- Email: [email protected]
Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens
Veterinary Immunology and Immunopathology 61 Ž1998. 331–341
Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens I.-J. Kim, K. Karaca 1, T.L. Pertile, S.A. Erickson, J.M. Sharma
)
Department of Veterinary PathoBiology, College of Veterinary Medicine, UniÕersity of Minnesota, St. Paul, MN 55108, USA Accepted 23 September 1997
Abstract We examined the effects of infectious bursal disease virus ŽIBDV. on splenic T cells and macrophages. In acute IBDV infection, splenocytes responded poorly to Con A stimulation. However, when T cells were isolated from whole spleen cells, purified T cells responded normally to Con A. This result indicated that functional T cells were present in the spleen but mitogen-induced proliferation of T cells was being suppressed by other cells. Previous studies indicated that soluble factors from suppressor cells may mediate this inhibition of T cell mitogenesis. We thus examined the effects of IBDV on spleen adherent cells. Reverse transcriptase–polymerase chain reaction ŽRT–PCR. was used to quantitate the expression of several cytokine genes in splenic macrophages. In acute IBDV infection, splenic macrophages exhibited enhanced gene expression of type I interferon ŽIFN., chicken myelomonocytic growth factor ŽcMGF., an avian homolog of mammalian IL-6, and 9E3rCEF4, an avian homolog of mammalian IL-8. Mitogen-stimulated spleen cell cultures also produced elevated levels of nitric oxide. The elevation of cytokine gene expression by macrophages occurred transiently during the acute phase of viral infection and coincided with in vitro inhibition of T cell mitogenic response of spleen cells. q 1998 Elsevier Science B.V. Keywords: Infectious bursal disease virus; Chicken; Macrophage; Cytokine; Immunosuppression; Polymerase chain reaction ŽPCR.
) Corresponding author. Tel.: q1 612 625 5276; fax: q1 612 625 5203; [email protected] 1 Current address: Tri-Bio Laboratory, 1400 Fox Hill Rd., State College, PA 16803, USA.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 3 5 - 9
e-mail:
332
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
1. Introduction Infectious bursal disease virus ŽIBDV., a double-stranded RNA virus, is a member of the Birnaviridae family ŽMuller et al., 1979.. The virus targets B lymphocytes in the bursa of Fabricius ŽKaufer and Weiss, 1980. which results in a reduction of the number of circulating B cells and serum immunoglobulin levels ŽIvanyi and Morris, 1976; Giambrone et al., 1977; Hirai et al., 1981; Rodenberger et al., 1994.. Subsequent bursal damage persists for a long period ŽChevile, 1968; Winterfield et al., 1972.. Infection of chickens with IBDV also results in suppression of cell-mediated immune responses and subsequently increases susceptibility to secondary infections. Previous studies indicated that the proliferative responses of peripheral blood lymphocytes and spleen cells to T-cell mitogens were transiently inhibited during the first week of IBDV infection ŽConfer et al., 1981; Sharma et al., 1989.. However, removal of adherent cells from spleen cells partially restored the mitogenic response to normal levels, and co-culture of normal spleen cells with supernatant of splenocytes from IBDV-exposed chickens inhibited T cell responses to Con A ŽSharma and Fredricksen, 1987.. These results suggested that adherent cells may produce suppressive factors upon IBDV infection. However, the immunomodulatory mechanism of spleen adherent cells following IBDV infection is not known and the soluble factors have not been identified. In the present study, we had two objectives: Ža. to confirm that spleens of IBDV-exposed chickens had mitogen-responsive T cells at the peak of mitogenic inhibition, and Žb. to determine if IBDV infection modulated spleen adherent cells. The spleen adherent cells are referred here as macrophages since the majority of adherent cells are monocytes and macrophages.
2. Materials and methods 2.1. Chickens and Õirus One-day-old specific pathogen-free ŽSPF. chicks were purchased from HyVac ŽAdel, IA.. The chickens were housed in Horsfall–Bauer-type isolation units. Feeds and water were provided ad libitum. The virulent IM strain of IBDV was propagated and titrated in chicken embryonated eggs ŽWinterfield et al., 1972.. The virus had a titer of 10 6 50% embryo infectious dose ŽEID50 . per ml. 2.2. Experimental plan Chickens were divided into two groups; Group 1 was inoculated with 1000 EID50 of IM-IBDV by the eyedrop route at 18 days of age, and Group 2 served as unexposed controls. At 18 h, 1, 2, 3, 4, 5, and 8 days post-inoculation ŽPI., three chickens from each group were sampled and spleens were harvested aseptically. Virus infection was confirmed by the presence of gross bursal necrosis in chickens of the virus-exposed group but not of those in the unexposed control group.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
333
Single cell suspensions of the spleens were prepared as described previously ŽPertile et al., 1996.. Lymphoid cells were separated by Histopaque-1083 ŽSigma Chemical Co., St. Louis, MO. and resuspended in RPMI-1640 medium supplemented with 2 mM L-glutamine, 50 m grml gentamycin and 10% FBS. Cell viability was assessed by trypan blue exclusion. 2.3. Pan-purification of T lymphocytes and lymphocyte proliferation assay T lymphocytes were purified by panning as previously described ŽMage et al., 1977; Pertile et al., 1996.. Briefly, spleen cells were incubated at 398C for 3 h to remove adherent cells. Ten million per milliliter of non-adherent cells was incubated with 5 m grml of CT3, mouse anti-chicken CD3-monoclonal antibody ŽSouthern Biotechnology, Birmingham, AL., at 48C for 30 min. The cells were washed and resuspended in RPMI-1640 supplemented with 10% FBS. Eight ml of the cell suspension containing CT3-interacting cells was added to a 100-mm tissue culture plate coated with goat anti-mouse polyvalent immunoglobulins. After 2 h at 48C incubation, unbound cells were washed off and the antibody-bound cells were removed by a cell scraper. The cell purity was assessed by fluorescence-activated cell sorter ŽFACS. analysis using fluorescein isothiocyanate ŽFITC.-conjugated goat anti-mouse polyclonal antibody ŽSouthern Biotechnology Associates.. The cells were consistently 88–95% pure T cells. The procedure for lymphoproliferation assay was described elsewhere ŽKaraca et al., 1996.. 2.4. IL-2 bioassay IL-2 was measured from spleen cell culture supernatant as described previously ŽPertile et al., 1996.. The concentration of IL-2 in the supernatant was determined by corresponding units of serial dilutions of an IL-2 standard. Conditioned medium prepared by stimulation of T cells with Con A Ž5 m grml. served as the IL-2 standard. One unit of IL-2 was defined as the quantity equal to 50% of the maximum absorbence value of the IL-2 standard. 2.5. Nitrite accumulation assay Nitrite concentration in supernatants of Con A-treated and untreated cultures was determined as previously described ŽGreen et al., 1982; Pertile et al., 1996.. A total of 100 m l of each sample was added to 96-well plates in triplicate followed by addition of 100 m l Greiss reagent to each well. Greiss reagent consisted of a 1:1 mixture of 1% sulfanilamide ŽSigma. in 5% phosphoric acid and 0.1% naphthylenediamine dihydrochloride ŽSigma. in deionized water. The plate was read at 570 nm in a microplate reader. The concentration of nitrite in the supernatant was determined by using a standard curve of sodium nitrite. 2.6. Total RNA isolation Total RNA was isolated from splenic adherent cells by using a Trizol reagent ŽGibco-BRL, Gaitherberg, MD. following the protocol provided by the manufacturer.
