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Increase in γδ T cells in the blood of cattle persistently infected with Bovine Leukemia Virus following administration of recombinant bovine IFN-γ
Veterinary Immunology and Immunopathology 101 (2004) 61–71
Increase in gd T cells in the blood of cattle persistently infected with Bovine Leukemia Virus following administration of recombinant bovine IFN-g Kenji Murakami*, Hiroshi Sentsui, Yasuo Inoshima, Shigeki Inumaru National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan Received 19 November 2003; received in revised form 15 March 2004; accepted 4 April 2004
Abstract To examine the effect of recombinant bovine interferon-g (rbIFN-g) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-g. All cattle were febrile after inoculation with IFN-g and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4þ and CD8þ T cells were decreased for 2– 3 days and then their numbers were recovered. The number of gd T cells increased after the fever. In contrast, the number of IgMþ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-g administration. These results suggest that IFN-g induces the up-regulation of gd T cells, decreases the number of IgMþ lymphocytes and suppresses the growth of BLV in BLVinfected cattle in vivo. # 2004 Elsevier B.V. All rights reserved. Keywords: BLV; Persistent lymphocytosis; Cytokine; IFN-g
1. Introduction Interferon (IFN) has various biological activities, including not only antiviral actions but also inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation and activation of various types of cells in the immune system (Balkwill and Taylor-Papadimitriou, 1978; Gastl and Huber, 1988; Morris et al.,
Abbreviations: PL, persistent lymphocytosis; EBL, enzootic bovine leukosis; BLV, bovine leukemia virus; AGID, agar gel immunodiffusion * Corresponding author. Tel.: þ81 298 38 7841; fax: þ81 298 38 7907. E-mail address: [email protected] (K. Murakami)
1987). On the basis of their antigenicity and biological and physicochemical properties, three distinct types, IFN-a, b and g, have been identified. These are fibroepithelial IFN produced by fibroblasts and epithelial cells, leukocyte IFN produced by leukocytes, and immune IFN produced by T lymphocytes on an immune-specific basis, respectively. Although IFN-g displays most of the biological activities that have been ascribed to the other interferons, it has 10- to 1000-fold lower specific antiviral activity and is 100– 10,000 times as active as an immunomodulator than the other classes of interferon (Pace et al., 1985). Activation of immunocytes such as macrophages and lymphocytes is expected to control chronic disease, including tumors, and IFN-g therapy is anticipated for it.
0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2004.04.016
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Enzootic bovine leukosis (EBL) is a disease complex of cattle associated with a B lymphocytotropic retrovirus, bovine leukemia virus (BLV). In BLV infection, abnormal regulation of B cell development results in sustained non-malignant, polyclonal expansion of CD5þ B cells, referred to as persistent lymphocytosis (PL) and the adult enzootic form of bovine lymphoma with variable progression (Burny et al., 1988; Depelchin et al., 1989; Meirom et al., 1993). However, little is known regarding the effect of IFN-g on lymphoma induced by BLV infection. Recently, a large amount of recombinant bovine IFN-g (rbIFN-g) has been produced by a baculovirus expression system (Murakami et al., 2001). The rbIFN-g successfully inhibits the syncytium formation of BLV in vitro (Sentsui et al., 2001). In the present study, we tried to investigate the effect of rbIFN-g on BLV-infected cattle with persistent lymphocytosis (PL) and those clinically normal in vivo. In particular, the reactions of rbIFN-g on gd T cells and IgMþ lymphocytes, which are targeted by BLV, were examined using flow cytometry.
2. Materials and methods 2.1. Animals and experimental design Identification, age and details of the five cattle (Holstein) used are listed in Table 1. Three BLV-
infected but clinically normal cow (H1–H3) and two cattle with PL (P1–P2), confirmed by hematological examination were subjected to the test. The number of leukocytes per microliter of blood was measured using an automatic blood cell counter (Coulter, Miami, FL). Lymphocytes were identified on Wright’s stained blood smears and the numbers of lymphocytes per microliter were estimated by multiplying the percentage of lymphocytes in 200 leukocytes counted for the white blood cell (WBC) differential distribution by the total leukocyte count. PL cattle were diagnosed according to the European key (Bendixen, 1965). The total lymphocyte counts in PL cattle used in this study ranged from 11,660 to 13,285/ml. Cattle H1–H3 were intraperitoneally injected with 100 units/g of body weight of rbIFN-g once. The dose of IFN-g was calculated according to a previously reported procedure (Higuchi et al., 2002). The effect of IFN-g administration was examined for 1 week (one-shot administration group). Cattle P1 and P2 were also intraperitoneally injected with the same dose of IFN-g three times every 3 days and the effects were examined for 2 weeks (three-shot administration group). Body temperature was measured twice daily over the experimental period. Hematological examinations were carried out every day using ethylene diamine-tetra acetic acid (EDTA)-treated blood taken from the jugular vein. BLV-specific antibodies in these cattle were also detected by the agar gel
Table 1 Outline and diagnosis of examined cases Lymphocytese
T cellsf (%)
IgMþcellsf (%)
Hematologic diagnosisg
Cattle for one-shot administration of bovine IFN-g H1 1Y þ þ 9000 H2 4Y þ þ 10400 H3 4Y þ þ 13100
6361 8429 9620
58.8 36.6 53.0
38.4 62.2 43.8
BLV-infected but healthy BLV-infected but healthy BLV-infected but healthy
Cattle for three-shot serial administration of bovine IFN-g P1 1Y þ þ 16300 P2 4Y þ þ 18800
13285 11660
39.6 25.6
60.0 55.2
PL PL
Case No.
