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Journal of Virology, July 2003, p. 7444-7451, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7444-7451.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Cytokine Profiles in Adult Mink Infected with Aleutian Mink Disease Parvovirus

P. V. Jensen, Y. Castelruiz, and B. Aasted^*

Laboratory of Virology and Immunology, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, 1870 Frederiksberg C, Copenhagen, Denmark

Received 7 November 2002/ Accepted 3 April 2003

Top Abstract Introduction Materials and Methods Results Discussion References

 
The aim of this study was to examine the levels of gamma interferon (IFN-)-, interleukin 4 (IL-4)-, and IL-8-producing cells in peripheral blood mononuclear cells from mink infected with the Aleutian mink disease parvovirus (ADV). As expected, ADV-infected mink developed high plasma gamma globulin values (hypergammaglobulinemia) and enhanced quantities of CD8-positive (CD8⁺) cells in the blood during the infection. We quantified the percentages of IFN-- and IL-4-positive lymphocytes and IL-8-positive monocytes up to week 38 after virus challenge. The results clearly indicated marked increases in the percentages of IFN-- and IL-4-producing lymphocytes during ADV infection. The total number of IL-8-producing monocytes in the blood of ADV-infected mink stayed fairly constant during the infection. In order to characterize the phenotype of the cytokine-producing cells, we performed double-labeling fluorescence-activated cell sorter (FACS) experiments with CD8 surface labeling in one channel and cytokine intracellular staining in the other. We found that most IFN- and IL-4 in ADV-infected mink was produced by CD8⁺ cells, while in the uninfected mink, these cytokines were primarily produced by a cell type that was not CD8 (possibly CD4-positive cells). We also observed that IL-8 was almost exclusively produced by monocytes. All of the above findings led us to conclude that both Th1- and Th2-driven immune functions are found in mink plasmacytosis.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Aleutian mink disease (AD), also known as mink plasmacytosis, is a common and economically important disease in mink. It is caused by a persistent infection with Aleutian mink disease virus (ADV), a nondefective parvovirus (16). In newborn mink, ADV infection may cause atypical interstitial pneumonia (10). This was observed just 20 years ago (34). The classical (adult) form of AD was described in 1956 by Hartsough and Gorham (30), and the disease is characterized by development of hypergammaglobulinemia (plasmacytosis), elevated levels of CD8-positive (CD8⁺) lymphocytes, viral persistency, and immune complex formation (1, 2, 15, 44). The most common pathological findings are glomerulonephritis and arteritis, and severely affected mink often die of renal failure (44). ADV cannot be neutralized in vivo despite the presence of very high concentrations in serum of antibody to virus capsid proteins (8, 45). In fact, antibody-mediated enhancement of infection has been observed in connection with ADV infection (3, 14, 15, 32, 43). With regard to the identity of the in vivo ADV replicating cell(s) it is generally agreed that the virus-permissive cells probably are Fc receptor-positive cells (7, 11, 15, 32).

There is only one existing report on cytokine levels during ADV infection (15). Using reverse transcription-PCR technology, this study reported higher interleukin 6 (IL-6) mRNA levels in lymph nodes from ADV-infected mink (10 and 60 days after infection) than from uninfected mink. It also found biologically active IL-6 in supernatants from short-term lymph node cultures from ADV-infected mink but not in cultures from uninfected mink. The study proposed that AD might be caused by a chronic, inappropriate production of IL-6 and perhaps of other cytokines.

