Department of Veterinary Microbiology and Pathology, Washington State University, PO Box 647040, Pullman, WA 99164, USA1
Author for correspondence: Diana Stone. Fax +1 509 335 8529. e-mail dstone{at}vetmed.wsu.edu
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Abstract |
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Introduction |
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One hallmark of HTLV and BLV infection is the incorporation of [3H]thymidine by peripheral blood lymphocytes when cultured in the absence of exogenous antigen or mitogen (Itoyama et al., 1988 ; Prince et al., 1991a
, b
; Lal et al., 1992
; Muscoplat et al., 1974
; Richardson et al., 1997
; Lal & Rudolph, 1991
), referred to as spontaneous lymphocyte proliferation. Because both clinical and lymphocyte phenotypic changes in HTLV and BLV infection are associated with spontaneous lymphocyte proliferation, it is suggested that this proliferation is an in vitro correlate of the disease process (Itoyama et al., 1988
; Mann et al., 1994
). The transition from asymptomatic infection to PL in BLV-infected cattle is well defined and there is a clear association between PL and spontaneous lymphocyte proliferation (Thorn et al., 1981
; Muscoplat et al., 1974
). BLV-induced PL in cattle is also characterized by increased in vivo viral gene expression and a marked increase in provirus-containing B lymphocytes (Mirsky et al., 1993
, 1996
; Gaynor et al., 1996
). Although viral gene expression is restricted in vivo, BLV proteins and virus particles are detected within 3 to 6 h after peripheral blood mononuclear cells (PBMC) from PL cattle are put into culture (Jensen et al., 1990
; Baliga & Ferrer, 1977
; Dijilali et al., 1987
) and thus, prior to peak spontaneous lymphocyte proliferation at 72 h (Jensen et al., 1990
; Baliga & Ferrer, 1977
). In addition, suppression of in vitro virus expression by either anti-BLV antibody or protein kinase C inhibitors decreases spontaneous proliferation (Trueblood et al., 1998
; Thorn et al., 1981
; Jensen et al., 1992
). Thus, the spontaneous lymphocyte proliferation associated with BLV-induced PL in cattle provides an excellent animal model system to investigate retroviral gene expression and mechanisms of lymphocyte activation and expansion associated with disease progression.
The expansion of B lymphocytes in BLV-induced PL could be due to increased proliferation and/or increased lifespan. To investigate the interaction between BLV expression, cell cycle progression and cell survival, we analysed lymphocyte subset DNA profiles, apoptosis and BLV expression during the time-frame of spontaneous lymphocyte proliferation on a single-cell basis using flow cytometric analysis. Contrary to expectations we found that proliferating lymphocytes did not contain detectable viral proteins and that lymphocytes with detectable expression of viral proteins were more likely to remain in G0/G1 of the cell cycle and were spared from apoptosis.
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Methods |
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Monoclonal antibodies.
Mouse monoclonal antibodies (MAbs) were obtained from the Washington State University Monoclonal Antibody Center, Pullman, WA, USA. Listed are the MAbs used in this study, the determinants recognized by these MAbs on bovine mononuclear cells and the MAb isotype. Cell populations were characterized with MAbs directed against the bovine monocyte/granulocyte lineage (DH59B, IgG1), CD14 (CAM36A, IgG1), bovine B lymphocytes (sIgM) (BIg73A, IgG1; PIg45A, IgG2b), the bovine T cell receptor (TCR) (CACT61A, IgM; GB21A, IgG2b), bovine (bo) CD4 lymphocytes (CACT138A, IgG1; GC50A1, IgM), boCD8 lymphocytes (CACT80C, IgG1; 7C2B, IgG2a), boCD3 lymphocytes (MMIA, IgG1), boCD2 (CACT95A, IgG1) and boCD5 lymphocytes (B29A, IgG2a). MAbs used for T lymphocyte depletion by complement-mediated lysis were boCD8 (7C2B, IgG2a), boCD4 (GC50A1, IgM), boCD2 (B26A4, IgM) and bovine
TCR (GB21A, IgG2b). For CD4 depletion alone MAb boCD4 (GC50A1) was used and for CD8 depletion alone MAb boCD8 (7C2B) was used. Mouse MAbs to BLV capsid protein p24 were obtained as a gift from D. Portetelle, Faculty of Agronomy, Gembloux, Belgium (4'G9, IgG1) and from Veterinary Medical Research Diagnostics (VMRD) (Pullman, WA, USA) (BLV3, IgG1). The gp51 epitope of the BLV envelope glycoprotein was identified with a mouse MAb to the G antigen (BLV1, IgG1; VMRD).