a
Agea
gp51b
BLVc
WBCd
Y, years. BLV antibody gp51 was detected by the agar gel immunodiffusion (AGID) test. c BLV was determined by syncytium assay. d White blood cell (WBC) count/ml. e Lymphocyte count/ml whole blood. f T cells were stained with anti-bovine CD3 and IgM cells were stained with anti-bovine IgM monoclonal antibody. g PL, persistent lymphocytosis. b
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immunodiffusion (AGID) test to examine the presence of serum antibodies to gp51 of BLV, and BLV was detected by syncytium assay (Itohara and Mizuno, 1984; Kono et al., 1982). Percentage reductions of IgMþ lymphocytes and syncytia were calculated as follows: {1 [mean number (after fever)/mean number (pre-inoculation)]} 100. 2.2. Recombinant bovine interferon-g (rbIFN-g) and monoclonal antibodies
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the FITC-conjugated second antibody for 30 min at 4 8C. Cells were fixed with 0.5% paraformaldehyde/ PBS before analysis. Control cells were stained only with the second antibody. Stained cells were analyzed on an Epics XL flow cytometer (Coulter). Peripheral blood lymphocytes (PBLs) were distinguished from PBMCs by their characteristic orthogonal and forward light-scattering properties as revealed by flow cytometry (Hoffman et al., 1980). 2.4. Statistics
The rbIFN-g was prepared using a baculovirus expression system (Murakami et al., 2001). The recombinant baculovirus-infected insect cell culture fluid was used as a source of rbIFN-g after ultracentrifugation (45,000 g) and filtration. Suspension cultures of recombinant baculovirus-infected insect cells were cultivated in serum-free medium and the rbIFN-g was accumulated. The culture fluids had a titer of 105 units/ml and a specific activity of 2 103 units/mg of protein as assayed using the modified virus plaque reduction assay for vesicular stomatitis virus (Langford et al., 1981). Monoclonal antibodies (mAbs) for bovine lymphocytes used in this study were as follows: MM1A (mouse IgG anti-bovine CD3), CACT138A (mouse IgG anti-bovine CD4), CACT80C (mouse IgG antibovine CD8), 86D (mouse IgG anti-bovine gd-TCR), BIG73A (mouse IgG anti-bovine IgM), and TH14B (mouse IgG anti-bovine MHC class II) were kindly supplied by Dr. W.C. Davis of Washington State University (Pullman, WA) or purchased from Veterinary Medical Research and Development (VMRD) (Pullman, WA). An FITC-conjugated F(ab0 )2 fragment of goat anti-mouse IgG þ IgM (H þ L) (Jackson Immunoresearch Laboratories, West Grove, PA) was used as the second antibody. 2.3. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation for 30 min at 2,500 rpm with 800 g on 60% Percoll (Pharmacia Biotech, Uppsala, Sweden) and washed twice in phosphatebuffered saline (PBS). For immunofluorescence staining, 106 cells were incubated with an mAb for 30 min at 4 8C, washed three times in PBS containing 0.1% bovine serum albumin (BSA), and then treated with
Body temperatures of more than 39.5 8C were considered indicative of fever. The experiment period was divided into three terms: (i) pre-IFN-g inoculation period; (ii) febrile period; and (iii) post-febrile period. The absolute number of PBL, antibody positive cells and the number of syncytia in each period were calculated, and data were expressed as mean and S.D. The significance of differences between the mean of the (i) pre- and (iii) post-febrile periods was determined by Student’s t-test.
3. Results 3.1. Clinical symptoms of cattle with rbIFN-g administration Two of the three cattle (H2 and H3) receiving oneshot administration of rbIFN-g group and all of the cattle in the three-time administration group were immediately febrile (39.5–41.5 8C) after inoculation of IFN-g, and then the fever receded within 2 days. To evaluate IFN-g in the serum, the biological activity of IFN-g was measured by a modified plaque reduction assay in the serum of every cow from just after inoculation until this experiment terminated. However, biological activity of IFN-g was not detectable in the sera (data not shown). 3.2. Change of lymphocyte subsets by rbIFN-g administration The absolute numbers of PBL, CD4þ T cells, CD8þ T cells, gd T cells, IgMþ cells, and MHC class IIþ cells were compared between the pre-inoculation and post-febrile period in each animal using a Coulter
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counter, Giemsa staining blood smears and flow cytometric analysis. There results and the significance of statistics are shown in Table 2. PBL were immediately decreased significantly just after the first IFN-g administration in four of the five cattle (Fig. 1A and B). However, when IFN-g administration was repeated, the decrease of PBL became a little milder or was not evident. No
decrease of PBL numbers was observed in P2 after the third IFN-g administration (Fig. 1B). The numbers of CD4þ T cells were increased by IFN-g administration compared to the pre-administration period in four of the five cattle and this phenomenon was significantly evident in H1 and P2 (P < 0:05 and P < 0:01, respectively). The numbers of CD8þ T cells were decreased just after IFN-g inoculation, but