As cytokines act as mediators for the immune system, the study of cytokines in connection with the classical ADV infection might give us a better understanding of this infection. Such analyses have until now been difficult to perform due to the lack of specific monoclonal antibodies against mink cytokines. The recent cross-reactivity study of monoclonal antibodies against cytokines from various animal species by Pedersen et al. (42) has revealed a few antibodies which are cross-reactive to mink cytokines (gamma interferon [IFN-], IL-4, and IL-8), enabling us to study these important mediators of immunity in ADV infection. The aim of this project, therefore, was to examine the levels of IFN--, IL-4-, and IL-8-producing cells in peripheral blood mononuclear cells (PBMCs) from adult mink infected with ADV.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Mink and experimental design. Eighteen healthy 8-month-old female mink of black genetic background (non-Aleutian genotype) were used in the study. Twelve mink were experimentally infected with ADV (5 x 10⁵ 50% infectious doses [ID₅₀] injected intraperitoneally), and six mink remained uninfected. The mink were kept in separate cages and given a standard diet. On day zero and 1, 2, 3, 5, 8, 13, 19, 26, and 38 weeks after the day of infection, each mink was anesthetized with 50 mg of ketamine (Ketaminol Vet; Intervet Scandinavia AS, Skovlunde, Denmark) and 3 mg of xylazin (Narcoxyl Vet; A/S Rosco, Taastrup, Denmark) administered intramuscularly. Blood samples were drawn from the heart and transferred to heparinized tubes (Nunc, Glostrup, Denmark).

During the experimental period, two control mink and four infected mink died (one control and one infected mink in week 8, one control and one infected mink in week 13, and the last two infected mink in weeks 20 and 27 after infection). Macroscopic pathological examinations were performed on all dead mink. These examinations indicated that three of the infected mink probably died of AD complications, while the other three mink deaths probably were caused by consequences of the heart punctures. The experimental procedures were carried out in accordance with the requirements of the Danish Animal Care and Ethics Committee.

Virus. Organ-produced material from Utah-1 ADV-infected mink (United Vaccines, Madison, Wis.) was used for the experimental infection. The titer of this material was 10⁷ ID₅₀/ml (3). The ADV-G strain (16) was used as the antigen source for the countercurrent electrophoresis (Antigen Laboratory, Danish Fur Breeders Association, Glostrup, Denmark).

Antibodies. For the cytokine-staining procedure, a cross-reactive monoclonal antibody to bovine IFN- (isotype immunoglobulin G1 [IgG1]; catalog no. MCA 1783; Serotec, Oxford, United Kingdom), a cross-reactive monoclonal antibody to bovine IL-4 (isotype IgG2a; catalog no. MCA 1820; Serotec), and a cross-reactive monoclonal antibody to ovine IL-8 (isotype IgG2a; catalog no. MCA 1660) were used as primary antibodies. Information about the cross-reactivities was published recently (42). Nonspecific monoclonal murine IgG1 (code no. X 0931; DAKO, Glostrup, Denmark) was used as a negative control. A known cross-reactive monoclonal antibody to human CD8 (2) was used for the staining of mink CD8⁺ cells. For the staining of mink CD3⁺ cells, a monoclonal antibody (antibody 165) with reactivity to mink CD3 was used (22). In all of the above-mentioned indirect staining techniques, fluorescein isothiocyanate (FITC)-labeled rabbit F(ab')₂ antibody to mouse immunoglobulins (code no. F 0313; DAKO) was used as the secondary antibody.

FACS double-labeling experiments (cytokines versus CD8 or CD3 phenotype) were performed using biotinylated antibodies to CD8 or CD3 combined with the following secondary reagents: FITC-labeled streptavidin (code no. F 0422; DAKO) and R-phycoerythrin-conjugated rabbit F(ab')₂ antibody to mouse immunoglobulins (code no. R 0439; DAKO) (the latter was for cytokine staining).

Countercurrent electrophoresis. Countercurrent electrophoresis was performed as previously described by Aasted et al. (4) using 0.7% agarose gels (HSA agarose; Litex, Copenhagen, Denmark).

Plasma electrophoresis. Plasma gammaglobulin quantitations were carried out by agarose electrophoresis (LSA agarose; Litex) using the Tris-barbital buffer system (53). The amido black staining procedure was used, and the protein fractions were quantified by densitometry (2202 ultrascan densitometer; LKB, Bromma, Sweden).