Lymphocyte isolation, subset depletion, culture and proliferation assays.
Blood was collected by jugular venipuncture in acid citrate dextrose and PBMC were isolated by density gradient centrifugation as previously described (Stone et al., 1995 ). Viable cells determined by trypan blue dye exclusion were plated at 5x105 cells per well in round-bottom 96-well plates (Sarstedt) in complete culture medium (RPMI-1640, antibiotics, 2 mM L-glutamine, 0·05 mM 2-mercaptoethanol and 10 mM HEPES buffer supplemented with 20% FBS). Lymphocytes were cultured at 37 °C in 5% CO2. For lymphoproliferation assays 0·5 µCi [methyl-3H]thymidine (6·7 Ci/mmole; NEN Life Science Products) was added at the initiation of culture for 24 h assays and for the last 18 h of culture for 72 h assays. Cells were harvested on an automated 96-well plate harvester (Tomtec) and the amount of radioactivity was determined by liquid scintillation spectroscopy (Wallac). Data are expressed as the mean and standard deviation (SD) of six replicate samples.
Complement-mediated depletion of T lymphocyte subsets was performed according to our previously published procedure (Stone et al., 1996 ).
Flow cytometry.
Single-colour surface staining of cells for flow cytometric analysis was performed using a standard protocol as previously described (Stone et al., 1995 ). The BLV internal antigen p24 was stained by first fixing cells in 2% formaldehyde in PBS for 20 min on ice, washing once in PBS, followed by permeabilization in 0·2% Tween 20 for 15 min at 37 °C. Following a second wash in PBS, cells were stained with MAb to BLV p24 (4'G9 or BLV3) by the standard protocol excluding fixation as the final step. Cells were either analysed for single fluorescence by resuspending the cells in PBS with 0·1% sodium azide as the final step or processed further as described below for detection of apoptotic nuclei by the TUNEL method or stained with propidium iodide for cell cycle analysis. Stained cells were enumerated using a FACSort flow cytometer (Becton Dickinson Immunocytometry Systems) and analysed with CELLQuest or Macintosh Attractors software. Percentages of live, blast-size, and both live and blast-size cell populations were calculated using the forward scatter (FSC) and right angle side scatter (SSC) properties of 2% formaldehyde-fixed cells and either CELLQuest or Macintosh Attractors software. Populations within PBMC were identified as either blast-size or non-blast-size based on the greater linear FSC of blast-size cells. Within the same cultures of PBMC, the live and dead cell populations were separated based on the increased log SSC of dead cells.
Cell cycle analysis.
Cells were stained with FITC using MAbs to detect sIgM (Blg73A) and CD3 (MMIA), permeabilized and stained for cell cycle analysis as previously described (Stone et al., 1995 ). Surface staining, fixation and permeabilization were carried out in a 96-well plate. Briefly, following staining for surface markers, cells were fixed with 2% formaldehyde in PBS for 20 min on ice, washed in PBS, permeabilized in 0·2% Tween 20 for 15 min at 37 °C and washed again in PBS. Cells were pooled from the 96-well plate to give 12x106 cells per 0·5 ml in 10 µg/ml propidium iodide with at least 22 Kunitz U/ml of RNase A. BLV p24 antigen was stained by the procedure described above followed by addition of the propidium iodide/RNase A reagent. For cell cycle analysis 2000040000 events were collected. Cells were gated to exclude cell debris and include all lymphocyte populations, including those with increased granularity typical of dead cells. Doublets were excluded from the final analysis using linear plots of FL2-A vs FL2-W. Cells were identified as either G2/M cells with a DNA content of 4n or as cells with a DNA content of less than 4n. The 2% formaldehyde fixation procedure used prior to permeabilization prevents leakage of small molecular mass DNA fragments from the nucleus so that total DNA content includes fragmented, apoptotic DNA (Darzynkiewicz et al., 1995
).
Incorporation and detection of BrdU.