334
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
Adherent cells were obtained as above. The quantity of isolated RNA was normalized by b-actin gene expression with dot blot. 2.6.1. ReÕerse transcription (RT) RT was performed by using Superscript RT Kit ŽGibco-BRL. according to manufacturer’s protocol. Briefly, 1 m g of total RNA was reverse transcribed in a reaction containing 20 mM Tris–HCl, pH8.3, 50 mM KCl, 2.5 mM MgCl 2 , 200 m M dNTP, 10 m M DTT, 0.5 m g oligo ŽdT. with 200 Urm l of M-MLV reverse transcriptase at 378C for 1 h. A tube with no reverse transcriptase was included in the RT reaction as a negative control. First strand-cDNAs were stored at y808C until used. 2.6.2. Amplification of cDNA and quantitation of cytokine gene expression The technique to detect cytokine mRNA was adapted from previous report ŽRottman et al., 1995.. Specific cytokine gene segments were amplified by Taq polymerase Ž2 U. in a reaction containing 50 mM KCl, 2.5 mM MgCl 2 , 10 mM Tris–HCl pH 9.0, 200 m M dNTP and 50 pM primers. The PCR was conducted for 30 cycles as follows; denaturation at 948C for 1 min, annealing at 658C for 30 s, polymerization at 728C for 1 min, and at the last cycle, the reaction was extended at 728C for 10 min. The primer sequences used were as follows: for b-actin, 5X-TCTGGTGGTACCACAATGTACCCT3X Ž sense . , 5X -ACCAGTAATTGGTACCGGATAATA-3X Ž antisense . ; chicken myelomonocytic growth factor ŽcMGF., 5X-TTCCTGCACAAGAACCTGGAG-3X Žsense., 5X-CTCCACCAACGTCGTGATT-3X Žantisense.; type I IFN, 5X-ATGGCTGTGCCTGCAAGCCCA-3X Ž sense . , 5X -AGTGCGCGTGTTGCCTGTCA-3X Ž antisense . ; 9E3rCEF4 Ž avian IL-8 . , 5X -GGCCTCCTCCTGGTTTCAGCTGC-3X Ž sense . , 5X GCACTCCAGGGGAGCAGG-3X Žantisense.. The PCR products were electrophoresed with 1.5% agarose gel and stained with ethidium bromide. The quantity of the PCR
Fig. 1. The mitogenic response of spleen cells. Splenocytes from virus-free and IBDV-exposed chickens were stimulated with Con A Ž5 m grml.. Cells cultured without Con A had mean CPM, 1843"762 Žmean"S.D... The result represents the mean of three chickens per group at each time intervals. Asterisks indicate statistical significance.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
335
Table 1 The IL-2 production by spleen cells IL-2 ŽUrmla . in spleen-cell cultures containing
Groups
Medium Virus-free IBDV-exposed a b
6"0.2 8"0.1
b
Con A Ž5 m grml. 160"9.0 7"2.0
One unit of IL-2 is defined as 50% of the maximum absorbance value of the IL-2 standard at 570 nm. The values are the means Ž"S.D.. of three pools of spleens. Each pool contained three spleens.
Table 2 The mitogenic response of fractionated spleen cells Delta CPM a Ž=1000. Unfractionated splenocytes Experiment 1 Virus-free IBDV
425.6"20.6 b 14.3"7.6 Ž3.4. c
Experiment 2 Virus-free IBDV x% Of two experiments
543.5"12.5 51.5"22.8 Ž9.5. 6.5
Panned T lymphocytes 90.8"6.016 93.2"28.8 Ž102.6.
110.6"11.4 59.1"17.2 Ž53.4. 74.5
a
Delta CPMsCPM of cells stimulated with Con A Ž5 m grml.yCPM of unstimulated cells. The CPM values are mean of nine chickens Žthree pools of three chickens each."S.D. c Percent of virus-free controls. b
products were analyzed by a densitometer ŽComputing Densitometer, Molecular Dynamics, Sunnyvale, CA. and quantitated by ImageQuan software ŽMolecular Dynamics. and IP Lab gel program ŽSignal Analytic Co... To confirm consistency of the quantity of RNA, 5% and 10% of first strand cDNA were used for cytokine gene expression ŽcMGF, type I IFN, and 9E3rCEF4.. b-actin expression was included to show that the differences of gene expressions were not due to differences in the concentration of the first strand cDNA between groups for PCR. The results were expressed as ratio of average counts of IBDV-exposed chicken to that of virus-free control chicken; IBDV: control ratio s Ž Cytokine RT–PCR sample 1 average count of IBDV-exposed chicken. - Ž cytokine background count . r Ž Cytokine RT –PCR sample 1 average count of virus-free chicken. - Ž cytokine background count .
336
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
337
Fig. 2 Cytokine expression in spleen macrophages. Macrophages were separated from spleen cells at 18, 24, 72 and 192 h after IBVD inoculation. Total RNA was isolated from macrophages and RT–PCR were performed with primers specific for: Ža. type I IFN, 582 bp; Žb. cMGF, 388 bp; and Žc. 9E3rCEF4, 683 bp. The PCR products were resolved on 1.5% agarose gel and photographed. Each sample was tested for PCR at 5% and 10% cDNA concentration, respectively. In Ža., Žb. and Žc., the results of the PCR reaction for samples from virus-exposed and virus-free chicken are shown in duplicate lanes. For each sample, the lane on the left contains results with 5% cDNA and the lane on the right with 10% cDNA. The quantitive gene expression is presented in Žd., Že. and Žf. as ratio of one average count of PCR reaction of IBVD-exposed chickens to that of the virus-free controls. M; 100 bp DNA ladder marker, q; a positive control of each cytokine gene.
