Lactic dehydrogenase virus (LDV ) infection inhibits allergic eosinophil reaction in the airway

Research in Veterinary Science 2002, 72, 131±136 doi:10.1053/rvsc.2001.0534, available online at http://www.idealibrary.com on

Lactic dehydrogenase virus (LDV) infection inhibits allergic eosinophil reaction in the airway M. MORIMOTO*, T. OHJI*, H. IWATAy, T. HAYASHI*z Laboratories of *Veterinary Pathology and yVeterinary Hygiene, Faculty of Agriculture, Yamaguchi University, Yamaguchi, Japan SUMMARY The effects of interferon (IFN)-g induced by virus infection on eosinophil reaction in allergic airway inflammation are not yet clear. We investigated the effects of lactic dehydrogenase virus (LDV) infection, which increases IFN-g production with no viral infection or replication in respiratory epithelium, on allergic airway hypersensitivity. LDV infection suppressed antigen-induced eosinophil recruitment into the airway in sensitized mice. IL-5 gene expression in bronchoalveolar lavage (BAL) cells was significantly suppressed in LDV-infected mice compared with uninfected controls. The numbers of total T cells and CD4‡ T cells were significantly reduced in LDV-infected mice compared with controls. The present results suggested that the increase in production of IFN-g by viral infection suppresses the eosinophil reaction, and this suppressive effect may be mediated by inhibition of the recruitment of CD4‡ T cell and IL-5 production. # 2002 Published by Elsevier Science Ltd

ALLERGIC inflammation involves the attraction and activation of a variety of cell types to the site of antigen stimulation (Arm and Lee 1992). Particular attention has focused on eosinophils, since their recruitment in elevated numbers into the airways in asthma after allergen exposure may lead to long-term damage of the bronchial mucosa (Bousquet et al 1990, Azzawi et al 1992). There is increasing evidence that Th2 cytokines are correlated with airway eosinophil infiltration and disease severity (Sanderson 1992, Coyle et al 1995), mediate the allergic response, and regulate the function of eosinophils (Walker et al 1991, Corrigan et al 1995). Interferon (IFN)-g from Th1 cells is a major downmodulator of Th2 cell function. (Iwamoto et al 1993, Zuany-Amorim et al 1994). IFN-g plays a critical role in protection against viruses, and viral infection can induce Th1 responses (Mosmann and Sad 1996). These observations suggested that exposure of the host to a virus that preferentially stimulates Th1 cells may suppress the antigen-induced allergic eosinophil reaction. In contrasty, viral respiratory tract infections can cause increased bronchial reactivity in normal subjects, and respiratory viruses are the most important trigger for the symptoms of acute asthma (Empey et al 1976, Busse 1991). Using a murine model of respiratory virus infection, acute virus infection was shown to be associated with airway hyper-responsiveness as well as enhanced responses to subsequent sensitisation to allergen, and increases in production of Th-1-type cytokines such as IFN-g (Schwarze et al 1997). Several studies have indicated that IFN-g is an activator of

eosinophils prolonging their survival (Yamaguchi et al 1988, Schwarze et al 1997), so viral-induced IFN-g may enhance the eosinophil reaction in airway of antigeninduced allergic reactions. However, it seems difficult to evaluate the effects of Th-1-type cytokines induced by virus infection on allergic airway reaction, because respiratory virus infection may cause changes in the function of the respiratory epithelium that affect allergic inflammation (Schwarze et al 1997). Although endogenous IFN-g induced by virus inhibit eosinophil infiltration into peritoneal cavity, the effects of endogenous IFN-g induced by virus infection on an eosinophil-mediated allergic airway inflammation are currently unknown. Lactic dehydrogenase virus (LDV) is a member of the Arteriviridae. LDV is a unique virus that persistently infects mice and modulates host inflammatory and immune responses without any pathological or clinical changes (Notkins 1965). LDV infection activates host Th1 responses, but neither infection nor replication of the virus have been found in the respiratory epithelium (Anderson et al 1995, Rowson and Mahy 1985). These findings suggest that mice infected with LDV would be a useful system in which to investigate the effects of Th-1type cytokines induced by virus infection on airway responses of eosinophils to subsequent sensitisation to allergen. The results of this study provide a useful information to analyse the induction mechanisms of airway hypersensitivity in animals and also in human.