Fig. 1. Changes in the numbers of peripheral blood lymphocytes (PBLs) in BLV-infected cattle after administration of recombinant bovine IFN-g. The number of PBL is indicated before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of PBL before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
PBL/mm3 One-shot administration of IFN-g H1 Pre-inoculation 6452.8 417.3 After fever 6986.0 997.9 H2 Pre-inoculation 8452.0 423.0 After fever 6839.0 144.0** H3 Pre-inoculation After fever
CD4a
CD8a
CD4/CD8
1588.2 216.7 2503.3 997.2*
1055.1 93.2 1408.9 432.0
1.57 0.21 1.81 0.25
889.0 70.1 670.3 207.6
853.0 146.5 757.6 107.0
1.03 0.10 0.91 0.37
9550.0 257.4 2584.7 307.7 7804.2 809.1** 2947.0 450.7
Three-shot administration of IFN-g P1 Pre-inoculation 12931.6 391.9 2058.0 242.3 After fever 10122.0 1766.5* 2216.8 215.4 P2 Pre-inoculation 11607.0 785.9 After fever 11635.0 473.7
184.7 17.5 1.48 0.26 1720.3 486.9** 1.78 0.30
1698.6 91.0 1551.0 346.0
1.22 0.20 1.50 0.43
1202.3 202.5 571.0 104.1 2.07 0.02 2813.7 260.5** 1656.2 124.5** 1.69 0.08**
gdT cella
535.3 157.3 719.4145.3*
IgMa
2692.1 151.4 2016.9 344.0**
MHC IIa
380.7 110.2 342.7 147.7
274.6 11.7 5227.0 229.6 3987.0 1597.1 1154.6 143.1** 1969.3 236.6** 2698.0 346.3 413.8 51.3 312.2 75.9
324.3 164.8 1999.6 1550.0 330.3 103.9 765.2 82.1**
4423.2 569.4 1997.2 152.4 2395.8 314.1** 1500.2 481.7*
Syncytia
496.6 135.7 380.0 299.0 1503.2 668.8 94.2 174.7** 819.3 313.7 318.6 141.7*
7870.6 784.0 6152.3 1032.9 1235.3 228.3 2937.1 557.3** 1897.5 582.8** 61.6 23.4** 6231.3 931.7 6720.6 1320.8 3116.7 1265.0* 4025.7 862.1*
2013.3 316.2 473.0 378.6**
The CD4þ, CD8þ, gd T cell, IgMþ cell, and MHC class IIþ counts were calculated from the percentage of anti-CD4, anti-CD8, anti-gd, anti-IgM and anti-MHC class II positive cells among PBL out of the total lymphocytes/mm3. Data show the mean S.D. Asterisk indicates significance relative to pre-inoculation. * P < 0:05. ** P < 0:01. a
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Table 2 Flow cytometric analysis and syncytium assay in BLV-infected cattle at pre-inoculation of bovine IFN-g and in the post-febrile period
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significantly increased in H3 and P2 after the febrile period (P < 0:01, respectively). Only in P2 did the CD4/CD8 ratio significantly decrease in the postfebrile period (P < 0:01). The number of gd T cells was increased 2 or 3 days after rbIFN-g administration and was maintained 1.3- and 4.2-folds higher in H1 and H2 in the post-febrile period (P < 0:05 and 0.01, respectively) (Table 2 and Fig. 2A). When rbIFN-g administration was repeated three times, one animal (P1) showed a high increase in the gd
T cell number immediately after rbIFN-g administration, but not after the third administration. The gd T cell number was, however, drastically increased after the fever (Fig. 2B). In another cow, the number also significantly increased after the fever (P2, P < 0:01) although less remarkably than in P1. IgMþ cell numbers were also significantly decreased after rbIFN-g administration in all cattle (Fig. 3A and B). A decrease in the IgMþ cell number was the main factor of the fluctuation of PBL numbers of H2, H3
Fig. 2. Changes in the numbers of gd T cells in BLV-infected cattle after administration of recombinant bovine IFN-g. gd T cells were stained with anti-gd TCR mAb and analyzed by flow cytometry before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of gd T cells before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
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Fig. 3. Changes in the numbers of IgMþ lymphocytes in BLV-infected cattle after administration of recombinant bovine IFN-g. IgMþ lymphocytes were stained with anti-IgM mAb and analyzed by flow cytometry before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of IgMþ lymphocytes before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
and P1 after IFN-g administration. Interestingly, there was no obvious decrease in the PBL number in two cattle, H1 and P2. One was a non-PL cow (H1), and her IgMþ cell number was decreased by 25.0%. The other was a PL cow (P2) and her IgMþ cell number was decreased by 50.8%. In contrast, CD4þ, CD8þ and gd T cell numbers were increased in both cattle. The cells presenting MHC class II
antigen showed patterns similar to those of IgMþ cells. 3.3. Decrease of syncytium formation after rbIFN-g administration In the cattle treated with rbIFN-g once, syncytium numbers of their PBL were significantly decreased but
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K. Murakami et al. / Veterinary Immunology and Immunopathology 101 (2004) 61–71
Fig. 4. Changes in the number of syncytia in BLV-infected cattle after administration of recombinant bovine IFN-g. PBL were obtained from BLV-infected cattle before and after one-shot (A) or three-shot (B) serial administration of recombinant bovine IFN-g. The cells were subjected to syncytium assay. The numbers of syncytia were counted under a microscope. The mean number S.D. is shown on the ordinate (H1, n ¼ 3; H2, n ¼ 5; H3, n ¼ 3; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
recovered within 1 week (Fig. 4A). In the cattle receiving IFN-g administration three times, syncytium numbers were reduced for at least 2 weeks (Fig. 4B).