Flow cytometry. The flow-cytometric intracellular-staining method has recently been described in detail (5, 42). Briefly, diluted heparinized blood was overlaid on Lymfoprep (Nycomed, Oslo, Norway) or Ficoll-Paque (Amersham Biosciences, Uppsala, Sweden), and the PBMCs were isolated by density gradient centrifugation (1,200 x g; 20 min). The isolated PBMCs were counted, and cell cultures were set up with 2 million cells per ml of RPMI 1640 (Gibco Life Technologies, New York, N.Y.) supplemented with 10% fetal calf serum (Gibco Life Technologies), 2% penicillin-streptomycin (Gibco Life Technologies), 10 µg of brefeldin A (Sigma, St. Louis, Mo.)/ml, 1 µg of ionomycin (Sigma)/ml, and 20 ng of phorbol myristate acetate (Sigma)/ml. The cells were cultured for 4 h (37°C; 5% CO₂), washed, and treated with 4% paraformaldehyde (Merck, Darmstadt, Germany) in phosphate-buffered saline.

Staining for intracellular cytokines was performed in the presence of 0.1% saponin (Sigma) as previously described (42). Finally, the cells were analyzed on a FACS Calibur flow cytometer (Becton Dickinson, Fullerton, Calif.).

FACS double stainings were performed using the following four-step procedure: staining with (i) cytokine antibody, (ii) R-phycoerythrin-conjugated antibody to mouse immunoglobulin, (iii) biotinylated CD8 or CD3, and (iv) FITC-labeled streptavidin, with proper wash procedures in between.

The FACS quantifications of cytokine-positive cells were based on lymphocyte and monocyte gatings on a forward-scatter-versus-side-scatter diagram (regions 1 and 2 [see Fig. 2 ]). Fluorescent histogram markers were defined and quantitated positively from negatively stained cells. The percent cytokine-positive cells was calculated after subtraction of eventual positive signals from the isotype-matched immunoglobulin control preparations.

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FIG. 2. Antibody staining profiles by flow cytometry using a nonreactive monoclonal murine IgG1 antibody (Control IgG1), a monoclonal antibody to IL-8 (IL-8), a monoclonal antibody to IL-4 (IL-4), and a monoclonal antibody to IFN- (IFN-). The staining reactions were performed on purified PBMCs isolated from an uninfected mink (A) and an ADV-infected mink (B). The histogram FL1 plots are presented on the basis of lymphocytes (gate R1 in the forward-scatter- side-scatter dot plot) and monocyte (gate R2) cell populations. The quantifications of cytokine-positive cells using the M1 markers were as follows; (i) uninfected mink, 5.1% IL-4- and 6.6% IFN--producing lymphocytes, and 66% IL-8-producing monocytes; (ii) ADV-infected mink, 15.8% IL-4- and 21.8% IFN--producing lymphocytes, and 32.1% IL-8- and 5.6% IFN--producing monocytes. The control (IgG1) preparations expressed <1% positive values on all lymphocyte markers and <2% positive values on all monocyte markers.

Statistical analysis. Cytokine, plasma gamma globulin, and CD8 profiles of ADV-infected and uninfected mink were evaluated. The means and standard deviations of the infected and uninfected mink groups were calculated for each experimental day. Then, statistical evaluation was performed by analyzing whether the variances of the two groups were equal (F test), and a t test was used to compare the means of the two groups (different formulas were used, depending on whether the variances tested as equal or not). Finally, correlations between plasma gamma globulin levels and cytokine profiles and between the number of CD8⁺ cells and cytokine profiles were evaluated. Test probabilities lower than 5% were considered significant. Statistical analysis was performed with Excel 2000 (Microsoft Corp., Redmond, Wash.) software.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Characterization of ADV infection. Eighteen healthy 8-month-old female mink of black genetic background were used. Twelve mink were infected with 5 x 10⁵ ID₅₀ of ADV, and six mink were left uninfected. One week after virus challenge, plasma samples from all mink were examined by countercurrent electrophoresis for the presence of antibodies to ADV, and all infected mink tested positive while the uninfected mink were all negative. At the end of the experiment (38 weeks after virus challenge), plasma samples from all mink were retested with results identical to those for the test done in week 1 after virus challenge. Blood samples were taken on the day of virus challenge and 1, 2, 3, 5, 8, 13, 19, 26, and 38 weeks after the challenge.

All plasma samples were electrophoresed in agarose gels and quantitated for relative yield of gamma globulins. Plasma gamma globulin levels from ADV-infected mink increased with time after challenge compared to basal levels found in the uninfected-mink group (Fig. 1A). This increase was significant at week 2 after challenge (P < 0.01) and from then on (P < 0.001).

