyclonal rabbit antirecombinant human IL-2 (rhIL-2) antibody, purified by ammonium sulfate precipitation (EP-100; Genzyme, Cambridge, Mass.), was used at 50 μg/ml to neutralize bovine IL-2 in culture supernatants and to inhibit spontaneous proliferation of PBMC (55). The anti-rhIL-2 antibody did not inhibit proliferation of an IL-2-independent human B-lymphocyte cell line, Raji (American Type Culture Collection, Rockville, Md.), indicating that it is nontoxic. Ammonium sulfate precipitated polyclonal rabbit antibody, R9415F, against Babesia bovis was used as the negative control antibody.

Polyclonal bovine immunoglobulin from a BLV-infected persistently lymphocytotic cow (anti-BLV antibody) and from a healthy uninfected cow (bovine control antibody) was isolated with a Protein G-Sepharose column (GammaBind Plus Sepharose; Pharmacia Biotech, Piscataway, N.J.) according to the manufacturer’s protocol. The immunoglobulin preparations were dialyzed in phosphate-buffered saline (PBS; pH 7.2) and used at 200 μg/ml.

Flow cytometry.

Single- and dual-color stainings for cell surface marker characterization were performed by using a standard protocol as previously described (51).

Prior to staining for BLV p24 with MAb 4′G9, cells were fixed and permeabilized. For fixation, 10⁶ cells/well were suspended in 200 μl of 2% formaldehyde in PBS and incubated on ice for 20 min. Cells were washed once in PBS, resuspended in 200 μl of 0.2% Tween 20 in PBS, and incubated at 37°C for 15 min. Following a wash in PBS, cells were stained by the standard protocol except that after the final wash cells were resuspended in PBS with 0.1% sodium azide rather than 2% formaldehyde in PBS.

Data were acquired using a Becton Dickinson FACscan flow cytometer and analyzed with CELLQUEST software (Becton Dickinson Immunocytochemistry Systems, San Jose, Calif.). Data were collected on 5,000 cells per sample for phenotyping PBMC and isolated cell populations and on 10,000 cells per sample for determination of IL-2R and viral p24 expression.

PBMC isolation and culture conditions.

Whole blood was collected by jugular venipuncture into acid citrate dextrose, and PBMC were isolated by density gradient sedimentation as previously described (51). Viable cells were counted by trypan blue dye exclusion. PBMC were suspended at 4 × 10⁶ cells/ml in complete RPMI (RPMI 1640 supplemented with 10% fetal bovine serum, 25 mM HEPES buffer, 2 mM l-glutamine, 5.5 × 10⁻² mM 2-mercaptoethanol, and antibiotic-antimycotic), placed in 25-cm² tissue culture flasks, and incubated at 37°C and 5% CO₂. For mitogen-stimulated cultures, ConA (5 μg/ml) was added to the medium. Preliminary experiments indicated that stimulation with ConA maximized differences in cytokine mRNA expression between groups compared to stimulation with Staphylococcus aureus Cowan strain I, lipopolysaccharide, pokeweed mitogen, or phorbol 12-myristate 13-acetate plus ionomycin.

Total RNA and culture supernatants were collected from ConA-stimulated PBMC at 1.5, 3, 6, 12, 18, 22, and/or 46 h poststimulation. Supernatants were treated with methyl α-d-mannopyranoside (20 mg/ml; Sigma, St. Louis, Mo.) to inactivate residual ConA, filtered through 0.2-μm filters, and stored frozen at −20°C until use.

T-lymphocyte isolation and culture conditions.

T lymphocytes were isolated from persistently lymphocytotic cows by sequential passage of PBMC over two nylon wool columns essentially as described previously (26). Eluted cells were pelleted and resuspended in complete RPMI. Viable cells were counted by trypan blue dye exclusion. Yields varied from 3 to 10% of starting PBMC. Isolated T lymphocytes contained ≤9% of cells staining for sIgM and ≤1% of cells staining with a monocyte/macrophage marker. The cell surface phenotypes of isolated T lymphocytes used to produce supernatants for the experiments in this report were 80 to 91% CD2⁺, 38 to 61% CD4⁺, 72 to 88% CD5⁺, 10 to 38% CD8⁺, and 10 to 27% TCRγδ⁺.

Supernatants were collected from ConA-stimulated isolated T lymphocytes from three persistently lymphocytotic cows. T lymphocytes were suspended at 2 × 10⁶ cells/ml in complete RPMI plus ConA (5 μg/ml) and incubated at 37°C and 5% CO₂ in 25-cm² tissue culture flasks. Culture supernatants were collected at 72 h poststimulation, treated with methyl α-d-mannopyranoside (20 mg/ml), filtered, and stored in aliquots at −20°C.