2.7. Statistical analysis Student’s t-test was employed to determine significance of differences between individual samples of virus-free and IBDV-exposed groups. P - 0.05 was considered to be statistically significant. 3. Results 3.1. Responses of chickens exposed to IBDV Spleen cells from IBDV-exposed chickens responded poorly to Con A. At 4- and 5-day PI, the spleen cell response to Con A in the virus-infected group was only 3.4% of the virus-free group Ž P - 0.05, Fig. 1.. Suppression of the mitogenic response was transient and spleen cells regained normal responsiveness by one week after virus inoculation.
338
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
In addition to being hyporesponsive to Con A, T cells present in the spleen cell suspension from IBDV-exposed chickens were also deficient in production of IL-2 ŽTable 1.. The levels of IL-2 produced by IBDV-exposed chickens was 4% of the levels produced by the virus-free group. Thus, T cell responsiveness was downregulated during IBDV infection. Previous data have indicated that removal of adherent cells from spleen cells restored the response of cells to PHA ŽSharma and Lee, 1983.. In this study, we examined the function integrity of T cells by pan-purifying T cells from spleens of IBDV-exposed chickens at the time when the mitogenic hyporesponsiveness was maximal. In two separate experiments, pan-purified T cells from IBDV-exposed chickens responded vigorously to Con A after in vitro stimulation ŽTable 2.. In Experiment 1, the proliferation was 102% of the response of T cells purified from IBDV-negative chickens and in Experiment 2, the response was 53% of the IBDV-negative chickens. This was a significant enhancement when compared to the mitogenic response of the unfractionated spleen cells of IBDV-exposed chickens which was less than 10% of the response of IBDV-negative chickens. 3.1.1. Enhancement of cytokine gene expression in adherent cells We examined the macrophages for the expression of three cytokines, i.e. type I IFN, cMGF, and 9E3rCEF4. These cytokines are known to be produced by macrophages and may play a crucial role in acute phase responses in mammals ŽCavaillon, 1994.. Thus, we considered an enhanced cytokine-expression in macrophages as an indication of macrophage activation that results from IBDV infection.
Fig. 3. The nitrite accumulation in supernatant of splenic cells from virus-free and IBDV-exposed groups. Splenocytes were cultured in the presence of Con A Ž5 m grml. as described in Materials and Methods. Supernatant from medium-treated cells had background level of nitrite Ž3.2"0.3.. The concentration of nitrite in the supernatant was determined by using a standard curve of sodium nitrite. ) sSignificantly different Ž P - 0.05. from levels of virus-free control group.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
339
Semi-quantitative PCR analysis revealed that spleen macrophages had an increased expression of type I IFN, cMGF, and 9E3rCEF4 during the first week of infection with IBDV ŽFig. 2.. The enhancement of the cytokine gene expression was detected without additional in vitro stimulation of the cells. Type I IFN expression was increased approximately 11-fold within 24 h of virus exposure. The expression of cMGF gradually increased starting at 18 h PI and reached a peak at 3 days PI. The peak level was 12-fold higher than the level in the control chickens. Enhancement of type I IFN and cMGF expressions was followed by marked increase of 9E3rCEF4 expression. Expression of 9E3rCEF4 in the virus-exposed group was apparent at 3 days PI and then rapidly subsided. At 3 days PI, the 9E3rCEF4 expression in macrophages from the IBDV-exposed chickens was eight times higher than the expression in macrophages from the virus-free control chickens. b-actin expression in each PCR reaction was included and showed that each tube contained consistent amount of cDNA for PCR. 3.1.2. Nitric oxide (NO) production We cultured spleen cells in vitro and examined the supernatant for NO. Significant levels of nitrite were detected in supernatant of Con A-stimulated spleen cell cultures from the virus-exposed group Ž P - 0.05, Fig. 3.. However, the upregulation of NO was transient and was detected only at 1 day PI.
4. Discussion In the present study, we have confirmed that although spleen cells from IBDV-exposed chicken did not respond to T-cell mitogen in vitro, pan-purified T cells from these spleens proliferated vigorously when stimulated with Con A. A previous study had shown that soluble factors produced by suppressor macrophages may be involved in hyporesponsiveness of lymphocytes ŽSharma and Fredricksen, 1987.. We now demonstrate that IBDV infection resulted in enhanced expression of several cytokines and elevated levels of NO in splenic macrophages. Modulation of macrophage function occurred during the acute phase of IBDV infection and coincided with inhibition of T-cell mitogenesis noted in spleen cells. These results provide circumstantial evidence that activation of macrophages by IBDV may have induced spleen cells to exhibit suppressive activity. However, the mechanism of suppression was not investigated. In this study, we examined cytokine gene expression and nitric oxide production. Unfortunately, only a small number of cytokines have been cloned and characterized in chickens ŽRautenschlein and Sharma, 1997.. We used the PCR technique to examine the profile of cytokines in macrophages. The mammalian homologues of three cytokines, i.e. type I-IFN, cMGF Ža homolog of IL-6; Leutz et al., 1989., and 9E3rCEF4 Ža homolog of IL-8; Stoeckle and Barker, 1990; Barker et al., 1993. were examined. These cytokines are known to be involved in inflammatory responses ŽHarada et al., 1994.. The expression of all three cytokines was up-regulated during the acute phase of IBDV infection ŽFig. 2.. The enhanced gene expression occurred without in vitro stimulation of macrophages. Thus, the up-regulation of cytokines was induced in vivo by IBDV infection.