4. Discussion The effect of recombinant bovine IFN-g on BLVinfected cattle was examined. All cattle were febrile
after inoculation with IFN-g and then recovered within 48 h. The numbers of PBL, CD4þ and CD8þ T cells were decreased for 2–3 days and then recovered in most of the cattle. Our previous study on the effect of IFN-g in cattle without BLV infection also showed that cattle were febrile temporarily after injection of IFN-g and the numbers of PBL, CD4þ, CD8þ T cells and B cells were decreased for 1–3 days and then recovered (Shimizu et al., Annu. Meet. Jpn. Soc. Vet. Sci., 127th,
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1999). These side effects, including fever and mild hematologic depression, were similar to those of IFN administration to humans (Tamura et al., 1987). As cattle were injected with IFN-g, the number of gd T cells temporarily increased within 1 h in normal cattle without BLV infection (Shimizu et al., Annu. Meet. Jpn. Soc. Vet. Sci., 127th, 1999). In this study, the number of gd T cells also increased after the fever and remained at a significantly high level in some cattle infected with BLV. Therefore, it was suggested that gd T cells were activated by IFN-g in vivo. IgMþ cell numbers and syncytium numbers were conversely decreased, accompanied by the increase of gd T cells. Because the syncytium assay was done using PBL in these experiments, syncytium numbers might have decreased with the ratio of IgMþ cells in PBL. However, the reduction percentage of syncytium numbers was higher than that of IgMþ cell numbers in PL cattle (Fig. 5). Although the two phenomena might be unrelated, our present working hypothesis is that gd T cells might have been activated by IFN-g and eliminated BLV-infected IgMþ cells from these cattle. gd T cells possess strong MHC-independent cytolytic activity and, in some instances, preferentially kill
Fig. 5. Percentage reduction of IgMþ lymphocytes and numbers of syncytia at the post-febrile period after recombinant bovine IFN-g. Values shown are percentage reduction of IgMþ lymphocyte numbers and numbers of syncytia as described in Section 2.1.
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autologous tumor cells and virus-infected cells (Haas et al., 1993). Based on these effector functions, it is conceivable that gd T cells might exhibit cytolytic reactivity against BLV-infected cells. The lymphoid systems of cattle and sheep contain large gd T lymphocyte subpopulations, in striking contrast to the lymphoid systems of the human and mouse (Mackay and Hein, 1989; Mackay et al., 1986). Approximately 20–30% of gd T cells express CD8, although this occurs at a lower level than for ab T cells (Haas et al., 1993). In the spleen and lymph nodes, gd T cells are localized in regions of cellular traffic, and they exhibit cytotoxic activity against allogenic cells (Mackay and Hein, 1989). This information also supports the idea that gd T cells activated by IFN-g can effectively eliminate BLV-infected cells in ruminants. The number of gd T cells was preserved in the PBL from BLVinfected but healthy sheep and sheep with lymphoma compared to those from normal sheep, and the accumulation of gd T cells was also observed in tumorous lymph nodes (Murakami et al., 1994). Recently, the continued presence of BLV transcripts was reported in significant numbers of B cells (Rovnak and Casey, 1999). Thus, it is possible that gd T cells recognise BLV on the B cell surface as an allogenic antigen, are up-regulated and exclude BLV-infected B lymphocytes. At present, the immune modulating capabilities of IFN-g remain to be proven. Anti-viral functions of IFN are classified into two types, those with direct action and indirect action. IFN directly suppresses cell proliferation and/or kill virus-infected cells. The number of IgMþ cells and syncytia might be depressed by IFN-g administration via direct action, because our in vitro experiment using rbIFN-g and persistently BLV-infected FLK showed successful inhibition of syncytium formation (Sentsui et al., 2001). However, in the present in vivo experiments, immunomodulatory effects, which enhance the cytotoxic activity of macrophages, cytotoxic T cells and natural killer (NK) cells, would be also considerable in the antiBLV reaction of IFN-g. Although IFN-g acts to increase the intensity of class I and class II MHC expression on PBL in vitro, it inhibits MHC class II expression on B cells (Mond et al., 1986; Walrand et al., 1989). Our result showed that the number of class II-positive cells in PBL was not up-regulated by IFN-g inoculation in vivo, because
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the animals we used had plenty of B cells in PBL induced by BLV infection. Clinical trials of recombinant IFN-g on BLVinfected cattle are expected to be effective. The activation of gd T cells as well as that of CD8þ T cells may be an important approach for the therapy of BLV-infected cattle, because there are large amounts of gd T cells in ruminants compared to other species. In humans, in vivo, combination therapy using recombinant IFN-a or -g and anticancer drugs has been reported to achieve remarkable tumor regression in adult T cell leukemia (ATL) (Ezaki et al., 1991; Makidono et al., 1986). To develop effective therapeutics for PL and EBL induced by BLV infection, further basic as well as clinical studies should be designed to explore and assess the agents’ ability to control this disease and its causative virus BLV.