B-lymphocyte isolation.

B lymphocytes were isolated by positive selection of PBMC by using anti-CD21 MAb GB25A and goat anti-mouse IgG-coated magnetic beads (Dynabeads M-450; Dynal, Oslo, Norway) following the manufacturer’s instructions for the indirect cell separation technique. MAb GB25 was added at 0.3 μg/10⁶ PBMC, and magnetic beads were added at 6 × 10⁵ beads/10⁶ PBMC for persistently lymphocytotic cows. For uninfected cows, GB25A was added at 0.25 μg/10⁶ PBMC and magnetic beads were added at 4.5 × 10⁵ beads/10⁶ PBMC. Recovered B lymphocytes were approximately 25 to 30% of starting PBMC for persistently lymphocytotic cows and 5 to 15% for uninfected cows. The isolated B-lymphocyte populations from persistently lymphocytotic and uninfected cows had ≤1% of cells staining for CD2, CD4, CD8, TCRγδ, or a monocyte/macrophage marker. Isolated B lymphocytes from persistently lymphocytotic cows had >90% of cells staining for sIgM, and isolated B lymphocytes from uninfected cows had >70% of cells staining for sIgM.

RNase protection assay.

Total RNA was extracted from both adherent and nonadherent ConA-stimulated PBMC by using TRIzol reagent (Gibco BRL, Grand Island, N.Y.) according to the manufacturer’s instructions. RNA was resuspended at 1 μg/μl in RNase free water and stored at −80°C until use. Prior to use, RNA samples were treated with RNase-free DNase to remove any contaminating DNA. Cytokine mRNA expression was measured in total RNA samples by using an RNase protection assay kit (RPAII; Ambion, Austin, Tex.) as directed by the manufacturer. High-specific-activity ³²P-labeled riboprobes were synthesized by using the Maxiscript system (Ambion), with linearized plasmids containing bovine cytokine cDNA or human cyclophilin cDNA as templates. Lou Gasbarre, USDA Helminthic Diseases Laboratory, Beltsville, Md., kindly provided the IL-2, IL-4, and IFN-γ templates, which consist of segments of 480, 370, and 470 bp, respectively, of bovine cytokine cDNA inserted into pGEM vectors (Promega, Madison, Wis.). The bovine IL-10 template consists of a 483-bp bovine IL-10 cDNA insert in the vector PCRII (Invitrogen, San Diego, Calif.) (19). Prior to use, the cytokine template plasmids were amplified, sequenced to confirm the size and integrity of the insert, and linearized. The cyclophilin template is a commercially available, prelinearized pTRIPLEscript plasmid containing a conserved 103-bp fragment of human cyclophilin cDNA (Ambion). Probes were purified by 5% polyacrylamide–8 M urea gel electrophoresis prior to use in the RNase protection assay.

In the assay, each reaction, consisting of 15 μg of total sample RNA and 40,000 cpm each of one cytokine probe and the cyclophilin internal control probe, was precipitated and annealed overnight at 45°C in 80% formamide buffer. Negative control samples consisted of 15 μg of yeast RNA. The unannealed single-stranded RNA and excess probe were digested with RNase, and the remaining protected fragments were precipitated, resuspended, and run on a 5% polyacrylamide gel. Bands representing the protected fragments were then visualized by autoradiography. Cytokine mRNA expression was determined by densitometry of autoradiographs in the linear range of sensitivity, using an IS-1000 Digital Imaging System densitometer (Alpha Innotech, San Leandro, Calif.). The cytokine densitometry readings were normalized for the amount of RNA present in each sample, using the band density of the constitutively expressed internal standard, cyclophilin, with the following formula: (cytokine densitometry reading/cyclophilin densitometry reading) × 1,000 = corrected densitometry reading. Due to low expression of IL-4 mRNA, the cytokine and internal control readings were made by using autoradiographs at different exposure times to keep the densitometry readings within the linear range.

IL-2 functional assay.