z

Animals

Corresponding author: Laboratory of Veterinary Pathology, Faculty of Agriculture, Yamaguchi University, 1-1677 Yoshida, Yamaguchi, 753-8515, Japan. Fax: ‡ 81-83-933-5890; E-mail: [email protected]

0034-5288/02/020131 ‡ 06 $35.00/0

MATERIALS AND METHODS Specific pathogen-free, male BALB/cA Jcl mice (Clea Japan Inc., Tokyo, Japan) were used. Autoclaved food # 2002 Published by Elsevier Science Ltd

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M. Morimoto, T. Ohji, H. Iwata, T. Hayashi

pellets (MF: Oriental Yeast, Tokyo, Japan) and tap water were supplied ad libitum. Animals that had been infected with LDV for more than 1
5 months were used. Groups of more than five mice of the same age and approximately the same weight were used for each experiment. Virus A stock preparation of LDV (kindly supplied by Dr A. L. Notkins, NIDR, NIH, USA) was used throughout the experiments (Hayashi et al 1988). Mice were infected with virus at the age of 5 weeks by intraperitoneal injection (i.p.) of 104
5 median infectious doses. Immunization was started 4 weeks after LDV infection (Hayashi et al 1988).

processed and stained according to standard procedures. Histopathology was evaluated 24 and 96 hours after antigen challenge. The slides were evaluated for eosinophil and mononuclear cell infiltration into each peribronchial or perivascular area by a pathologist blind to the treatment groups. The score of peribronchial or perivascular inflammation was graded on a subjective scale of 0, 1, 2 or 3 corresponding to of absent, mild, marked or severe, respectively. The grade of peribronchial or perivascular inflammation was expressed as the average score calculated by the following equation: score index ˆ total score/number of bronchi or vessels. About 25 different bronchi or vessels were examined per lung. PCR

Immunisation, challenge and elicitation of hypersensitivity LDV-infected and non-infected (control) mice were actively sensitised by subcutaneous (s.c.) injection of 20 mg of ovalbumin (OVA: Grade V; Sigma Chem. Co., St. Louis, MO) adsorbed to 1
2 mg of aluminum hydroxide (Alum, SERVA, Heidelberg, Germany) diluted with 0
1 ml of phosphate-buffered saline (PBS). Ten days later, animals received the same dose of ovalbumin in the presence of aluminium hydroxide. Mice were intranasally challenged with 50 ml of OVA in PBS 4 days later.

Bronchoalveolar lavage (BAL) and blood sampling

primers for mRNA amplification of

PCR

product

The following oligonucleotide primers were used: specific forward primer for IL-5, 5 0 -GACAAGCAATGAGACGATGAGGCTTC-3 0 (171 through 197), specific reverse primer for IL-5, 5 0 -GGCTACATTACCAGTTTGAGGCCAG-3 0 (657 through 681; numbers in parentheses refer to the region of the sequence) (Campbell et al 1988). In addition to the oligonucleotide primers mentioned above, the following oligonucleotide primers for the house-keeping gene hypoxanthine-guanine phosphoribosyl transferase (HPRT) were also synthesised to verify that equal amounts of RNA were added in each PCR reaction within an experiment: specific forward primer for HPRT, 5 0 -GTTGGATACAGGCCAGACTTTGTTG-3 0 (601 through 625), specific reverse primer for HPRT, 5 0 -GCTTCAACTTGCGCTCATCTTAGGC-3 0 (739 through 763; numbers in parentheses refer to the region of the sequence) (Konecki et al 1982).