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Increase in gd T cells in the blood of cattle persistently infected with Bovine Leukemia Virus following administration of recombinant bovine IFN-g Kenji Murakami*, Hiroshi Sentsui, Yasuo Inoshima, Shigeki Inumaru National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki 305-0856, Japan Received 19 November 2003; received in revised form 15 March 2004; accepted 4 April 2004
Abstract To examine the effect of recombinant bovine interferon-g (rbIFN-g) on cattle persistently infected with bovine leukemia virus (BLV), BLV-infected cattle were inoculated intraperitoneally with IFN-g. All cattle were febrile after inoculation with IFN-g and then recovered within 48 h. Flow cytometric analysis showed that the numbers of CD4þ and CD8þ T cells were decreased for 2– 3 days and then their numbers were recovered. The number of gd T cells increased after the fever. In contrast, the number of IgMþ lymphocytes remained low for about 1 week. Moreover, the numbers of syncytia produced by peripheral blood lymphocytes decreased and remained low compared to that before IFN-g administration. These results suggest that IFN-g induces the up-regulation of gd T cells, decreases the number of IgMþ lymphocytes and suppresses the growth of BLV in BLVinfected cattle in vivo. # 2004 Elsevier B.V. All rights reserved. Keywords: BLV; Persistent lymphocytosis; Cytokine; IFN-g
1. Introduction Interferon (IFN) has various biological activities, including not only antiviral actions but also inhibition of cell growth and proliferation, regulation of the expression of specific genes, modulation of cell differentiation and activation of various types of cells in the immune system (Balkwill and Taylor-Papadimitriou, 1978; Gastl and Huber, 1988; Morris et al.,
Abbreviations: PL, persistent lymphocytosis; EBL, enzootic bovine leukosis; BLV, bovine leukemia virus; AGID, agar gel immunodiffusion * Corresponding author. Tel.: þ81 298 38 7841; fax: þ81 298 38 7907. E-mail address: [email protected] (K. Murakami)
1987). On the basis of their antigenicity and biological and physicochemical properties, three distinct types, IFN-a, b and g, have been identified. These are fibroepithelial IFN produced by fibroblasts and epithelial cells, leukocyte IFN produced by leukocytes, and immune IFN produced by T lymphocytes on an immune-specific basis, respectively. Although IFN-g displays most of the biological activities that have been ascribed to the other interferons, it has 10- to 1000-fold lower specific antiviral activity and is 100– 10,000 times as active as an immunomodulator than the other classes of interferon (Pace et al., 1985). Activation of immunocytes such as macrophages and lymphocytes is expected to control chronic disease, including tumors, and IFN-g therapy is anticipated for it.
0165-2427/$ – see front matter # 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2004.04.016
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Enzootic bovine leukosis (EBL) is a disease complex of cattle associated with a B lymphocytotropic retrovirus, bovine leukemia virus (BLV). In BLV infection, abnormal regulation of B cell development results in sustained non-malignant, polyclonal expansion of CD5þ B cells, referred to as persistent lymphocytosis (PL) and the adult enzootic form of bovine lymphoma with variable progression (Burny et al., 1988; Depelchin et al., 1989; Meirom et al., 1993). However, little is known regarding the effect of IFN-g on lymphoma induced by BLV infection. Recently, a large amount of recombinant bovine IFN-g (rbIFN-g) has been produced by a baculovirus expression system (Murakami et al., 2001). The rbIFN-g successfully inhibits the syncytium formation of BLV in vitro (Sentsui et al., 2001). In the present study, we tried to investigate the effect of rbIFN-g on BLV-infected cattle with persistent lymphocytosis (PL) and those clinically normal in vivo. In particular, the reactions of rbIFN-g on gd T cells and IgMþ lymphocytes, which are targeted by BLV, were examined using flow cytometry.
2. Materials and methods 2.1. Animals and experimental design Identification, age and details of the five cattle (Holstein) used are listed in Table 1. Three BLV-
infected but clinically normal cow (H1–H3) and two cattle with PL (P1–P2), confirmed by hematological examination were subjected to the test. The number of leukocytes per microliter of blood was measured using an automatic blood cell counter (Coulter, Miami, FL). Lymphocytes were identified on Wright’s stained blood smears and the numbers of lymphocytes per microliter were estimated by multiplying the percentage of lymphocytes in 200 leukocytes counted for the white blood cell (WBC) differential distribution by the total leukocyte count. PL cattle were diagnosed according to the European key (Bendixen, 1965). The total lymphocyte counts in PL cattle used in this study ranged from 11,660 to 13,285/ml. Cattle H1–H3 were intraperitoneally injected with 100 units/g of body weight of rbIFN-g once. The dose of IFN-g was calculated according to a previously reported procedure (Higuchi et al., 2002). The effect of IFN-g administration was examined for 1 week (one-shot administration group). Cattle P1 and P2 were also intraperitoneally injected with the same dose of IFN-g three times every 3 days and the effects were examined for 2 weeks (three-shot administration group). Body temperature was measured twice daily over the experimental period. Hematological examinations were carried out every day using ethylene diamine-tetra acetic acid (EDTA)-treated blood taken from the jugular vein. BLV-specific antibodies in these cattle were also detected by the agar gel
Table 1 Outline and diagnosis of examined cases Lymphocytese
T cellsf (%)
IgMþcellsf (%)
Hematologic diagnosisg
Cattle for one-shot administration of bovine IFN-g H1 1Y þ þ 9000 H2 4Y þ þ 10400 H3 4Y þ þ 13100
6361 8429 9620
58.8 36.6 53.0
38.4 62.2 43.8
BLV-infected but healthy BLV-infected but healthy BLV-infected but healthy
Cattle for three-shot serial administration of bovine IFN-g P1 1Y þ þ 16300 P2 4Y þ þ 18800
13285 11660
39.6 25.6
60.0 55.2
PL PL
Case No.