The IL-2-dependent bovine T-cell clone G4.2E8 was obtained by limiting dilution cloning of ConA-stimulated PBMC as previously described (3). Cryopreserved clonal cells were thawed, and viable cells were recovered by density gradient centrifugation. The cells were maintained at 10⁶ cells/ml in complete RPMI containing 20 U of rhIL-2 (Boehringer Mannheim, Indianapolis, Ind.) per ml. Cells were split every 3 to 4 days and refed with rhIL-2-containing medium. Three to four days after the last stimulation with rhIL-2, the clone was used to assay IL-2 activity in culture supernatants of ConA-stimulated PBMC or ConA-stimulated isolated T lymphocytes. For the assay, the cells were washed in IL-2-free complete medium and plated in round-bottom 96-well plates at 3 × 10⁴ cells per well in 50 μl of complete medium. All supernatants were treated with 20 mg of methyl α-d-mannopyranoside per ml and filtered through 0.2-μm filters prior to use. Supernatants to be tested were serially diluted, and 50 μl of each dilution was added to duplicate wells. Duplicate wells containing serial dilutions of rhIL-2, from 200 to 0.001 U/ml, were used to formulate a standard curve. Assay plates were incubated, pulsed with [³H]thymidine (NEN Life Science Products, Boston, Mass.), and harvested as described below. Data are expressed as the mean counts per minute of duplicate wells.

IFN-γ protein ELISA.

IFN-γ protein in culture supernatants from ConA-stimulated PBMC was measured using a bovine IFN-γ-specific enzyme-linked immunosorbent assay (ELISA; IDEXX, Westbrook, Maine) according to the manufacturer’s instructions. Duplicate wells of serially diluted samples (1:1 to 1:1,000) were measured. A standard curve was formulated by using serially diluted supernatant from Mycobacterium tuberculosis purified protein derivative-specific bovine T-cell clone C97.3G11, determined to contain 440 U of IFN-γ/ml in a vesicular stomatitis virus neutralization assay as previously described (5). Negative control wells consisted of complete medium treated with 20 mg of methyl α-d-mannopyranoside per ml.

Lymphocyte proliferation assays.

Spontaneous proliferation of PBMC and the proliferative response of isolated B lymphocytes to rhIL-2, rhIL-10, and T-lymphocyte supernatant were measured in 3-day lymphoproliferation assays. Assays were done in 96-well round-bottom tissue culture plates with 2 × 10⁵ cells/well in complete RPMI (total volume of 100 μl/well). For B-lymphocyte stimulation, serial dilutions of rhIL-2 (100 to 0.001 U/ml), rhIL-10 (100 to 0.001 ng/ml; R&D Systems, Minneapolis, Minn.), and T-lymphocyte supernatant (reciprocal dilutions of 10⁰ to 10⁵) were made in complete RPMI and added to the wells. The proliferative response of B lymphocytes to combinations of rhIL-2 and rhIL-10 (20, 5, or 0.25 U of rhIL-2/ml with 100 or 3 ng of rhIL-10 per ml) was also tested. In some wells, polyclonal rabbit anti-rhIL-2 antibody or polyclonal rabbit R9415F control antibody was added at 50 μg/ml. Assay plates were incubated at 37°C and 5% CO₂ for 3 days. [³H]thymidine was added at 0.5 μCi/well 18 h prior to the termination of culture. Cells were harvested on an automated 96-well plate harvester (TomTec Inc., Orange, Conn.), and [³H]thymidine uptake was measured by liquid scintillation spectroscopy (Wallac Inc., Gaithersburg, Md.). Data are expressed as the mean counts per minute of duplicate or quadruplicate wells.

RESULTS

IL-2 and IL-10 mRNA expression is altered in ConA-stimulated PBMC from persistently lymphocytotic BLV-infected cows. (i) mRNA expression.

To determine if a B-lymphocyte-stimulatory cytokine environment could be contributing to the development or maintenance of persistent lymphocytosis, we used an RNase protection assay to measure the expression of IL-2, IL-4, IL-10, and IFN-γ mRNAs in ConA-stimulated PBMC isolated from persistently lymphocytotic BLV-infected, nonlymphocytotic BLV-infected, and uninfected cows. Mean IL-2 mRNA expression in PBMC from persistently lymphocytotic cows was significantly greater than that of nonlymphocytotic BLV-infected cows (P < 0.05) and uninfected cows (P < 0.01), using a one-tailed Student t test (Fig. 1A). Similarly, IL-10 mRNA expression was significantly increased in ConA-stimulated PBMC from persistently lymphocytotic cows in pairwise comparison to either nonlymphocytotic or uninfected cows by using a one-tailed Student t test (P < 0.05) (Fig. 1B). No significant differences were seen in IL-4 expression between the three groups of cows (Fig. 1C). IFN-γ mRNA expression in PBMC from persistently lymphocytotic cows did not differ significantly from that in nonlymphocytotic BLV-infected cows; however, IFN-γ mRNA expression in PBMC from infected cows was significantly higher (P < 0.05) than that of uninfected cows (Fig. 1D). Identical trends in IL-2 and IL-10 mRNA expression were noted in three independent experiments.