Mice were anesthetised with sodium pentobarbital (Nembutal; Abbott Laboratories, North Chicago, IL), and killed by exsanguination from the heart using a heparinised syringe. Fresh blood was used for determination of eosinophil count. The number of eosinophils was counted after staining with Hinkelmann's solution, as described previously (Morimoto et al 1999a). BAL fluids were obtained by cannulating the trachea through a small incision and performing lavage with three aliquots of 1 ml of sterile saline. BAL cells were washed once with HBSS containing 2 per cent FCS by centrifugation at 200 g at 4 C. The pelleted BAL cells were resuspended and total cell counts were determined by counting in a hemocytometer. For differential counts, cytocentrifuge preparations of BAL fluids were stained with May±GruÈnwald's±Giemsa, and the eosinophils, neutrophils, lymphocytes and macrophages in the stained slides were enumerated by counting at least 500 cells. The total number of each cell type was determined by multiplying the percentage of that cell type in the BAL by the total number of cells. The results were expressed as the number of each cell population in 1 ml of BAL fluid. The remaining BAL cells were used for reverse-transcription (RT) and polymerase chain reaction (PCR) analysis or flow cytometric analysis.

BAL cells were collected and pooled from four to seven mice, and used for RT-PCR. Total RNA was extracted using an RNeasyTM Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Coupled RT-PCR was used to quantify tissue RNA levels of IL-5 (Morimoto et al 1999a), because RT-PCR is more sensitive than Northern blot hybridisation (Tono et al 1992). Briefly, the RNA samples were reversetranscribed with AMV Reverse Transcriptase Firststrand cDNA Synthesis Kit (Life Sciences Inc, St. Petersburg, USA) and cytokine-specific primers were used to amplify cytokines. The optimum number of cycles for PCR amplification of HPRT and IL-5 were determined experimentally and were defined as the number of cycles that would achieve a detectable concentration well below saturating conditions. Primers for the house-keeping gene HPRT were used to verify that equal amounts of RNA were added in each PCR.

Histopathology

Flow cytometry

The lungs were removed from mice and fixed in 10 per cent buffered formalin. Then samples were

BAL cells were washed with PBS containing 2 per cent fetal bovine serum and 0
1 per cent NaN3. BAL cells

Isolation of total

RNA

and

RT-PCR

and allergic eosinophil reaction in the airway

were pelletted and resuspended in 20 ml of normal mouse serum to block non-specific binding of antibodies. After preincubation, cells were incubated with fluorescein-conjugated anti-CD4 antibody (PharMingen, San Diego, CA, USA) in staining buffer (PBS, 2 per cent fetal bovine serum, 0
1 per cent NaN3) for 30 minutes on ice. After washing aliquats of 5  104 BAL cells were analysed with an EPICS-XL flow cytometer (Coulter Electronics Inc., Hialeah, FL, USA). Lymphocytes were identified as relatively small, non-granular cells by their forward- and side-scatter characteristics (Garlisi et al 1995). We gated this area and counted the numbers of CD4-positive cells. The total number of CD4-positive T cells in each mouse was determined as a percentage of that cell type relative to the total number of BAL cells. The results are expressed as the number of CD4 T cells in 1 ml of BAL fluid. Statistical analysis Student's t-test (two-tailed) was used to evaluate the significance of differences; a P value less than 0
05 was considered significant.