a
Agea
gp51b
BLVc
WBCd
Y, years. BLV antibody gp51 was detected by the agar gel immunodiffusion (AGID) test. c BLV was determined by syncytium assay. d White blood cell (WBC) count/ml. e Lymphocyte count/ml whole blood. f T cells were stained with anti-bovine CD3 and IgM cells were stained with anti-bovine IgM monoclonal antibody. g PL, persistent lymphocytosis. b
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immunodiffusion (AGID) test to examine the presence of serum antibodies to gp51 of BLV, and BLV was detected by syncytium assay (Itohara and Mizuno, 1984; Kono et al., 1982). Percentage reductions of IgMþ lymphocytes and syncytia were calculated as follows: {1 [mean number (after fever)/mean number (pre-inoculation)]} 100. 2.2. Recombinant bovine interferon-g (rbIFN-g) and monoclonal antibodies
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the FITC-conjugated second antibody for 30 min at 4 8C. Cells were fixed with 0.5% paraformaldehyde/ PBS before analysis. Control cells were stained only with the second antibody. Stained cells were analyzed on an Epics XL flow cytometer (Coulter). Peripheral blood lymphocytes (PBLs) were distinguished from PBMCs by their characteristic orthogonal and forward light-scattering properties as revealed by flow cytometry (Hoffman et al., 1980). 2.4. Statistics
The rbIFN-g was prepared using a baculovirus expression system (Murakami et al., 2001). The recombinant baculovirus-infected insect cell culture fluid was used as a source of rbIFN-g after ultracentrifugation (45,000 g) and filtration. Suspension cultures of recombinant baculovirus-infected insect cells were cultivated in serum-free medium and the rbIFN-g was accumulated. The culture fluids had a titer of 105 units/ml and a specific activity of 2 103 units/mg of protein as assayed using the modified virus plaque reduction assay for vesicular stomatitis virus (Langford et al., 1981). Monoclonal antibodies (mAbs) for bovine lymphocytes used in this study were as follows: MM1A (mouse IgG anti-bovine CD3), CACT138A (mouse IgG anti-bovine CD4), CACT80C (mouse IgG antibovine CD8), 86D (mouse IgG anti-bovine gd-TCR), BIG73A (mouse IgG anti-bovine IgM), and TH14B (mouse IgG anti-bovine MHC class II) were kindly supplied by Dr. W.C. Davis of Washington State University (Pullman, WA) or purchased from Veterinary Medical Research and Development (VMRD) (Pullman, WA). An FITC-conjugated F(ab0 )2 fragment of goat anti-mouse IgG þ IgM (H þ L) (Jackson Immunoresearch Laboratories, West Grove, PA) was used as the second antibody. 2.3. Flow cytometric analysis Peripheral blood mononuclear cells (PBMCs) were obtained by centrifugation for 30 min at 2,500 rpm with 800 g on 60% Percoll (Pharmacia Biotech, Uppsala, Sweden) and washed twice in phosphatebuffered saline (PBS). For immunofluorescence staining, 106 cells were incubated with an mAb for 30 min at 4 8C, washed three times in PBS containing 0.1% bovine serum albumin (BSA), and then treated with
Body temperatures of more than 39.5 8C were considered indicative of fever. The experiment period was divided into three terms: (i) pre-IFN-g inoculation period; (ii) febrile period; and (iii) post-febrile period. The absolute number of PBL, antibody positive cells and the number of syncytia in each period were calculated, and data were expressed as mean and S.D. The significance of differences between the mean of the (i) pre- and (iii) post-febrile periods was determined by Student’s t-test.
3. Results 3.1. Clinical symptoms of cattle with rbIFN-g administration Two of the three cattle (H2 and H3) receiving oneshot administration of rbIFN-g group and all of the cattle in the three-time administration group were immediately febrile (39.5–41.5 8C) after inoculation of IFN-g, and then the fever receded within 2 days. To evaluate IFN-g in the serum, the biological activity of IFN-g was measured by a modified plaque reduction assay in the serum of every cow from just after inoculation until this experiment terminated. However, biological activity of IFN-g was not detectable in the sera (data not shown). 3.2. Change of lymphocyte subsets by rbIFN-g administration The absolute numbers of PBL, CD4þ T cells, CD8þ T cells, gd T cells, IgMþ cells, and MHC class IIþ cells were compared between the pre-inoculation and post-febrile period in each animal using a Coulter
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counter, Giemsa staining blood smears and flow cytometric analysis. There results and the significance of statistics are shown in Table 2. PBL were immediately decreased significantly just after the first IFN-g administration in four of the five cattle (Fig. 1A and B). However, when IFN-g administration was repeated, the decrease of PBL became a little milder or was not evident. No
decrease of PBL numbers was observed in P2 after the third IFN-g administration (Fig. 1B). The numbers of CD4þ T cells were increased by IFN-g administration compared to the pre-administration period in four of the five cattle and this phenomenon was significantly evident in H1 and P2 (P < 0:05 and P < 0:01, respectively). The numbers of CD8þ T cells were decreased just after IFN-g inoculation, but