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FIG. 1. Evolution of infection parameters following ADV challenge in infected () and uninfected () mink. (A) Plasma gamma globulin quantities as percentages of total plasma protein. (B) CD8+ T cells as percentages of PBMCs. (C) Correlation between plasma gamma globulin quantities and CD8+ cell levels (P < 0.001; r = 0.68; n = 106).

Heparinized blood samples were used for determination of the relative number of CD8⁺ lymphocytes by flow cytometry (Fig. 1B). A significant increase in the percentage of CD8⁺ T cells at week 2 postinfection and thereafter was noted compared to the level observed in the uninfected mink (minimum significance, P < 0.05).

When all plasma gamma globulin values from the ADV-infected mink were plotted against the corresponding CD8 values (Fig. 1C), a highly significant correlation was found (P < 0.001; r = 0.68; n = 106).

IFN-, IL-4, and IL-8 intracellular staining in mink PBMCs. Figure 2 illustrates flow-cytometric antibody-staining profiles of IFN-, IL-4, and IL-8 performed in purified PBMCs isolated from one uninfected and one ADV-infected mink. The infected mink was selected due to the clear distinction between lymphocyte- and monocyte-produced IFN- by FACS. No other selection parameters were taken into account. Staining profiles with a nonreactive IgG1 isotype control monoclonal antibody are also included in the figure (top rows). It is evident from the figure that IL-4 signals were considerably weaker than the signals found for IFN- and IL-8. IL-4-positive cells were almost exclusively lymphocytes, while IL-8-positive cells belonged almost exclusively to the monocyte cell population. Therefore, this report does not contain data on the IL-4-producing monocytes and IL-8-producing lymphocytes. Cells positive for IFN- were distributed in both the lymphocyte and the monocyte gates.

IFN- and IL-4 levels in blood lymphocytes during ADV infection. The percentages of IFN-- and IL-4-positive lymphocytes were quantitated in blood samples from ADV-infected and uninfected mink (Fig. 3). The results clearly indicate marked increases in the percentages of IFN-- and IL-4-producing lymphocytes during ADV infection. The IFN- increase became statistically significant as early as 2 weeks after infection (P < 0.03 between control and infected animals). After 1.5 months, this difference became highly significant (P < 0.01). A significant increase in the percentage of IL-4-producing lymphocytes came much later and was first evident one-half year after infection (P < 0.05 in the peripheral blood lymphocytes). By week 38, this difference was highly significant (P < 0.01).

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FIG. 3. Percentages of IFN--positive (A) and IL-4-positive (B) peripheral blood lymphocytes from infected () and uninfected () mink with time after ADV challenge. Briefly, PBMCs were purified and cultured for 4 h in the presence of the strong cellular stimulants phorbol myristate acetate and ionomycin. The culture conditions also included the drug brefeldin A, which inhibits the Golgi complex and thus prevents proteins from being secreted. This allows cytokines and other intracellular translated products to concentrate intracellularly. After cell culturing, the cells were fixed with paraformaldehyde, and the cell membrane was perforated with saponin. The "opened" cells were then stained intracellularly for expressed cytokines and finally analyzed by FACS. The lymphocytes were defined as cells within the R1 gate (Fig. 2).

Correlation studies between CD8⁺ T cells and cytokines. Figure 4 illustrates the correlation between the percentage of CD8⁺ T cells and the percentages of IL-4- and IFN--producing lymphocytes (Fig. 4A and B, respectively). In both cases, a high and very significant correlation was found (for CD8⁺- IL-4, P < 0.001, r = 0.60, and n = 106; for CD8⁺- IFN-, P < 0.001, r = 0.74, and n = 106).

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FIG. 4. Correlations between the percentage of CD8+ lymphocytes and the percentage of IL-4-positive lymphocytes (P < 0.001; r = 0.60; n = 106) (A) and the percentage of IFN--positive lymphocytes (P < 0.001; r = 0.74; n = 106) (B). The CD8-positive lymphocytes were defined by surface staining of PBMCs, while IL-4- and IFN--producing lymphocytes were defined as described in the legend to Fig. 3. Lymphocytes were defined as cells within the R1 gate (Fig. 2).