FIG. 1.

Mean cytokine mRNA expression in 18-h ConA-stimulated PBMC. IL-2 (A), IL-10 (B), IL-4 (C), and IFN-γ (D) mRNA expression (determined in an RNase protection assay) are presented as mean corrected densitometry units. IL-2 data are for seven persistently lymphocytotic (PL), nine nonlymphocytotic BLV-infected (BLV+), and nine uninfected (BLV−) cows; IL-10 and IL-4 data are for seven PL, six BLV(+), and four BLV(−) cows; IFN-γ data are for seven PL, eight BLV(+), and nine BLV(−) cows. Error bars are standard errors. Bars marked with different letters are significantly different (P < 0.05) by pairwise comparison using a one-tailed Student t test.

(ii) Expression kinetics.

To determine if the kinetics of cytokine mRNA expression were also altered in persistently lymphocytotic cows, IL-2, IL-4, IL-10, and IFN-γ mRNA expression was measured in ConA-stimulated PBMC from four persistently lymphocytotic, two nonlymphocytotic BLV-infected, and two uninfected cows at 1.5, 3, 6, 12, 18, 22, and 46 h poststimulation, using an RNase protection assay (Fig. 2). For IL-2 and IL-10, the mean peak mRNA expression in nonlymphocytotic and uninfected cows occurred at (IL-2) or before (IL-10) 3 h poststimulation. In contrast, in persistently lymphocytotic cows mean peak IL-2 mRNA expression was at 6 h and mean peak IL-10 mRNA expression was at 18 h. In addition to delayed peak expression, persistently lymphocytotic cows maintained elevated expression of IL-2 and IL-10 mRNAs over a longer time period than nonlymphocytotic and uninfected cows.

FIG. 2.

Kinetics of cytokine mRNA expression. IL-2 (A), IL-10 (B), IL-4 (C), and IFN-γ (D) mRNA expression (determined in an RNase protection assay) in PBMC stimulated for 3, 6, 12, 18, 22, or 46 h with ConA are presented for four persistently lymphocytotic (○), two nonlymphocytotic BLV-infected (▵), and two uninfected (□) cows. Data are presented as the percent change in the mean corrected densitometry units from the 3-h time point using the formula [(mean mRNA expression at time point x − mean mRNA expression at 3 h)/mean mRNA expression at 3 h] × 100 = mean percent change.

The kinetics of IL-4 mRNA expression were similar in all three groups of cows, with early peak expression at 6 h poststimulation and rapid decline of expression to very low levels by 12 h poststimulation. As for IL-4, differences were not noted in the kinetics of IFN-γ mRNA expression between groups.

(iii) IL-2 functional activity.

Assays to quantify bovine cytokines are available for IL-2 and IFN-γ. Therefore, results of IL-2 and IFN-γ mRNA expression experiments were confirmed in quantitative functional and protein assays, respectively. The bovine IL-2-dependent T-lymphocyte clone G4.2E8 was used to measure IL-2 activity in supernatants from ConA-stimulated PBMC from persistently lymphocytotic, nonlymphocytotic BLV-infected, and uninfected cows at 46 h poststimulation. In correlation with mRNA levels, persistently lymphocytotic cows had significantly (P < 0.05) increased IL-2 functional activity in ConA-stimulated PBMC supernatant compared to either nonlymphocytotic or uninfected cows (Fig. 3A). IL-2 mRNA expression at 18 h after ConA stimulation and IL-2 activity in 46-h poststimulation PBMC culture supernatants were significantly (P < 0.01) positively correlated (r = 0.71) (Fig. 3B).

FIG. 3.

Correlation of IL-2 functional activity and IFN-γ protein level to mRNA expression. (A) IL-2 activity in supernatants from 46-h ConA-stimulated PBMC was measured by determining [³H]thymidine uptake by the IL-2-dependent T-lymphocyte clone G4.2E8. Bars represent the mean uptake stimulated by supernatants from 9 persistently lymphocytotic (PL), 11 nonlymphocytotic BLV-infected (BLV+), and 12 uninfected (BLV−) cows. Error bars are standard errors. Bars marked with different letters are significantly different (P < 0.05) by pairwise comparison using a one-tailed Student t test. (B) The IL-2 mRNA expression at 18 h (measured by RNase protection assay) was plotted against the [³H]thymidine uptake of G4.2E8 cells induced by 46-h ConA-stimulated PBMC supernatant for each of seven PL, nine BLV(+), and nine BLV(−) cows. The correlation coefficient of the relationship between these two variables is 0.71 (P < 0.01). (C) IFN-γ protein in supernatants from 46-h ConA-stimulated PBMC was measured by a bovine IFN-γ-specific ELISA. Bars represent the mean optical density at 650 nm (O.D. 650) of supernatants from 8 PL, 8 BLV(+), and 11 BLV(−) cows. (D) The IFN-γ mRNA expression at 18 h (measured by RNase protection assay) was plotted against the bovine IFN-γ-specific ELISA reading of the supernatant for each of seven PL, eight BLV(+), and nine BLV(−) cows. The correlation coefficient of the relationship between these two variables is 0.77 (P < 0.01).