RESULTS The intranasally injection of ovalbumin into sensitised mice induced a marked increase in the number of neutrophils 24 hours after OVA challenge. Ovalbumin administration also induced a marked increase in the number of eosinophils in BAL fluid 96 hours after antigen challenge, and an increase in the number of total cells compared to that of 24 hours after antigen challenge. LDV infection reduced the number of eosinophils and lymphocytes accumulated in BAL fluid. However, LDV infection did not affect the number of neutrophils or macrophages (Table 1). There were no significant differences in the number of eosinophils in the peripheral blood between control and LDV-infected mice before and 24 hours after antigen challenge. However, the number of eosinophils was significantly decreased in LDV-infected mice compared to controls 96 hours after OVA challenge (Fig 1). OVA administration induced infiltration of eosinophils and mononuclear cells into the peribronchiolar

133

and perivascular region 24 and 96 hours after ovalbumin challenge respectively. LDV infection did not affect the grade of eosinophil or mononuclear cell infiltration into the peribronchiolar or perivascular region 24 hours after antigen challenge. The grade of eosinophil accumulation in control mice was increased 96 after antigen challenge compared with that at 24 after antigen challenge. However, LDV infection slightly reduced the grade of eosinophil accumulation in the peribronchiolar or perivascular regions 96 hours after antigen challenge, and significantly decreased the grade of eosinophil accumulation compared with that in controls (Table 2). The levels of IL-5 gene expression were determined in BAL cells from control and LDV-infected mice 96 hours after OVA challenge using the RT-PCR method. The house-keeping gene HPRT cDNA from BAL cell equivalents was amplified by PCR in control and LDV-infected groups. The IL-5 band was recognisable in control mice, but not in LDV-infected mice. Thus, LDV infection reduced the level of IL-5 gene expression by BAL cells (Fig 2). To determine the number of CD4 ‡ T cells in the BAL fluid of challenged mice, cells in BAL samples were stained and analysed by flow cytometry. LDV infection significantly reduced both the percentage of CD4‡ T cells in BAL lymphocytes and the number of CD4‡ T cells in BAL fluid (Fig 3).

Number of peripheral blood eosinophils ( × 103 /mm3)

LDV

8 6 4

*

2 0

0 24 96 Hours after antigen challenge

FIG 1: Number of eosinophils in the peripheral blood before and 24 and 96 hours after ovalbumin challenge in control and lactic dehydrogenase (LDV )-infected mice. Each group consisted of five mice. Data shown are mean (SEM). Significantly different from the values of control mice (*P < 0
05).Open bars: control; closed bars: LDV.

TABLE 1: Changes in total cell numbers and distribution in BAL fluids of sensitised mice

Control LDV infection

Hours postchallenge

Total

24 96 24 96

Number of cells ( 10 4) Macrophages

Neutrophils

Eosinophils

Lymphocytes

52
1 (4
9) 109
5 (16
6)

23
6 (2
7) 24
3 (2
1)

24
6 (3
8) 0
8 (0
2)

1
6 (0
5) 71
1 (14
5)

2
4 (0
5) 11
8 (1
9)

54
4 (7
8) 36
8 (4
6*)

22
3 (2
6) 23
7 (2
7)

29
3 (4
7) 0
8 (0
4)

1
1 (0
6) 7
5 (1
5*)

1
7 (0
3) 4
8 (0
8*)

Cells were counted at 24 and 96 hours after injection of 10 mg of OVA. Data shown are mean ( SEM) of five mice in each group. *Significantly different from the corresponding values for controls (*P < 0
05).

134

M. Morimoto, T. Ohji, H. Iwata, T. Hayashi

TABLE 2: Histopathological index of peribronchial and perivascular accumulation of leukocytes Hours postchallenge

Control LDV infection

Eosinophils

Mononuclear cells

Peribronchial

Perivascular

Peribronchial

Perivascular

24 96

1
1 (0
2) 1
7 (0
1)

1
6 (0
3) 2
2 (0
1)

0
9 (0
3) 1
6 (0
2)

0
8 (0
2) 1
9 (0
2)

24 96

1
1 (0
1) 0
7 (0
2*)

1
7 (0
2) 1
0 (0
2*)

0
9 (0
2) 1
2 (0
3)

1
0 (0
2) 1
6 (0
2)

Cells were counted at 24 and 96 hours after injection of 10 mg of ovalbumin.The cell infiltration was graded: 0, negative; 1, weak; 2, moderate; 3, strong. Histopathological index of individual mice was expressed as an average of grade in 10 regions. Data shown are mean  SEM of five mice in each group.*Significantly different from the corresponding values for controls (*P < 0
05).