IFN-, IL-4, and IL-8 levels in PBMCs during ADV infection. The percentages of IFN-- and IL-8-positive monocytes were also quantified in the blood samples (Fig. 5A and B). The results indicate an increase in the percentage of IFN--producing monocytes with time after infection. This increase was not as evident as for lymphocytes, and the difference was first significant 3 months after infection (P < 0.02). On the other hand, there was a clear decrease in the relative amount of IL-8-producing monocytes with time after infection. This difference was already evident 2 weeks after infection (P < 0.01). Taking into account that ADV-infected mink on average had twice the number of PBMCs found in the uninfected-mink group (Fig. 5C), the absolute number of IL-8-producing monocytes in the blood was fairly constant throughout the experiment. The level of IL-8-positive lymphocytes was <2% in all blood samples (data not presented).

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FIG. 5. (A and B) Percentages of IFN--positive (A) and IL-8-positive (B) monocytes from infected () and uninfected () mink with time after ADV challenge. (C) Monocytes as a percentage of purified PBMCs in the same ADV-infected and -uninfected mink as in panels A and B. The cellular preparations, culture conditions, and intracellular-staining technique are explained in the legend to Fig. 3. Monocytes were defined as cells within the R2 gate (Fig. 2).

Identification of cytokine-producing cells. In order to characterize the phenotype of the cytokine-producing PBMCs, we designed FACS double-labeling experiments performed on blood samples from the last day of the study (week 38). Technically, we were able to test for CD8⁺ and CD3⁺ T cells (FL1 channel) and for the three studied cytokines, IL-4, IL-8, and IFN- (FL2 channel). Figure 6 presents FL1-FL2 quadrant plots on gated lymphocytes (R1 gate) and gated monocytes (R2 gate). Data from only two mink, one uninfected and one ADV infected, are shown in the figure. The infected mink chosen was the most mildly affected. The reason for including data from this mink was that it gave the most distinct FACS staining results. In more severely affected mink, the FACS staining profiles of the four major cell populations (represented in the four quadrants) fused together, making the quadrant plots difficult to diffract.

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FIG. 6. Double antibody staining reactions by flow cytometry on PBMCs from one uninfected mink (A; plots 1 to 4) and one mildly infected mink (B; plots 5 to 16) 38 weeks after ADV challenge. CD8+ T cells were detected using the green (FL1) channel, and IFN--, IL-4-, and IL-8-positive cells were detected using the red (FL2) channel. When quadrant statistics were used, the following percentage values were obtained (upper left quadrant/upper right quadrant/lower left quadrant/lower right quadrant): plot 1, 0.4/0.3/68/31; plot 2, 4/0.5/65/31; plot 3, 2.8/1.1/67/29; plot 4, 3/2/62/33; plot 5, 0.2/0.4/61/38; plot 6, 5.9/0.7/61/33; plot 7, 3.7/6.6/63/27; plot 8, 4.3/8.9/56/31; plot 9, 0.1/0.1/63/36; plot 10, 1.0/0.2/63/35; plot 11, 2.4/6.6/63/28; plot 12, 3.9/8.4/59/29; plot 13, 0.9/1.3/44/54; plot 14, 48/2.5/38/12; plot 15, 7.5/4.3/73/16; plot 16, 6.3/12/30/51.

The double-staining experiment (Fig. 6) produced six major findings: (i) IL-8 was produced almost exclusively by monocytes (plot 14 compared to plot 10); (ii) IL-4 was produced almost exclusively by lymphocytes (plot 11 compared to plot 15); (iii) in uninfected mink, most IL-4 was produced by a non-CD8 phenotypic cell (possibly CD4 cells) (plot 3, 2.8% CD8^- versus 1.1% CD8⁺); (iv) in ADV-infected mink, most IL-4 was produced by a CD8⁺ cell type (plot 11, 2.5% CD8^- versus 6.6% CD8⁺); (v) in uninfected mink, most IFN- was produced by a non-CD8 phenotypic cell (possibly CD4 cells) (plot 4, 3.0% CD8^- versus 2.0% CD8⁺); and finally, (vi) in ADV-infected mink, most IFN- was produced by a CD8⁺ cell type (plot 12, 3.9% CD8^- versus 8.4% CD8⁺). The average cytokine values for all eight infected mink on the final test day were as follows: for IL-4-producing cells, 4.1% CD8^- T cells and 17.4% CD8⁺ T cells; for IFN--producing cells, 5.2% CD8^- T cells and 20.3% CD8⁺ T cells.