A commercially available bovine IFN-γ ELISA (IDEXX) was used to measure IFN-γ protein in PBMC culture supernatants from persistently lymphocytotic BLV-infected, nonlymphocytotic BLV-infected, and uninfected cows at 46 h after ConA stimulation (Fig. 3C). No significant difference was present between the mean IFN-γ protein level in persistently lymphocytotic and nonlymphocytotic BLV-infected cows. However, the mean IFN-γ protein level of the uninfected cows was lower than that of both groups of infected cows (P < 0.05). The IFN-γ mRNA expression at 18 h was significantly (P < 0.01) positively correlated (r = 0.77) to the IFN-γ protein level in 46-h culture supernatants from ConA-stimulated PBMC (Fig. 3D).

rhIL-2 increases IL-2R and BLV p24 expression in isolated B lymphocytes from persistently lymphocytotic BLV-infected cows.

Since IL-2 and IL-10 upregulate IL-2Rα on human B-lymphocytes (14, 22), we used flow cytometric analysis to quantitate bovine IL-2Rα on isolated B lymphocytes from persistently lymphocytotic and uninfected cows (Fig. 4A). Additionally, since IL-2 is a potent activator of B lymphocytes (34), and BLV expression is increased in activated B lymphocytes in vitro (1, 25, 29), BLV viral capsid protein (p24) expression was also measured (Fig. 4B). B lymphocytes were stained with anti-IL-2R antibody GB112A or anti-BLV p24 antibody 4′G9 after culture for 3 days in the presence of either no cytokine, rhIL-2 (20 U/ml), rhIL-10 (100 ng/ml), or a combination of rhIL-2 (20 U/ml) and rhIL-10 (100 ng/ml). rhIL-2 upregulated IL-2Rα expression on the surface of B lymphocytes isolated from both persistently lymphocytotic and uninfected cows (Fig. 4A). Similarly, although BLV p24 is detected in unstimulated B lymphocytes from persistently lymphocytotic cows, suggesting that BLV genes can be expressed in the absence of added T-lymphocyte-derived cytokines, rhIL-2 increased BLV p24 expression two- to threefold (Fig. 4B). rhIL-10 alone or in concert with rhIL-2 did not consistently increase IL-2R or BLV p24 expression. However, in contrast to persistently lymphocytotic cows, rhIL-10 inhibited IL-2R expression in B lymphocytes from an uninfected cow when present alone and blocked the stimulatory effect of rhIL-2 when present in combination (Fig. 4A). Data presented in Fig. 4 represent one of two experiments with similar results.

FIG. 4.

Effects of IL-2 and IL-10 on IL-2R and BLV p24 expression. IL-2R (A) and BLV p24 (B) expression were measured in isolated B lymphocytes from two persistently lymphocytotic cows (1583 and 1713) and one uninfected cow (1736) incubated for 3 days in medium alone or medium supplemented with either rhIL-2 (20 U/ml), rhIL-10 (100 ng/ml), or rhIL-2 (20 U/ml) plus rhIL-10 (100 ng/ml). B lymphocytes were stained with MAb GB112A (for IL-2R) or 4′G9 (for BLV p24), and the percent positive staining cells determined by flow cytometric analysis. Data represent one of two experiments with similar results.

rhIL-2 and IL-2-containing T-lymphocyte supernatant induce proliferation of B lymphocytes from persistently lymphocytotic BLV-infected cows.