340
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
IBDV causes a highly immunosuppressive disease in chickens ŽGiambrone et al., 1977.. Both cellular and humoral responses are compromised ŽSharma et al., 1989.. The cytokines produced by macrophages in the IBDV-exposed chickens may play a role in mediating immunosuppression. In mice, cytokines and other soluble factors derived from macrophages have been shown to suppress immune responses Žreviewed by Cavaillon, 1994.. In lymphocytic choriomeningitis virus-induced immunosuppression, Type I IFN contributed to the suppression of immune response by stimulating cytotoxic T cells that destroyed antigen-presenting cells and prevented antibody production ŽOdermatt et al., 1991.. Recently, we have noted that IBDV infection results in a rapid influx of CD8 q cells in bursal follicles ŽTanimura and Sharma, 1997.. The role of cytokines in proliferation of CD8 q cells and the possible role of these cells in mediating immunosuppression need to be established. Suppressive effects of macrophage-derived NO on T-cell mitogenic response have been well demonstrated in rats ŽFecho et al., 1994.. Addition of N G -monomethyl-Larginine ŽNMMA., a potent inhibitor of NO production, to the cultures resulted in reversal of the suppressor effect of supernatants of the rat splenocyte cultures. We have recently shown that co-culture of normal splenocytes with the adherent cells from reovirus-infected chickens resulted in inhibition of mitogen-induced proliferation of normal splenocytes ŽPertile et al., 1996.. Removal of adherent cells from the cultures resulted in a partial restoration of T cell responsiveness to Con A in reovirus-exposed chickens. Our w 3 HxTdR incorporation data of pan-purified T cells during IBDV infection showed a similar result to that of T cells during reovirus infection. Our study has established that IBDV modulates macrophage in chickens. Cytokine expressions by macrophages were up-regulated and the up-regulation coincided with inhibition of T-cell mitogen responsiveness. Acknowledgements We thank Drs. S. Reddy, M. Suresh, and S. Rautenschlein, and Miss C. St. Hill for advice and discussions. References Barker, K.A., Hample, A., Stoeckle, M.Y., 1993. Transformation-associated cytokine 9E3rCEF4 is chemotatic for chicken peripheral blood mononuclear cells. J. Virol. 67, 3528–3533. Cavaillon, J.M., 1994. Cytokines and macrophages. Biomed. Pharmacother. 48, 445–453. Chevile, N.F., 1968. Studies of the pathogenesis of Gumboro Disease in bursa of Fabricius, spleen and thymus of the chicken. Am. J. Pathol. 51, 515–551. Confer, A.W., Springer, W.T., Shane, S.M., Donovian, J.F., 1981. Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal disease virus. Am. J. Vet. Res. 452, 2109–2113. Fecho, K., Maslonek, K.A., Coussons-Read, M.E., Dykstra, L.A., Lysle, D.T., 1994. Macrophage-derived nitric oxide is involved in the depressed Conconavalin A responsiveness of splenic lymphocytes from rats administered morphine in vivo. J. Immunol. 152, 5845–5852. Giambrone, J.J., Dawe, D.L., Eidson, C.S., 1977. Specific suppression of the bursa-dependent immune system of chickens with infectious bursal disease virus. Am. J. Vet. Res. 38, 581–583.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
341
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannebaum, S.R., 1982. Analysis of nitrate, nitrite and w 15 Nxnitrate in biological fluids. Anal. Biochem. 126, 131–138. Harada, A., Sekido, N., Akahoshi, T., Wada, T., Mukaida, N., Matsushima, K., 1994. Essential involvement of interleukin-8 ŽIL-8. in acute inflammation. J. Leukocyte Biol. 56, 559–563. Hirai, K., Funakoshi, T., Nakai, T., Shimakura, S., 1981. Sequential changes in the number of surface immunoglobulin-bearing B lymphocytes in infectious bursal disease virus-infected chickens. Avian Dis. 25, 484–496. Ivanyi, J., Morris, R., 1976. Immunodeficiency in chicken: IV. An immunological study of infectious bursal disease. Clin. Exp. Immunol. 28, 1015–1021. Karaca, K., Kim, I.-J., Reddy, S.K., Sharma, J.M., 1996. Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens. J. Immunol. Methods 192, 97–103. Kaufer, I., Weiss, E., 1980. Significance of bursa of Fabricius as target organ in infectious bursal disease of chickens. Infect. Immun. 27, 364–367. Leutz, A., Damm, K., Sterneck, E., Kowenz, E., Ness, S., Frank, R., Gausepohl, H., Pan, Y.-C.E., Smart, J., Hayman, M., Graf, T., 1989. Molecular cloning of the chicken myelomonocytic growth factor ŽcMGF. reveals relationship to interleukin 6 and granulocyte colony stimulating factor. EMBO J. 8, 175–181. Mage, M.G., McHugh, L.L., Rothstein, T.L., 1977. Mouse lymphocytes with and without surface immunoglobulin; preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. J. Immunol. Methods 15, 47–56. Muller, H., Scholtissek, C., Becht, H., 1979. The genome of infectious bursal disease virus consists of two segments of double-stranded RNA. J. Virol. 31, 584–589. Odermatt, B., Eppler, M., Leist, T.P., Henagartner, H., Zinkernagel, R.M., 1991. Virus-triggered acquired immunodeficiency by cytotoxic T-cell dependent destruction of antigen-presenting cells and lymph follicle structure. Proc. Natl. Acad. Sci. USA 88, 8252–8256. Pertile, T.L., Karaca, K., Walser, M.M., Sharma, J.M., 1996. Suppressor macrophages mediate depressed lymphoproliferation in chickens infected with avian reovirus. Vet. Immunol. Immunopathol. 53, 129–145. Rautenschlein, S., Sharma, J.M., 1997. Cytokines. In: Pastret, P.P., Bazin, H., Govererts, A., Giebel, P.J. ŽEds.., Handbook of Vertebrate Immunology. Academic Press, Orlando Žin press.. Rodenberger, J., Sharma, J.M., Belzer, S.W., Nordgren, R.M., Naqi, S., 1994. Flow cytometric analysis of B cell and T cell subpopulations in specific pathogen-free chickens infected with infectious bursal disease virus. Avian Dis. 38, 16–21. Rottman, J.B., Freeman, E.B., Tonkonogy, S., Tompkins, M.B., 1995. A reverse transcription–polymerase chain reaction technique to detect feline cytokine genes. Vet. Immunol. Immunopathol. 45, 1–18. Sharma, J.M., Dohms, J.E., Metz, A.L., 1989. Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens. Avian Dis. 33, 112–124. Sharma, J.M., Fredricksen, T.L., 1987. Mechanism of T cell immunosuppression by infectious bursal disease virus of chickens. Proc. Avian Immunol. 238, 283–294. Sharma, J.M., Lee, L.F., 1983. Effects of infectious bursal disease virus on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression. Infect. Immun. 42, 747–754. Stoeckle, M.Y., Barker, K.A., 1990. Two burgeoning families of platelet factor 4-related proteins: mediators of inflammatory response. New Biol. 2, 313–323. Tanimura, N., Sharma, J.M., 1997. Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens. Avian Dis. 41, 638–645. Winterfield, R.W., Fadly, A.M., Bickford, A., 1972. Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis. 16, 623–632.