IL-5

HPRT

Control

LDV

100

50

*

0

Number of CD4+ T lymphocytes in BAL fluids (x 104ml–1)

Percentage of CD4+ T lymphocyte in BAL lymphocytes (percentage)

FIG 2: Effect oflactic dehydrogenase (LDV ) infectiononlevelof IL -5 mRNA in bronchoalveolar lavage (BAL) fluid cell pellets from sensitised mice killed 96 hours after ovalbumin challenge. BAL cells from LDV infected or control mice were pooled, and analysed by reverse transcriptase-polymerase chain reaction (RT-PCR) methods.

Control LDV

8

4 *

0 Control LDV

FIG 3: Effect of lactic dehydrogenase (LDV ) infection on the percentage of CD4 ‡ T cells and the number of CD4 ‡ T cells in bronchoalveolar lavage (BAL) fluid from sensitised mice sacrificed 96 hours after ovalbumin challenge.Data shown are mean (SEM) of five individual mice. Significantly different from the values of control mice (*P < 0
05).

DISCUSSION The present results showed that LDV infection inhibited antigen-induced eosinophil and lymphocyte accumulation in the BAL fluid of sensitised mice. LDV infection selectively affected the infiltration of eosinophils and lymphocytes into the bronchi. This result suggested that the influence of vascular permeability on cell infiltration into the bronchi after LDV infection was negligible, since neutrophil and macrophage infiltration 24 and 96 hours after ovalbumin challenge were unaffected. In addition, the immunomodulation by LDV

observed in the present study may not have been due to the direct cytocidal effect of LDV on eosinophils or T cells, since no infection or replication of the virus has been found in these cells (Anderson et al 1995). We also showed that the number of peripheral eosinophils was significantly reduced in LDV-infected compared to control mice. In addition, LDV infection inhibited eosinophil and mononuclear cell accumulation to the perivascular and bronchial regions 96 hours after antigen challenge. These findings suggested that the inhibitory effect of LDV infection on the antigen-induced eosinophil response might be the consequence of a direct and/or indirect effect on eosinophil growth and/or chemotaxis. It has been demonstrated in various studies that the allergic eosinophilia and tissue infiltration of eosinophils in sensitised mice are dependent on IL-5 production or secretion (Okudaira et al 1991), and IL-5 is known to affect the migration of eosinophils as a chemotactic factor (Yamaguchi et al 1988). In the present study, expression of the IL-5 gene could be detected in the BAL fluid in control mice but its expression was faint in that of LDV-infected mice. Although we did not measure the concentrations of IL-5 in the BAL fluid, the levels of expression of the IL-5 gene in lymphoid organs are proportional to the levels of IL-5 secretion (Zuany-Amorim et al 1996). Thus, our results suggested that the decrease of eosinophil accumulation in the BAL fluid by LDV infection is, in part, due to inhibition of IL-5 production or secretion, as a chemotactic factor from BAL cells. It has also been demonstrated in previous studies that the IL-5 production or secretion in allergic reactions is derived from T cells (Garlisi et al 1996). In this study, we showed that the number of lymphocytes in BAL fluids was significantly reduced in LDV-infected compared to control mice 96 hours after antigen challenge. This result suggested that the decrease in expression of the IL-5 gene in BAL fluid was due to the decrease in number of lymphocytes in BAL fluid. CD4 ‡ T cells have especially important roles in allergic eosinophilia and tissue infiltration of eosinophils in sensitised mice, since inhibition of CD4 ‡ T cell infiltration reduced eosinophil recruitment (Nakajima et al 1992, Zuany-Amorim et al 1996). In this study, we demonstrated that LDV infection reduced the proportion and number of CD4 ‡ T cells in BAL T cells. This