5-Bromo-2'-deoxyuridine (BrdU) (Roche Molecular Biochemicals) was added to the complete medium at a final concentration of 10 µg/ml at the onset of culture. Phycoerythyrin (PE) was used to identify MAbs binding to sIgM (BIg73A) and CD3 (MMIA). BrdU was detected according to published procedure (Tough & Sprent, 1996 ). Briefly, cells were surface-stained by the standard protocol omitting the final fixation step, collected from the 96-well plate into 12x75 mm tubes in 0·5 ml PBS and fixed in 1·2 ml 95% ice-cold ethanol for 30 min. After washing, cells were resuspended in 1·0 ml formaldehyde fixative (1% formaldehyde, 0·01% Tween 20 in PBS) for 30 min at room temperature. After washing, cells were resuspended in DNase I from bovine pancreas (Sigma) at 50 Kunitz U/ml in 4·2 mM MgCl2/0·15 M NaCl, pH 5·0 and incubated for 10 min at room temperature. Cells were washed in PBS followed by addition of 50 µl anti-BrdU-FITC (Roche Molecular Biochemicals) diluted to 5 µg/ml to resuspended cell pellets. Cells were incubated for 30 min at room temperature, washed, resuspended in PBS containing 0·1% sodium azide and analysed by flow cytometry.
Detection of apoptotic cells.
Apoptotic cells were detected using the TUNEL technique performed according to the manufacturers instructions (ApopTag Kit Fluorescein, Oncor) with the following modifications in fixation and permeabilization. Cells were fixed in 2% formaldehyde for 20 min on ice, followed by permeabilization in 0·2% Tween 20 for 15 min at 37 °C. After the permeabilization procedure cells were washed three times in PBS before proceeding with the manufacturers instructions for the TUNEL assay. Cell populations identified by PE staining for internal antigens (p24, IgM, CD3) followed the TUNEL-FITC reaction. The propidium iodide/RNase A reagent used to detect the stage of cell cycle concurrent with apoptosis was added following TUNEL-FITC staining.
Statistical analysis.
Analysis of variance was used to determine significant differences at P0·05 in the number of B and T lymphocytes in G2/M between PL, NPL and NEG cow groups at all time-points and between time-points for each of the PL, NPL and NEG groups. For comparison of the two sample means at t=0 and t=24 h within a disease group a one-tailed Student t-test was used. For lymphoproliferation assays significant differences between the six replicate samples from non-depleted and CD4-depleted PBMC for each cow were determined by analysis of variance at the P
0·05. Students t-test was used to test for significance in the percentage of p24-expressing lymphocytes at t=24 h and t=72 h as well as for the percentage of p24-expressing lymphocytes in G2/M at these time-points.
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Results |
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The majority of bovine peripheral blood B lymphocytes spontaneously enter G2/M of the cell cycle during short-term culture regardless of BLV infection or PL status and independent of any accessory cells
Next we evaluated lymphocyte subset marker expression and DNA content (propidium iodide staining) of cultured PBMC to determine the percentage of B and T lymphocytes entering G2/M of the cell cycle in relation to BLV infection and PL status. For clarity, the term proliferating cells will be restricted to cells that take up exogenous nucleotides during culture. Cells that enter G2/M of the cell cycle during culture as determined by a twofold increase in peak intensity propidium iodide staining will not be described as proliferating cells as this change from 2n to 4n total DNA content is not always accompanied by uptake of exogenous nucleotides (Jones et al., 1994 ; Spinozzi et al., 1995
). PBMC from BLV-seronegative, BLV-infected NPL, and BLV-infected PL cattle were cultured without stimulation and the percentages of B and T lymphocytes with 4n DNA content were determined at 0, 24 and 72 h (Table 3
). The 2% formaldehyde fixation that preceded permeabilization prevents leakage of fragmented DNA from the cell (Darzynkiewicz et al., 1995
). Thus, results reflected total DNA content of all cells, including cells that both synthesized DNA and underwent DNA fragmentation by apoptosis. Results showed that the percentage of B lymphocytes with G2/M DNA content in freshly isolated PBMC ranged from 13% to 23% but means did not differ significantly by BLV infection or PL status. By 24 h there was a significant increase from t=0 in the percentage of B lymphocytes with G2/M DNA content in all cultures (P
0·001, calculation not shown in Table 3
). The percentage of B lymphocytes in G2/M at 24 h was between 51% and 57% and did not differ significantly at P
0·05 by BLV infection or PL status. By 72 h the percentage of B lymphocytes in G2/M was significantly higher in PBMC cultures from PL cattle compared to cultures from BLV-infected NPL cattle or NEG cattle (P
0·01). When PBMC from four PL and three BLV-seronegative cattle were depleted of CD4 or CD8 T lymphocytes there was no significant change in the percentage of B lymphocytes in G2/M at 72 h (data not shown). Depletion experiments on PBMC from two PL cattle showed that depletion of CD2, CD4, CD8 and
T lymphocytes also had no effect, nor did the depletion of monocytes with and without T lymphocyte depletions (data not shown). In contrast to the movement of the majority of bovine B lymphocytes into G2/M, few if any T lymphocytes from NPL or BLV-seronegative cattle entered the cell cycle during the 72 h time-frame (Table 3
). A low but significant number of T lymphocytes from PL cattle were in G2/M by 72 h post-culture compared to the earlier time-points (P
0·03).