The CD3 double-staining experiment supported the conclusions from the CD8 double-staining experiments in all areas. The most important findings from these plots were that CD3-negative lymphocytes (B cells and NK cells) produced very little IL-4 and IFN- (plots not shown).

Top Abstract Introduction Materials and Methods Results Discussion References

 
AD is caused by a persistent parvovirus infection. The disease is characterized by hyperstimulation of the humoral immune system, which causes plasmacytosis and hypergammaglobulinemia, occasionally with extreme serum gamma globulin values higher than 100 mg/ml (44). Such a condition normally contains a monoclonal immunoglobulin component, and this component has antibody activity toward the ADV VP1/VP2 capsid (7). During development of AD, the quantity of CD8-positive T cells (probably cytotoxic T lymphocytes) increases as well. Levels of PBMCs above 80% can be observed (2, 3). The quantity of CD4-positive T lymphocytes during the disease process seems fairly normal (23). In spite of these active immune functions, the virus persists, and the reason for this is not fully understood, although there are theories (55). Persistent virus and antibody cause immune complex formation, and AD is known as a classical example of immune complex disease (type 3 hypersensitivity) characterized by immune complex formation, glomerulonephritis, and arteritis (44).

Generally, mink immunological research suffers from a lack of suitable reagents, which limits possible experimental designs for AD research. However, recently a few cross-reacting antibodies to mink IFN-, IL-4, and IL-8 were defined (42), which permitted us to perform this study. In earlier reports, useful antibodies for mink leukocyte phenotype characterizations were defined (6, 20, 22, 31).

The overall results from the present study indicated that the number of IFN-- and IL-4-positive cells increased during ADV infection and that these cytokines were produced mainly by lymphocytes. IFN- is known to be a cytokine produced by Th1 lymphocytes directing cell-mediated immune responses. It activates macrophages and plays an important role in antigen presentation (9, 18, 25, 39). This could explain the high number of CD8⁺ T cells seen during the infection. On the other hand, and according to the literature (19, 27, 39), IL-4 is mostly produced by Th2 cells (and mast cells), which direct humoral immune functions. We have to conclude from the present study that AD is associated with an increase of both type 1 (IFN-) and type 2 (IL-4) cytokine-producing cells. Some authors have reached similar conclusions when studying other chronic infections, like human immunodeficiency virus (HIV) (29) and feline immunodeficiency virus (46) diseases.

The enhanced level of IL-4-producing cells observed (Fig. 3) should be seen in connection to the only existing report on cytokine levels during ADV infection (15), which described higher IL-6 mRNA levels in lymph nodes from ADV-infected mink and also reported biologically active IL-6 in supernatants from short-term lymph node cultures from ADV-infected mink. Enhanced IL-4, as well as IL-6, production during the progression of AD explains quite well the development of plasmacytosis. However, the slow IL-4-producing kinetics found in the present study (Fig. 3) is somewhat surprising. First, after half a year of infection, significant enhancement of IL-4-producing cells was observed. This finding does not fully correlate with published reports (7, 17, 23, 44) of the much faster maturation curve of antibody production and of the development of hypergammaglobulinemia. The reason, we believe, is to be found in the fact that the present study focuses only on IL-4-producing PBMCs while local IL-4-producing cells in lymphoid organs like the lymph nodes and bone marrow would have been more appropriate to look at. In this context, it is also important to mention that the nonstructural protein (NS-1) from the human-pathogenic parvovirus B19 has been found to transactivate the IL-6 (37, 38) and tumor necrosis factor alpha (28) promoters. It is very likely that the ADV NS-1 protein acts in a similar way (52). Such a mechanism could also explain plasmacytosis development.