Enhanced expression of cytokine genes in spleen macrophages during acute infection with infectious bursal disease virus in chickens I.-J. Kim, K. Karaca 1, T.L. Pertile, S.A. Erickson, J.M. Sharma
)
Department of Veterinary PathoBiology, College of Veterinary Medicine, UniÕersity of Minnesota, St. Paul, MN 55108, USA Accepted 23 September 1997
Abstract We examined the effects of infectious bursal disease virus ŽIBDV. on splenic T cells and macrophages. In acute IBDV infection, splenocytes responded poorly to Con A stimulation. However, when T cells were isolated from whole spleen cells, purified T cells responded normally to Con A. This result indicated that functional T cells were present in the spleen but mitogen-induced proliferation of T cells was being suppressed by other cells. Previous studies indicated that soluble factors from suppressor cells may mediate this inhibition of T cell mitogenesis. We thus examined the effects of IBDV on spleen adherent cells. Reverse transcriptase–polymerase chain reaction ŽRT–PCR. was used to quantitate the expression of several cytokine genes in splenic macrophages. In acute IBDV infection, splenic macrophages exhibited enhanced gene expression of type I interferon ŽIFN., chicken myelomonocytic growth factor ŽcMGF., an avian homolog of mammalian IL-6, and 9E3rCEF4, an avian homolog of mammalian IL-8. Mitogen-stimulated spleen cell cultures also produced elevated levels of nitric oxide. The elevation of cytokine gene expression by macrophages occurred transiently during the acute phase of viral infection and coincided with in vitro inhibition of T cell mitogenic response of spleen cells. q 1998 Elsevier Science B.V. Keywords: Infectious bursal disease virus; Chicken; Macrophage; Cytokine; Immunosuppression; Polymerase chain reaction ŽPCR.
) Corresponding author. Tel.: q1 612 625 5276; fax: q1 612 625 5203; [email protected] 1 Current address: Tri-Bio Laboratory, 1400 Fox Hill Rd., State College, PA 16803, USA.
0165-2427r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 1 6 5 - 2 4 2 7 Ž 9 7 . 0 0 1 3 5 - 9
e-mail:
332
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
1. Introduction Infectious bursal disease virus ŽIBDV., a double-stranded RNA virus, is a member of the Birnaviridae family ŽMuller et al., 1979.. The virus targets B lymphocytes in the bursa of Fabricius ŽKaufer and Weiss, 1980. which results in a reduction of the number of circulating B cells and serum immunoglobulin levels ŽIvanyi and Morris, 1976; Giambrone et al., 1977; Hirai et al., 1981; Rodenberger et al., 1994.. Subsequent bursal damage persists for a long period ŽChevile, 1968; Winterfield et al., 1972.. Infection of chickens with IBDV also results in suppression of cell-mediated immune responses and subsequently increases susceptibility to secondary infections. Previous studies indicated that the proliferative responses of peripheral blood lymphocytes and spleen cells to T-cell mitogens were transiently inhibited during the first week of IBDV infection ŽConfer et al., 1981; Sharma et al., 1989.. However, removal of adherent cells from spleen cells partially restored the mitogenic response to normal levels, and co-culture of normal spleen cells with supernatant of splenocytes from IBDV-exposed chickens inhibited T cell responses to Con A ŽSharma and Fredricksen, 1987.. These results suggested that adherent cells may produce suppressive factors upon IBDV infection. However, the immunomodulatory mechanism of spleen adherent cells following IBDV infection is not known and the soluble factors have not been identified. In the present study, we had two objectives: Ža. to confirm that spleens of IBDV-exposed chickens had mitogen-responsive T cells at the peak of mitogenic inhibition, and Žb. to determine if IBDV infection modulated spleen adherent cells. The spleen adherent cells are referred here as macrophages since the majority of adherent cells are monocytes and macrophages.
2. Materials and methods 2.1. Chickens and Õirus One-day-old specific pathogen-free ŽSPF. chicks were purchased from HyVac ŽAdel, IA.. The chickens were housed in Horsfall–Bauer-type isolation units. Feeds and water were provided ad libitum. The virulent IM strain of IBDV was propagated and titrated in chicken embryonated eggs ŽWinterfield et al., 1972.. The virus had a titer of 10 6 50% embryo infectious dose ŽEID50 . per ml. 2.2. Experimental plan Chickens were divided into two groups; Group 1 was inoculated with 1000 EID50 of IM-IBDV by the eyedrop route at 18 days of age, and Group 2 served as unexposed controls. At 18 h, 1, 2, 3, 4, 5, and 8 days post-inoculation ŽPI., three chickens from each group were sampled and spleens were harvested aseptically. Virus infection was confirmed by the presence of gross bursal necrosis in chickens of the virus-exposed group but not of those in the unexposed control group.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
333
Single cell suspensions of the spleens were prepared as described previously ŽPertile et al., 1996.. Lymphoid cells were separated by Histopaque-1083 ŽSigma Chemical Co., St. Louis, MO. and resuspended in RPMI-1640 medium supplemented with 2 mM L-glutamine, 50 m grml gentamycin and 10% FBS. Cell viability was assessed by trypan blue exclusion. 2.3. Pan-purification of T lymphocytes and lymphocyte proliferation assay T lymphocytes were purified by panning as previously described ŽMage et al., 1977; Pertile et al., 1996.. Briefly, spleen cells were incubated at 398C for 3 h to remove adherent cells. Ten million per milliliter of non-adherent cells was incubated with 5 m grml of CT3, mouse anti-chicken CD3-monoclonal antibody ŽSouthern Biotechnology, Birmingham, AL., at 48C for 30 min. The cells were washed and resuspended in RPMI-1640 supplemented with 10% FBS. Eight ml of the cell suspension containing CT3-interacting cells was added to a 100-mm tissue culture plate coated with goat anti-mouse polyvalent immunoglobulins. After 2 h at 48C incubation, unbound cells were washed off and the antibody-bound cells were removed by a cell scraper. The cell purity was assessed by fluorescence-activated cell sorter ŽFACS. analysis using fluorescein isothiocyanate ŽFITC.-conjugated goat anti-mouse polyclonal antibody ŽSouthern Biotechnology Associates.. The cells were consistently 88–95% pure T cells. The procedure for lymphoproliferation assay was described elsewhere ŽKaraca et al., 1996.. 2.4. IL-2 bioassay IL-2 was measured from spleen cell culture supernatant as described previously ŽPertile et al., 1996.. The concentration of IL-2 in the supernatant was determined by corresponding units of serial dilutions of an IL-2 standard. Conditioned medium prepared by stimulation of T cells with Con A Ž5 m grml. served as the IL-2 standard. One unit of IL-2 was defined as the quantity equal to 50% of the maximum absorbence value of the IL-2 standard. 2.5. Nitrite accumulation assay Nitrite concentration in supernatants of Con A-treated and untreated cultures was determined as previously described ŽGreen et al., 1982; Pertile et al., 1996.. A total of 100 m l of each sample was added to 96-well plates in triplicate followed by addition of 100 m l Greiss reagent to each well. Greiss reagent consisted of a 1:1 mixture of 1% sulfanilamide ŽSigma. in 5% phosphoric acid and 0.1% naphthylenediamine dihydrochloride ŽSigma. in deionized water. The plate was read at 570 nm in a microplate reader. The concentration of nitrite in the supernatant was determined by using a standard curve of sodium nitrite. 2.6. Total RNA isolation Total RNA was isolated from splenic adherent cells by using a Trizol reagent ŽGibco-BRL, Gaitherberg, MD. following the protocol provided by the manufacturer.