Fig. 1. Changes in the numbers of peripheral blood lymphocytes (PBLs) in BLV-infected cattle after administration of recombinant bovine IFN-g. The number of PBL is indicated before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of PBL before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
PBL/mm3 One-shot administration of IFN-g H1 Pre-inoculation 6452.8 417.3 After fever 6986.0 997.9 H2 Pre-inoculation 8452.0 423.0 After fever 6839.0 144.0** H3 Pre-inoculation After fever
CD4a
CD8a
CD4/CD8
1588.2 216.7 2503.3 997.2*
1055.1 93.2 1408.9 432.0
1.57 0.21 1.81 0.25
889.0 70.1 670.3 207.6
853.0 146.5 757.6 107.0
1.03 0.10 0.91 0.37
9550.0 257.4 2584.7 307.7 7804.2 809.1** 2947.0 450.7
Three-shot administration of IFN-g P1 Pre-inoculation 12931.6 391.9 2058.0 242.3 After fever 10122.0 1766.5* 2216.8 215.4 P2 Pre-inoculation 11607.0 785.9 After fever 11635.0 473.7
184.7 17.5 1.48 0.26 1720.3 486.9** 1.78 0.30
1698.6 91.0 1551.0 346.0
1.22 0.20 1.50 0.43
1202.3 202.5 571.0 104.1 2.07 0.02 2813.7 260.5** 1656.2 124.5** 1.69 0.08**
gdT cella
535.3 157.3 719.4145.3*
IgMa
2692.1 151.4 2016.9 344.0**
MHC IIa
380.7 110.2 342.7 147.7
274.6 11.7 5227.0 229.6 3987.0 1597.1 1154.6 143.1** 1969.3 236.6** 2698.0 346.3 413.8 51.3 312.2 75.9
324.3 164.8 1999.6 1550.0 330.3 103.9 765.2 82.1**
4423.2 569.4 1997.2 152.4 2395.8 314.1** 1500.2 481.7*
Syncytia
496.6 135.7 380.0 299.0 1503.2 668.8 94.2 174.7** 819.3 313.7 318.6 141.7*
7870.6 784.0 6152.3 1032.9 1235.3 228.3 2937.1 557.3** 1897.5 582.8** 61.6 23.4** 6231.3 931.7 6720.6 1320.8 3116.7 1265.0* 4025.7 862.1*
2013.3 316.2 473.0 378.6**
The CD4þ, CD8þ, gd T cell, IgMþ cell, and MHC class IIþ counts were calculated from the percentage of anti-CD4, anti-CD8, anti-gd, anti-IgM and anti-MHC class II positive cells among PBL out of the total lymphocytes/mm3. Data show the mean S.D. Asterisk indicates significance relative to pre-inoculation. * P < 0:05. ** P < 0:01. a
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Table 2 Flow cytometric analysis and syncytium assay in BLV-infected cattle at pre-inoculation of bovine IFN-g and in the post-febrile period
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significantly increased in H3 and P2 after the febrile period (P < 0:01, respectively). Only in P2 did the CD4/CD8 ratio significantly decrease in the postfebrile period (P < 0:01). The number of gd T cells was increased 2 or 3 days after rbIFN-g administration and was maintained 1.3- and 4.2-folds higher in H1 and H2 in the post-febrile period (P < 0:05 and 0.01, respectively) (Table 2 and Fig. 2A). When rbIFN-g administration was repeated three times, one animal (P1) showed a high increase in the gd
T cell number immediately after rbIFN-g administration, but not after the third administration. The gd T cell number was, however, drastically increased after the fever (Fig. 2B). In another cow, the number also significantly increased after the fever (P2, P < 0:01) although less remarkably than in P1. IgMþ cell numbers were also significantly decreased after rbIFN-g administration in all cattle (Fig. 3A and B). A decrease in the IgMþ cell number was the main factor of the fluctuation of PBL numbers of H2, H3
Fig. 2. Changes in the numbers of gd T cells in BLV-infected cattle after administration of recombinant bovine IFN-g. gd T cells were stained with anti-gd TCR mAb and analyzed by flow cytometry before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of gd T cells before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
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Fig. 3. Changes in the numbers of IgMþ lymphocytes in BLV-infected cattle after administration of recombinant bovine IFN-g. IgMþ lymphocytes were stained with anti-IgM mAb and analyzed by flow cytometry before and after one-shot (A) or three-shot (B) serial administration of rbIFN-g. Columns indicate the number of IgMþ lymphocytes before IFN-g administration. The mean number S.D. is shown on the ordinate (H1, n ¼ 4; H2, n ¼ 3; H3, n ¼ 6; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
and P1 after IFN-g administration. Interestingly, there was no obvious decrease in the PBL number in two cattle, H1 and P2. One was a non-PL cow (H1), and her IgMþ cell number was decreased by 25.0%. The other was a PL cow (P2) and her IgMþ cell number was decreased by 50.8%. In contrast, CD4þ, CD8þ and gd T cell numbers were increased in both cattle. The cells presenting MHC class II
antigen showed patterns similar to those of IgMþ cells. 3.3. Decrease of syncytium formation after rbIFN-g administration In the cattle treated with rbIFN-g once, syncytium numbers of their PBL were significantly decreased but
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Fig. 4. Changes in the number of syncytia in BLV-infected cattle after administration of recombinant bovine IFN-g. PBL were obtained from BLV-infected cattle before and after one-shot (A) or three-shot (B) serial administration of recombinant bovine IFN-g. The cells were subjected to syncytium assay. The numbers of syncytia were counted under a microscope. The mean number S.D. is shown on the ordinate (H1, n ¼ 3; H2, n ¼ 5; H3, n ¼ 3; P1, n ¼ 3; P2, n ¼ 3). Points and bars represent the means S.D. for duplicate determinations.
recovered within 1 week (Fig. 4A). In the cattle receiving IFN-g administration three times, syncytium numbers were reduced for at least 2 weeks (Fig. 4B).