Interestingly, we found that in ADV-infected mink, the majority of IFN- and IL-4 was produced by CD8⁺ T cells, while in uninfected animals, the production of these cytokines was mainly by CD8^- cells (probably CD4 T cells). The staining method we used did not allow us to investigate to what extent the CD8⁺ cells were double-cytokine-producing cells. It has been demonstrated that very few CD8⁺ T cells express IFN- constitutively, but when they encounter antigenic peptides, the cells up-regulate cytokine genes, like the IFN- gene (47, 48). With this in mind, we suggest that CD8⁺ T cells from infected mink are continuously confronted with virus peptides and thus producing IFN-, which is in accordance with the ADV infection being persistent. The same finding has been described for HIV⁺ patients (56), for human hepatitis C virus (HCV) patients (54), and in lymphocytic choriomeningitis virus-infected mice (33). Moreover, it has been demonstrated that CD8⁺ T cells can produce many cytokines, including IFN-, as well as IL-4 and IL-5, after induction (24, 51), and a classification of these CD8⁺ cells into a Tc1 subset (producing Th1-like cytokines) and a Tc2 subset (producing Th2-like cytokines) has been proposed (26).

Most persistent viral infections are characterized by impairment of important cellular immune functions, such as cytokine production (50), by lack of cellular activation markers (36), or by down-regulation of perforins or granzymes (13). The mechanisms behind these impairments are often not fully understood. On the other hand, nonpersistent viral infections in the active immune phase are normally characterized by a high Th1 profile, including high production of IFN--producing cells and creation of high specific cytotoxic-T-lymphocyte activity (35, 54). ADV seems to represent a persistent viral infection which does not follow these rules. This study identified an augmented number of both Th1 and Th2 cytokine-producing cells during viral persistency. The outcome of such a condition seems to create immune complex disease (type III hypersensitivity disease).

There are only a few studies of veterinary-relevant animals in which IFN- and IL-4 have been found to be produced by the same cell type. When >60 CD4⁺ cell lines prepared from cattle infected by various parasites were analyzed with regard to IFN- and IL-4 production, quite a few were found to be double positive (21). In uninfected cattle, PBMC IFN- synthesis was found in both CD4⁺ and CD8⁺ T cells; however, IL-4 production was primarily found in the CD4⁺-T-cell population (49).

We also observed that, apart from lymphocytes, some cells in the monocyte population produced IFN-. A similar result has been documented in other studies, where macrophages secreted IFN- under appropriate stimulation (40, 41, 46).

This study focused only on phenotypic characterization of CD8 and CD3. An earlier study also focused on B cells in blood and lymphoid organs but found that the CD8-positive T cells were in all cases the most interesting cell type to study (23). This was our reason for focusing on CD8- and CD3-positive cells in the present study. Phenotype studies from other persistent viral infections (Epstein-Barr virus, HCV, cytomegalovirus, and HIV) have focused on markers like CD27, CD28, CD38, CD45RA, CD69, bcl-2, and CCR7 (12). These studies leave the impression that memory and effector T cells cannot be reliably classified on the basis of a particular differentiation phenotype (12). With a focus on CD27 and CD28 expression, antigen-specific memory CD8⁺ T cells were shown to be enriched in different phases depending on the infection: CD27⁺/CD28⁺ cells (early experienced cells) were found to be enriched primarily in Epstein-Barr virus and HCV, CD27⁺/CD28^- (intermediate experienced cells) were found in HIV, and CD27^-/CD28^- (late experienced cells) were found in cytomegalovirus. Such phenotypic characterizations are important to focus on in future AD research, as soon as antibodies are found.

 
This study was supported by the Danish Veterinary and Agricultural Research Council.

Pernille Bach is gratefully acknowledged for technical expertise.

 
* Corresponding author. Mailing address: Laboratory of Virology and Immunology, Department of Veterinary Microbiology, The Royal Veterinary and Agricultural University, Stigbøjlen 7, 1870 Frederiksberg C, Copenhagen, Denmark. Phone: 45 35282727. Fax: 45 35282742. E-mail: bas{at}kvl.dk.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Virology, July 2003, p. 7444-7451, Vol. 77, No. 13
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.13.7444-7451.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

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