334
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
Adherent cells were obtained as above. The quantity of isolated RNA was normalized by b-actin gene expression with dot blot. 2.6.1. ReÕerse transcription (RT) RT was performed by using Superscript RT Kit ŽGibco-BRL. according to manufacturer’s protocol. Briefly, 1 m g of total RNA was reverse transcribed in a reaction containing 20 mM Tris–HCl, pH8.3, 50 mM KCl, 2.5 mM MgCl 2 , 200 m M dNTP, 10 m M DTT, 0.5 m g oligo ŽdT. with 200 Urm l of M-MLV reverse transcriptase at 378C for 1 h. A tube with no reverse transcriptase was included in the RT reaction as a negative control. First strand-cDNAs were stored at y808C until used. 2.6.2. Amplification of cDNA and quantitation of cytokine gene expression The technique to detect cytokine mRNA was adapted from previous report ŽRottman et al., 1995.. Specific cytokine gene segments were amplified by Taq polymerase Ž2 U. in a reaction containing 50 mM KCl, 2.5 mM MgCl 2 , 10 mM Tris–HCl pH 9.0, 200 m M dNTP and 50 pM primers. The PCR was conducted for 30 cycles as follows; denaturation at 948C for 1 min, annealing at 658C for 30 s, polymerization at 728C for 1 min, and at the last cycle, the reaction was extended at 728C for 10 min. The primer sequences used were as follows: for b-actin, 5X-TCTGGTGGTACCACAATGTACCCT3X Ž sense . , 5X -ACCAGTAATTGGTACCGGATAATA-3X Ž antisense . ; chicken myelomonocytic growth factor ŽcMGF., 5X-TTCCTGCACAAGAACCTGGAG-3X Žsense., 5X-CTCCACCAACGTCGTGATT-3X Žantisense.; type I IFN, 5X-ATGGCTGTGCCTGCAAGCCCA-3X Ž sense . , 5X -AGTGCGCGTGTTGCCTGTCA-3X Ž antisense . ; 9E3rCEF4 Ž avian IL-8 . , 5X -GGCCTCCTCCTGGTTTCAGCTGC-3X Ž sense . , 5X GCACTCCAGGGGAGCAGG-3X Žantisense.. The PCR products were electrophoresed with 1.5% agarose gel and stained with ethidium bromide. The quantity of the PCR
Fig. 1. The mitogenic response of spleen cells. Splenocytes from virus-free and IBDV-exposed chickens were stimulated with Con A Ž5 m grml.. Cells cultured without Con A had mean CPM, 1843"762 Žmean"S.D... The result represents the mean of three chickens per group at each time intervals. Asterisks indicate statistical significance.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
335
Table 1 The IL-2 production by spleen cells IL-2 ŽUrmla . in spleen-cell cultures containing
Groups
Medium Virus-free IBDV-exposed a b
6"0.2 8"0.1
b
Con A Ž5 m grml. 160"9.0 7"2.0
One unit of IL-2 is defined as 50% of the maximum absorbance value of the IL-2 standard at 570 nm. The values are the means Ž"S.D.. of three pools of spleens. Each pool contained three spleens.
Table 2 The mitogenic response of fractionated spleen cells Delta CPM a Ž=1000. Unfractionated splenocytes Experiment 1 Virus-free IBDV
425.6"20.6 b 14.3"7.6 Ž3.4. c
Experiment 2 Virus-free IBDV x% Of two experiments
543.5"12.5 51.5"22.8 Ž9.5. 6.5
Panned T lymphocytes 90.8"6.016 93.2"28.8 Ž102.6.
110.6"11.4 59.1"17.2 Ž53.4. 74.5
a
Delta CPMsCPM of cells stimulated with Con A Ž5 m grml.yCPM of unstimulated cells. The CPM values are mean of nine chickens Žthree pools of three chickens each."S.D. c Percent of virus-free controls. b
products were analyzed by a densitometer ŽComputing Densitometer, Molecular Dynamics, Sunnyvale, CA. and quantitated by ImageQuan software ŽMolecular Dynamics. and IP Lab gel program ŽSignal Analytic Co... To confirm consistency of the quantity of RNA, 5% and 10% of first strand cDNA were used for cytokine gene expression ŽcMGF, type I IFN, and 9E3rCEF4.. b-actin expression was included to show that the differences of gene expressions were not due to differences in the concentration of the first strand cDNA between groups for PCR. The results were expressed as ratio of average counts of IBDV-exposed chicken to that of virus-free control chicken; IBDV: control ratio s Ž Cytokine RT–PCR sample 1 average count of IBDV-exposed chicken. - Ž cytokine background count . r Ž Cytokine RT –PCR sample 1 average count of virus-free chicken. - Ž cytokine background count .
336
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
337
Fig. 2 Cytokine expression in spleen macrophages. Macrophages were separated from spleen cells at 18, 24, 72 and 192 h after IBVD inoculation. Total RNA was isolated from macrophages and RT–PCR were performed with primers specific for: Ža. type I IFN, 582 bp; Žb. cMGF, 388 bp; and Žc. 9E3rCEF4, 683 bp. The PCR products were resolved on 1.5% agarose gel and photographed. Each sample was tested for PCR at 5% and 10% cDNA concentration, respectively. In Ža., Žb. and Žc., the results of the PCR reaction for samples from virus-exposed and virus-free chicken are shown in duplicate lanes. For each sample, the lane on the left contains results with 5% cDNA and the lane on the right with 10% cDNA. The quantitive gene expression is presented in Žd., Že. and Žf. as ratio of one average count of PCR reaction of IBVD-exposed chickens to that of the virus-free controls. M; 100 bp DNA ladder marker, q; a positive control of each cytokine gene.