Persistent expansion of the CD5⁺ sIgM⁺ B-lymphocyte population is the defining feature of BLV-induced persistent lymphocytosis (10). To determine the potential contribution of increased IL-2 and/or IL-10 to this expansion, the ability of rhIL-2 and rhIL-10, alone and in combination, to stimulate proliferation of B lymphocytes from persistently lymphocytotic cows was measured in 3-day lymphocyte proliferation assays. rhIL-2 stimulated proliferation of isolated bovine B lymphocytes from both persistently lymphocytotic and uninfected cows in a dose-dependent manner (Fig. 5). The dose-dependent proliferative response of the IL-2-dependent T-lymphocyte clone G4.2E8 is included in Fig. 5 as a positive control. Although the [³H]thymidine uptake of B lymphocytes from persistently lymphocytotic cows was, on average, greater than that of uninfected cows at each rhIL-2 concentration, the background proliferation of B lymphocytes from persistently lymphocytotic cows was also higher. The dose-response curves are similar for the two groups, suggesting that B lymphocytes from the two groups of cows respond similarly to rhIL-2. In contrast to rhIL-2, rhIL-10, which is biologically active on bovine T lymphocytes (4), had no proliferative effect on bovine B lymphocytes at doses ranging from 100 to 0.001 ng/ml, either alone or in combination with rhIL-2, in 3-day lymphocyte proliferation assays (data not shown).

FIG. 5.

Dose-dependent proliferative response to rhIL-2. Isolated B lymphocytes from four persistently lymphocytotic cows (PL B cells), isolated B lymphocytes from four uninfected cows [BLV(−) B cells], and IL-2-dependent clonal bovine T-lymphocytes (G4.2E8 T cells) (mean of three separate experiments) were stimulated for 3 days with the indicated dilutions of rhIL-2. Each data point represents the mean [³H]thymidine uptake. Error bars are standard errors.

As for rhIL-2, bovine IL-2-containing supernatants of ConA-stimulated T lymphocytes isolated from three persistently lymphocytotic cows also induced dose-dependent proliferation of the IL-2-dependent T-lymphocyte clone and of isolated B lymphocytes from either persistently lymphocytotic or uninfected cows (data not shown).

B-lymphocyte proliferation in response to T-lymphocyte supernatant is IL-2 dependent.

Supernatants of ConA-stimulated T-lymphocytes from three persistently lymphocytotic cows induced proliferation of isolated B lymphocytes from three persistently lymphocytotic and three uninfected cows (data not shown). Based on the ability of rhIL-2 to stimulate B-lymphocyte proliferation, neutralizing anti-IL-2 antibody was used to determine whether bovine IL-2 in the stimulated T-lymphocyte supernatant was necessary for the observed B-lymphocyte proliferation. Polyclonal rabbit anti-rhIL-2 antibody completely inhibited the proliferative response to T-lymphocyte supernatant (Fig. 6). Addition of the control antibody, R9415F, had no effect on proliferation of B lymphocytes, and the rabbit anti-IL-2 antibody did not inhibit proliferation of an IL-2-independent B-lymphocyte cell line, Raji (data not shown). Therefore, the observed B-lymphocyte proliferation is dependent on bovine T-lymphocyte-derived IL-2.

FIG. 6.

Inhibition of B-lymphocyte proliferation by anti-IL-2 antibody. B lymphocytes isolated from a persistently lymphocytotic (PL) cow (1583), B lymphocytes isolated from an uninfected (−) cow (1649), and IL-2-dependent clonal T lymphocytes (G4.2E8) were stimulated with a 1:5 dilution of supernatant from ConA-stimulated T lymphocytes (T-cell Sup) isolated from a persistently lymphocytotic cow. Results of wells with B lymphocytes incubated in medium alone are included to indicate background proliferation (white bars). Fifty micrograms of polyclonal rabbit anti-IL-2 antibody (Ab) (EP100) or polyclonal rabbit control antibody (R9415F) per ml was added to wells at the beginning of culture. Results are expressed as mean [³H]thymidine uptake for quadruplicate wells. Error bars are standard errors representing within assay variation of quadruplicate wells.

Spontaneous proliferation of PBMC from persistently lymphocytotic BLV-infected cows is IL-2 and viral antigen dependent.

PBMC from persistently lymphocytotic cows spontaneously incorporate [³H]thymidine when placed in culture, predominantly due to proliferation of B lymphocytes (27, 28). In 14 individual experiments, spontaneous [³H]thymidine uptake of PBMC from persistently lymphocytotic cows was 2.5 to 50 times that of uninfected cows measured in the same assay (combined data presented in Fig. 7A). Since stimulated T lymphocytes produced B-lymphocyte-stimulatory IL-2 in the previous experiments, the hypothesis that the spontaneous proliferation characteristic of persistent lymphocytosis is IL-2 dependent was tested. Addition of polyclonal rabbit anti-rhIL-2 antibody at the beginning of the 3-day lymphocyte proliferation assay completely inhibited spontaneous proliferation of PBMC from persistently lymphocytotic cows (Fig. 7B), while control antibody had no effect, indicating that spontaneous proliferation of PBMC from persistently lymphocytotic cows is IL-2 dependent.