4. Discussion The effect of recombinant bovine IFN-g on BLVinfected cattle was examined. All cattle were febrile
after inoculation with IFN-g and then recovered within 48 h. The numbers of PBL, CD4þ and CD8þ T cells were decreased for 2–3 days and then recovered in most of the cattle. Our previous study on the effect of IFN-g in cattle without BLV infection also showed that cattle were febrile temporarily after injection of IFN-g and the numbers of PBL, CD4þ, CD8þ T cells and B cells were decreased for 1–3 days and then recovered (Shimizu et al., Annu. Meet. Jpn. Soc. Vet. Sci., 127th,
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1999). These side effects, including fever and mild hematologic depression, were similar to those of IFN administration to humans (Tamura et al., 1987). As cattle were injected with IFN-g, the number of gd T cells temporarily increased within 1 h in normal cattle without BLV infection (Shimizu et al., Annu. Meet. Jpn. Soc. Vet. Sci., 127th, 1999). In this study, the number of gd T cells also increased after the fever and remained at a significantly high level in some cattle infected with BLV. Therefore, it was suggested that gd T cells were activated by IFN-g in vivo. IgMþ cell numbers and syncytium numbers were conversely decreased, accompanied by the increase of gd T cells. Because the syncytium assay was done using PBL in these experiments, syncytium numbers might have decreased with the ratio of IgMþ cells in PBL. However, the reduction percentage of syncytium numbers was higher than that of IgMþ cell numbers in PL cattle (Fig. 5). Although the two phenomena might be unrelated, our present working hypothesis is that gd T cells might have been activated by IFN-g and eliminated BLV-infected IgMþ cells from these cattle. gd T cells possess strong MHC-independent cytolytic activity and, in some instances, preferentially kill
Fig. 5. Percentage reduction of IgMþ lymphocytes and numbers of syncytia at the post-febrile period after recombinant bovine IFN-g. Values shown are percentage reduction of IgMþ lymphocyte numbers and numbers of syncytia as described in Section 2.1.
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autologous tumor cells and virus-infected cells (Haas et al., 1993). Based on these effector functions, it is conceivable that gd T cells might exhibit cytolytic reactivity against BLV-infected cells. The lymphoid systems of cattle and sheep contain large gd T lymphocyte subpopulations, in striking contrast to the lymphoid systems of the human and mouse (Mackay and Hein, 1989; Mackay et al., 1986). Approximately 20–30% of gd T cells express CD8, although this occurs at a lower level than for ab T cells (Haas et al., 1993). In the spleen and lymph nodes, gd T cells are localized in regions of cellular traffic, and they exhibit cytotoxic activity against allogenic cells (Mackay and Hein, 1989). This information also supports the idea that gd T cells activated by IFN-g can effectively eliminate BLV-infected cells in ruminants. The number of gd T cells was preserved in the PBL from BLVinfected but healthy sheep and sheep with lymphoma compared to those from normal sheep, and the accumulation of gd T cells was also observed in tumorous lymph nodes (Murakami et al., 1994). Recently, the continued presence of BLV transcripts was reported in significant numbers of B cells (Rovnak and Casey, 1999). Thus, it is possible that gd T cells recognise BLV on the B cell surface as an allogenic antigen, are up-regulated and exclude BLV-infected B lymphocytes. At present, the immune modulating capabilities of IFN-g remain to be proven. Anti-viral functions of IFN are classified into two types, those with direct action and indirect action. IFN directly suppresses cell proliferation and/or kill virus-infected cells. The number of IgMþ cells and syncytia might be depressed by IFN-g administration via direct action, because our in vitro experiment using rbIFN-g and persistently BLV-infected FLK showed successful inhibition of syncytium formation (Sentsui et al., 2001). However, in the present in vivo experiments, immunomodulatory effects, which enhance the cytotoxic activity of macrophages, cytotoxic T cells and natural killer (NK) cells, would be also considerable in the antiBLV reaction of IFN-g. Although IFN-g acts to increase the intensity of class I and class II MHC expression on PBL in vitro, it inhibits MHC class II expression on B cells (Mond et al., 1986; Walrand et al., 1989). Our result showed that the number of class II-positive cells in PBL was not up-regulated by IFN-g inoculation in vivo, because
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the animals we used had plenty of B cells in PBL induced by BLV infection. Clinical trials of recombinant IFN-g on BLVinfected cattle are expected to be effective. The activation of gd T cells as well as that of CD8þ T cells may be an important approach for the therapy of BLV-infected cattle, because there are large amounts of gd T cells in ruminants compared to other species. In humans, in vivo, combination therapy using recombinant IFN-a or -g and anticancer drugs has been reported to achieve remarkable tumor regression in adult T cell leukemia (ATL) (Ezaki et al., 1991; Makidono et al., 1986). To develop effective therapeutics for PL and EBL induced by BLV infection, further basic as well as clinical studies should be designed to explore and assess the agents’ ability to control this disease and its causative virus BLV.
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