2.7. Statistical analysis Student’s t-test was employed to determine significance of differences between individual samples of virus-free and IBDV-exposed groups. P - 0.05 was considered to be statistically significant. 3. Results 3.1. Responses of chickens exposed to IBDV Spleen cells from IBDV-exposed chickens responded poorly to Con A. At 4- and 5-day PI, the spleen cell response to Con A in the virus-infected group was only 3.4% of the virus-free group Ž P - 0.05, Fig. 1.. Suppression of the mitogenic response was transient and spleen cells regained normal responsiveness by one week after virus inoculation.
338
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
In addition to being hyporesponsive to Con A, T cells present in the spleen cell suspension from IBDV-exposed chickens were also deficient in production of IL-2 ŽTable 1.. The levels of IL-2 produced by IBDV-exposed chickens was 4% of the levels produced by the virus-free group. Thus, T cell responsiveness was downregulated during IBDV infection. Previous data have indicated that removal of adherent cells from spleen cells restored the response of cells to PHA ŽSharma and Lee, 1983.. In this study, we examined the function integrity of T cells by pan-purifying T cells from spleens of IBDV-exposed chickens at the time when the mitogenic hyporesponsiveness was maximal. In two separate experiments, pan-purified T cells from IBDV-exposed chickens responded vigorously to Con A after in vitro stimulation ŽTable 2.. In Experiment 1, the proliferation was 102% of the response of T cells purified from IBDV-negative chickens and in Experiment 2, the response was 53% of the IBDV-negative chickens. This was a significant enhancement when compared to the mitogenic response of the unfractionated spleen cells of IBDV-exposed chickens which was less than 10% of the response of IBDV-negative chickens. 3.1.1. Enhancement of cytokine gene expression in adherent cells We examined the macrophages for the expression of three cytokines, i.e. type I IFN, cMGF, and 9E3rCEF4. These cytokines are known to be produced by macrophages and may play a crucial role in acute phase responses in mammals ŽCavaillon, 1994.. Thus, we considered an enhanced cytokine-expression in macrophages as an indication of macrophage activation that results from IBDV infection.
Fig. 3. The nitrite accumulation in supernatant of splenic cells from virus-free and IBDV-exposed groups. Splenocytes were cultured in the presence of Con A Ž5 m grml. as described in Materials and Methods. Supernatant from medium-treated cells had background level of nitrite Ž3.2"0.3.. The concentration of nitrite in the supernatant was determined by using a standard curve of sodium nitrite. ) sSignificantly different Ž P - 0.05. from levels of virus-free control group.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
339
Semi-quantitative PCR analysis revealed that spleen macrophages had an increased expression of type I IFN, cMGF, and 9E3rCEF4 during the first week of infection with IBDV ŽFig. 2.. The enhancement of the cytokine gene expression was detected without additional in vitro stimulation of the cells. Type I IFN expression was increased approximately 11-fold within 24 h of virus exposure. The expression of cMGF gradually increased starting at 18 h PI and reached a peak at 3 days PI. The peak level was 12-fold higher than the level in the control chickens. Enhancement of type I IFN and cMGF expressions was followed by marked increase of 9E3rCEF4 expression. Expression of 9E3rCEF4 in the virus-exposed group was apparent at 3 days PI and then rapidly subsided. At 3 days PI, the 9E3rCEF4 expression in macrophages from the IBDV-exposed chickens was eight times higher than the expression in macrophages from the virus-free control chickens. b-actin expression in each PCR reaction was included and showed that each tube contained consistent amount of cDNA for PCR. 3.1.2. Nitric oxide (NO) production We cultured spleen cells in vitro and examined the supernatant for NO. Significant levels of nitrite were detected in supernatant of Con A-stimulated spleen cell cultures from the virus-exposed group Ž P - 0.05, Fig. 3.. However, the upregulation of NO was transient and was detected only at 1 day PI.
4. Discussion In the present study, we have confirmed that although spleen cells from IBDV-exposed chicken did not respond to T-cell mitogen in vitro, pan-purified T cells from these spleens proliferated vigorously when stimulated with Con A. A previous study had shown that soluble factors produced by suppressor macrophages may be involved in hyporesponsiveness of lymphocytes ŽSharma and Fredricksen, 1987.. We now demonstrate that IBDV infection resulted in enhanced expression of several cytokines and elevated levels of NO in splenic macrophages. Modulation of macrophage function occurred during the acute phase of IBDV infection and coincided with inhibition of T-cell mitogenesis noted in spleen cells. These results provide circumstantial evidence that activation of macrophages by IBDV may have induced spleen cells to exhibit suppressive activity. However, the mechanism of suppression was not investigated. In this study, we examined cytokine gene expression and nitric oxide production. Unfortunately, only a small number of cytokines have been cloned and characterized in chickens ŽRautenschlein and Sharma, 1997.. We used the PCR technique to examine the profile of cytokines in macrophages. The mammalian homologues of three cytokines, i.e. type I-IFN, cMGF Ža homolog of IL-6; Leutz et al., 1989., and 9E3rCEF4 Ža homolog of IL-8; Stoeckle and Barker, 1990; Barker et al., 1993. were examined. These cytokines are known to be involved in inflammatory responses ŽHarada et al., 1994.. The expression of all three cytokines was up-regulated during the acute phase of IBDV infection ŽFig. 2.. The enhanced gene expression occurred without in vitro stimulation of macrophages. Thus, the up-regulation of cytokines was induced in vivo by IBDV infection.