FIG. 7.

Anti-IL-2 antibody and anti-BLV antibody inhibit spontaneous proliferation of PBMC from persistently lymphocytotic cows. (A) Spontaneous [³H]thymidine uptake in 3-day lymphoproliferation assays is plotted for 14 separate experiments including PBMC from five different persistently lymphocytotic (PL) and six different uninfected (BLV−) cows. (B) PBMC from four persistently lymphocytotic (PL) cows (1583, 1602, 1713, and 1833) were incubated for 3 days in medium alone or in medium with 50 μg of either polyclonal rabbit anti-IL-2 antibody (Ab) (EP100) or polyclonal rabbit control antibody (R9415F) per ml. (C) PBMC from three persistently lymphocytic (PL) cows (1583, 1622, and 1713) and 3 uninfected (−) cows (1616, 1728, and 1754) were incubated for 3 days in medium alone or medium with 200 μg of either polyclonal bovine anti-BLV antibody or polyclonal bovine control antibody per ml. Bars represent the mean [³H]thymidine uptake of quadruplicate wells. Error bars are standard errors representing within assay variation of quadruplicate wells.

To determine the role of antigen in spontaneous proliferation of PBMC from persistently lymphocytotic cows, the effect of purified immunoglobulin from a BLV-infected cow was examined. Anti-BLV antibody significantly inhibited spontaneous proliferation compared to the control antibody (purified immunoglobulin from an uninfected cow) (Fig. 7C). This finding confirms an earlier report in the literature (53) and suggests that spontaneous proliferation is dependent on viral antigen, as well as IL-2.

DISCUSSION

Since cytokines are critical in the stimulation and regulation of normal B lymphocytes, we hypothesize that a B-lymphocyte-stimulatory cytokine environment is required for the development and maintenance of the expanded B-lymphocyte population during BLV-induced persistent lymphocytosis. Consistent with this hypothesis, IL-2 and IL-10 mRNA expression in ConA-stimulated PBMC from cows with BLV-induced persistent lymphocytosis were significantly elevated. Increased IL-2 functional activity in supernatants from ConA-stimulated PBMC corroborates the elevated IL-2 mRNA expression, and altered kinetics of IL-2 and IL-10 mRNA expression provide additional support that cytokine expression is indeed changed during persistent lymphocytosis.

The finding of increased IL-2 expression confirms an earlier report of increased IL-2-like biologic activity in ConA-stimulated PBMC supernatants from persistently lymphocytotic cows (49) and a recent study in which IL-2 mRNA expression in unstimulated PBMC from persistently lymphocytotic BLV-ifected cows was increased approximately 20 times that of uninfected cows (44). Lymphocytes from persistently lymphocytotic cows were able to produce extremely high levels of IL-2 despite PBMC populations containing on average less than half as many T lymphocytes as PBMC from nonlymphocytotic or uninfected cows. Since IL-2 and IL-10 have been shown, alone and in synergy, to stimulate human B-lymphocyte proliferation and differentiation (14, 21, 22, 34, 47), the finding of elevated IL-2 and IL-10 expression supports the hypothesis that a B-lymphocyte-stimulatory cytokine environment predominates in BLV-infected cows with persistent lymphocytosis.

Increased expression of IL-2 and IL-10 suggests that these cytokines may contribute to expansion of the B-lymphocyte population during persistent lymphocytosis. Indeed, B lymphocytes from persistently lymphocytotic cows demonstrated dose-dependent proliferation to both rhIL-2 and bovine IL-2-containing T-lymphocyte culture supernatants. The necessity of IL-2 for the stimulatory activity of the T-lymphocyte culture supernatant was demonstrated by complete inhibition of B-lymphocyte proliferation with a polyclonal anti-IL-2 antibody. These results confirm that B lymphocytes from persistently lymphocytotic cows proliferate in response to bovine T-lymphocyte-derived IL-2 (36) and indicate that T lymphocytes may be involved in B-lymphocyte dysregulation during persistent lymphocytosis, as has previously been suggested (50). In addition to stimulating B-lymphocyte proliferation, rhIL-2 induced increased IL-2R and BLV expression, suggesting a positive feedback loop in which IL-2 produced by stimulated T lymphocytes activates viral expression in B lymphocytes, which feeds back to further stimulate IL-2 production. Similarly, IL-2-induced upregulation of IL-2R on B lymphocytes would further enhance their IL-2 responsiveness (14, 50).