LDV

and allergic eosinophil reaction in the airway

suggested that the inhibition of eosinophilia and eosinophils in BAL fluid by LDV infection resulted from the decrease in the number of CD4 ‡ T cells in BAL fluid. The distinct role of CD4 ‡ T cells in the recruitment of eosinophils is unknown, but previous studies demonstrated that CD4 ‡ T cells have the capacity to produce IL-5 (Hofstra et al 1999, Sasaki et al 2000). Therefore, the decrease in the level of production of IL-5 may be due to inhibition of the infiltration of CD4 ‡ T cells in LDV-infected mice. We found no significant differences in eosinophil reaction 24 hours after antigen challenge between control and LDV-infected mice. Immunization with OVA and alum induces not only Th2 cytokine production but also temporal Th1 cytokine (IFN-g) production (Aramaki et al 1995). In addition, we found that the level of IFN-g expression in LDV-infected mice was similar to that in control mice soon after antigen challenge immunized with OVA and alum (Morimoto et al 1999a). However, the expression level in LDV-infected mice was significantly higher than that in control mice several days after antigen challenge (Morimoto et al 1999b). Thus, one possible explanation for the small effect of LDV infection 24 hours after antigen challenge was that antigen-induced IFN-g may overlap LDVinduced IFN-g soon after antigen challenge, but LDVinduced IFN-g was effective several days after antigen challenge. Our findings that LDV infection inhibit the eosinophil and CD4 ‡ T cell infiltration into airway, and the production of IL-5 from BAL cells suggest that viral induced IFN-g is not an activator for eosinophil reaction in antigen induced allergic reaction ACKNOWLEDGMENTS This work was supported, by Grant-in-Aid of the Ministry of Education, Science, Sports and Culture (11660296 and 12306015). REFERENCES ANDERSON, G. W., ROWLAND, R. R. R., PALMER, G. A., EVEN, C. & PLAGEMANN, G. W. (1955) Lactate dehydrogenase-elevating virus replication persists in liver, spleen, lymph node, and testis tissues and results in accumulation of viral RNA in germinal centers, concomitant with polyclonal activation of B cells. Journal of Virology 69, 5177±5185 ARAMAKI, Y., SUDA, H. & TSUCHIYA, S. (1995) Interferon-gamma inductive effect of liposomes as an immunoadjuvant. Vaccine 13, 1809±1814 ARM, J. P. & LEE, T. H. (1992) The pathobiology of bronchial asthma. Advances in Immunolgy 51, 323±382 AZZAWI, M., JOHNSTON, S., MAJUMDAR, S., KAY, A. B. & JEFFERY, P. K. (1992) T lymphocytes and activated eosinophils in airway mucosa in fatal asthma and cystic fibrosis. American Review of Respiratory Disease 145, 1477±1482 BOUSQUET, J., CHANEZ, P., LACOSTE, J. Y., BARNEON, G., GHAVANIAN, N., ENANDER, I., VENGE, P., AHLSTEDT, S., SIMONY, L. J., GODARD, P. & MICHEL, F. B. (1990) Eosinophilic inflammation in asthma. New England Journal of Medicine 323, 1033±1039 BUSSE, W. W. (1991) Respiratory infections: their role in airway responsiveness and the pathogenesis of asthma. Journal of Allergy and Clinical Immunology 85, 671±683 CAMPBELL, H. D., SANDERSON, C. J., WANG, Y., HORT, Y., MARTINSON, M. E., TUCKER, W. Q., STELLWAGEN, A., STRATH, M. & YOUNG, I. G. (1988) Isolation, structure and expression of cDNA and genomic clones for murine eosinophil differentiation factor. Comparison with other eosinophilopoietic lymphokines and identity with interleukin-5. European Journal of Biochemistry 174, 345±352

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Accepted November 29, 2001