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Discussion |
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Because suppression of BLV expression results in decreased spontaneous lymphocyte proliferation (Trueblood et al., 1998 ; Jensen et al., 1992
), our results suggest that viral expression by a subset of B lymphocytes promotes the proliferation of B lymphocytes that do not express virus. One possibility is that secreted viral proteins activate T lymphocytes, cells not infected by BLV, that in turn promote B lymphocyte proliferation. This is consistent with previous reports that spontaneous lymphocyte proliferation is, in part, mediated by IL-2 (Trueblood et al., 1998
; Stone et al., 1994
, 1995
) and our observations that a subset of CD4 T lymphocytes proliferate during spontaneous proliferation, that CD4 T lymphocyte depletion significantly decreases spontaneous proliferation, and that T lymphocytes in the absence of B lymphocytes do not become blast-size or take up appreciable [3H]thymidine. Other retroviruses code for proteins with important biological effects on uninfected T lymphocytes (Marriott et al., 1991
, 1992
; Lindholm et al., 1990
; Li et al., 1995
, 1997
; Ensoli et al., 1993
, 1990
; Mann & Frankel, 1991
). In HTLV, the transactivating protein Tax is secreted (Lindholm et al., 1990
) and can activate uninfected T lymphocytes (Marriott et al., 1991
). In HIV, the transactivating protein Tat can activate uninfected, quiescent T lymphocytes, generating a population of cells more permissive to infection (Li et al., 1997
).
B lymphocyte proliferation in BLV may also be due to a direct effect of secreted viral proteins on B lymphocytes. Our observation that CD4 T lymphocyte depletion decreases but does not abolish spontaneous lymphocyte proliferation and a previous report that purified B lymphocytes from PL cattle also spontaneously proliferate (Trueblood et al., 1998 ) lend support to this possibility.
Results from this study also revealed that the majority of both normal bovine B lymphocytes and B lymphocytes from BLV-infected cattle spontaneously enter G2/M of the cell cycle and die by apoptosis within the first 24 h of unstimulated culture. Of interest was the lack of association between this early B lymphocyte entry into G2/M and the number of BrdU-staining cells or the amount of [3H]thymidine uptake. One possible explanation is that bovine B lymphocytes initially utilize internal stores of nucleotides for the salvage pathway of DNA synthesis. Alternatively or additionally, bovine B lymphocytes may utilize the de novo pathway of DNA synthesis when first put into culture.
Our data provide evidence that BLV expression increases survival of the virus-expressing cells themselves and may promote the proliferation of virus-non-expressing B lymphocytes. If the low level but persistent virus expression that is known to occur in vivo in BLV-infected cattle is similarly able to perturb B lymphocyte cell cycle progression, survival and proliferation, this would greatly promote virus expansion within the host. First, increased survival of the non-dividing, virus-producing cells would maximize virus production. Second, if BLV more efficiently infects and integrates into dividing cells as has been shown for other retroviruses (reviewed by Luciw & Leung, 1994 ) then virus-induced proliferation of uninfected B lymphocytes would expand the number of target cells for infection. Over time these processes would result in B lymphocyte expansion and increased numbers of virus-infected cells, the defining characteristics of BLV-infected PL cattle. Because PL cattle are the main source of transmission of virus to uninfected animals (Hopkins & DiGiacomo, 1997
; DiGiacomo, 1992
), these processes would promote spread of the virus within the population. BLV infection of an outbred population of animals under natural conditions offers an excellent animal model system to investigate the effects of virus expression on lymphocyte activation and proliferation. In particular, our results provide evidence that BLV, like HTLV and HIV, may have important biological functions on uninfected T lymphocytes that may promote virus spread and disease progression.
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Acknowledgments |
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References |
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Received 22 September 1999;
accepted 20 December 1999.