Interestingly, upregulation of IL-2 and IL-2R expression also occurs in HTLV-infected human T lymphocytes (45, 54). The tax gene product of HTLV transactivates IL-2 and IL-2R transcription, leading to increased production of IL-2 and increased expression of IL-2R in HTLV-infected human T lymphocytes (2, 18, 20). Upregulation of IL-2 and IL-2R expression may thereby contribute to the early stages of HTLV-induced disease by autocrine or paracrine stimulation of T lymphocytes. HTLV-1 Tax also transactivates other cytokines, including IL-10 (39), as well as other genes important in cell growth, such as c-fos (45, 54). A similar virus-induced increase in IL-2 expression has recently been reported in HIV type 1-infected cells. As for HTLV-1 Tax, the HIV type 1 transactivator, Tat, induces increased transcription of IL-2 through the CD28-responsive element in the IL-2 promoter and thereby may contribute to the chronic activation of the immune response seen in infected patients (17, 42). A similar mechanism could be proposed in BLV infection; however, the potential importance of BLV Tax in upregulating IL-2 expression is complicated by the fact that T-helper lymphocytes, the presumed source of IL-2, are not infected by BLV (37). Therefore, a sufficient quantity of soluble Tax would be necessary to transactivate IL-2 production by T lymphocytes.

Spontaneous incorporation of [³H]thymidine in cultured PBMC is a common feature in persistent BLV, HIV, and HTLV infections (23, 31, 40, 43). Since spontaneous lymphoproliferation correlates to clinical disease progression, it has been proposed that spontaneous lymphoproliferation is an in vitro reflection of disease in vivo (35). Spontaneous proliferation of PBMC from persistently lymphocytotic cows was inhibited by anti-BLV antibody. It has previously been shown that the inhibitory activity of anti-BLV serum copurifies with immunoglobulin and is reversed by absorption with BLV (53), suggesting spontaneous proliferation is viral antigen specific. Further, development of persistent lymphocytosis is linked to specific bovine major histocompatibility complex class II haplotypes (56), and spontaneous proliferation is partially inhibited by anti-major histocompatibility complex class I and II antibody (52), providing additional evidence for the contribution of viral antigen to spontaneous proliferation. In this report, it was shown that spontaneous proliferation is dependent on IL-2 as well as on BLV antigen. Therefore, spontaneous proliferation of PBMC from persistently lymphocytotic cows may be the result of viral antigen-induced IL-2 production by T lymphocytes, stimulating a positive feedback mechanism.

Although a direct effect of IL-10 on B-lymphocyte proliferation was not demonstrated, our results, as well as a previous report showing increased IL-10 mRNA expression in unstimulated PBMC during persistent lymphocytosis (44), suggests a role for IL-10 in BLV-induced disease. The mechanism of activation of B lymphocytes (e.g., through CD40 versus cross-linking of sIg) (47), the timing of addition of IL-10 (21), and the presence or absence of other cytokines (22) have all been shown to influence the B-lymphocyte response to IL-10. In addition to its effects on B lymphocytes, IL-10 is an important regulator of T lymphocytes and macrophages (38). Loss of T-helper function in HIV-infected individuals has been shown to be mediated, at least in part, by IL-10 (8). Similarly, the inhibitory effect of IL-10 on bovine T-helper function (4) could partially explain the recently reported decrease in BLV antigen-specific proliferation of CD4⁺ T lymphocytes from persistently lymphocytotic cows (41). Additionally, the ability of IL-10 to downregulate IL-2R, but not IL-2, expression in bovine T lymphocytes (7) suggests a mechanism by which elevated IL-2 could preferentially stimulate proliferation of B lymphocytes without a concurrent increase in T lymphocytes during persistent lymphocytosis. Therefore, although exogenous IL-10 did not directly effect B-lymphocyte proliferation or BLV expression in these experiments, additional parameters will be explored in future experiments to determine the possible contribution of elevated IL-10 to BLV-induced disease.

In summary, this report demonstrates that IL-2 and IL-10 expression was elevated during BLV-induced persistent lymphocytosis. Further, IL-2 enhanced proliferation, IL-2R expression, and BLV p24 expression in B lymphocytes from persistently lymphocytotic cows. Finally, IL-2 was shown to be necessary for the spontaneous proliferation characteristic of PBMC from persistently lymphocytotic cows. These findings strongly suggest that increased expression of IL-2 contributes to development and/or maintenance of BLV-induced persistent B lymphocytosis and provides insight into the mechanism of immune dysregulation during chronic retrovirus infection.