340
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
IBDV causes a highly immunosuppressive disease in chickens ŽGiambrone et al., 1977.. Both cellular and humoral responses are compromised ŽSharma et al., 1989.. The cytokines produced by macrophages in the IBDV-exposed chickens may play a role in mediating immunosuppression. In mice, cytokines and other soluble factors derived from macrophages have been shown to suppress immune responses Žreviewed by Cavaillon, 1994.. In lymphocytic choriomeningitis virus-induced immunosuppression, Type I IFN contributed to the suppression of immune response by stimulating cytotoxic T cells that destroyed antigen-presenting cells and prevented antibody production ŽOdermatt et al., 1991.. Recently, we have noted that IBDV infection results in a rapid influx of CD8 q cells in bursal follicles ŽTanimura and Sharma, 1997.. The role of cytokines in proliferation of CD8 q cells and the possible role of these cells in mediating immunosuppression need to be established. Suppressive effects of macrophage-derived NO on T-cell mitogenic response have been well demonstrated in rats ŽFecho et al., 1994.. Addition of N G -monomethyl-Larginine ŽNMMA., a potent inhibitor of NO production, to the cultures resulted in reversal of the suppressor effect of supernatants of the rat splenocyte cultures. We have recently shown that co-culture of normal splenocytes with the adherent cells from reovirus-infected chickens resulted in inhibition of mitogen-induced proliferation of normal splenocytes ŽPertile et al., 1996.. Removal of adherent cells from the cultures resulted in a partial restoration of T cell responsiveness to Con A in reovirus-exposed chickens. Our w 3 HxTdR incorporation data of pan-purified T cells during IBDV infection showed a similar result to that of T cells during reovirus infection. Our study has established that IBDV modulates macrophage in chickens. Cytokine expressions by macrophages were up-regulated and the up-regulation coincided with inhibition of T-cell mitogen responsiveness. Acknowledgements We thank Drs. S. Reddy, M. Suresh, and S. Rautenschlein, and Miss C. St. Hill for advice and discussions. References Barker, K.A., Hample, A., Stoeckle, M.Y., 1993. Transformation-associated cytokine 9E3rCEF4 is chemotatic for chicken peripheral blood mononuclear cells. J. Virol. 67, 3528–3533. Cavaillon, J.M., 1994. Cytokines and macrophages. Biomed. Pharmacother. 48, 445–453. Chevile, N.F., 1968. Studies of the pathogenesis of Gumboro Disease in bursa of Fabricius, spleen and thymus of the chicken. Am. J. Pathol. 51, 515–551. Confer, A.W., Springer, W.T., Shane, S.M., Donovian, J.F., 1981. Sequential mitogen stimulation of peripheral blood lymphocytes from chickens inoculated with infectious bursal disease virus. Am. J. Vet. Res. 452, 2109–2113. Fecho, K., Maslonek, K.A., Coussons-Read, M.E., Dykstra, L.A., Lysle, D.T., 1994. Macrophage-derived nitric oxide is involved in the depressed Conconavalin A responsiveness of splenic lymphocytes from rats administered morphine in vivo. J. Immunol. 152, 5845–5852. Giambrone, J.J., Dawe, D.L., Eidson, C.S., 1977. Specific suppression of the bursa-dependent immune system of chickens with infectious bursal disease virus. Am. J. Vet. Res. 38, 581–583.
I.-J. Kim et al.r Veterinary Immunology and Immunopathology 61 (1998) 331–341
341
Green, L.C., Wagner, D.A., Glogowski, J., Skipper, P.L., Wishnok, J.S., Tannebaum, S.R., 1982. Analysis of nitrate, nitrite and w 15 Nxnitrate in biological fluids. Anal. Biochem. 126, 131–138. Harada, A., Sekido, N., Akahoshi, T., Wada, T., Mukaida, N., Matsushima, K., 1994. Essential involvement of interleukin-8 ŽIL-8. in acute inflammation. J. Leukocyte Biol. 56, 559–563. Hirai, K., Funakoshi, T., Nakai, T., Shimakura, S., 1981. Sequential changes in the number of surface immunoglobulin-bearing B lymphocytes in infectious bursal disease virus-infected chickens. Avian Dis. 25, 484–496. Ivanyi, J., Morris, R., 1976. Immunodeficiency in chicken: IV. An immunological study of infectious bursal disease. Clin. Exp. Immunol. 28, 1015–1021. Karaca, K., Kim, I.-J., Reddy, S.K., Sharma, J.M., 1996. Nitric oxide inducing factor as a measure of antigen and mitogen-specific T cell responses in chickens. J. Immunol. Methods 192, 97–103. Kaufer, I., Weiss, E., 1980. Significance of bursa of Fabricius as target organ in infectious bursal disease of chickens. Infect. Immun. 27, 364–367. Leutz, A., Damm, K., Sterneck, E., Kowenz, E., Ness, S., Frank, R., Gausepohl, H., Pan, Y.-C.E., Smart, J., Hayman, M., Graf, T., 1989. Molecular cloning of the chicken myelomonocytic growth factor ŽcMGF. reveals relationship to interleukin 6 and granulocyte colony stimulating factor. EMBO J. 8, 175–181. Mage, M.G., McHugh, L.L., Rothstein, T.L., 1977. Mouse lymphocytes with and without surface immunoglobulin; preparative scale separation in polystyrene tissue culture dishes coated with specifically purified anti-immunoglobulin. J. Immunol. Methods 15, 47–56. Muller, H., Scholtissek, C., Becht, H., 1979. The genome of infectious bursal disease virus consists of two segments of double-stranded RNA. J. Virol. 31, 584–589. Odermatt, B., Eppler, M., Leist, T.P., Henagartner, H., Zinkernagel, R.M., 1991. Virus-triggered acquired immunodeficiency by cytotoxic T-cell dependent destruction of antigen-presenting cells and lymph follicle structure. Proc. Natl. Acad. Sci. USA 88, 8252–8256. Pertile, T.L., Karaca, K., Walser, M.M., Sharma, J.M., 1996. Suppressor macrophages mediate depressed lymphoproliferation in chickens infected with avian reovirus. Vet. Immunol. Immunopathol. 53, 129–145. Rautenschlein, S., Sharma, J.M., 1997. Cytokines. In: Pastret, P.P., Bazin, H., Govererts, A., Giebel, P.J. ŽEds.., Handbook of Vertebrate Immunology. Academic Press, Orlando Žin press.. Rodenberger, J., Sharma, J.M., Belzer, S.W., Nordgren, R.M., Naqi, S., 1994. Flow cytometric analysis of B cell and T cell subpopulations in specific pathogen-free chickens infected with infectious bursal disease virus. Avian Dis. 38, 16–21. Rottman, J.B., Freeman, E.B., Tonkonogy, S., Tompkins, M.B., 1995. A reverse transcription–polymerase chain reaction technique to detect feline cytokine genes. Vet. Immunol. Immunopathol. 45, 1–18. Sharma, J.M., Dohms, J.E., Metz, A.L., 1989. Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens. Avian Dis. 33, 112–124. Sharma, J.M., Fredricksen, T.L., 1987. Mechanism of T cell immunosuppression by infectious bursal disease virus of chickens. Proc. Avian Immunol. 238, 283–294. Sharma, J.M., Lee, L.F., 1983. Effects of infectious bursal disease virus on natural killer cell activity and mitogenic response of chicken lymphoid cells: role of adherent cells in cellular immune suppression. Infect. Immun. 42, 747–754. Stoeckle, M.Y., Barker, K.A., 1990. Two burgeoning families of platelet factor 4-related proteins: mediators of inflammatory response. New Biol. 2, 313–323. Tanimura, N., Sharma, J.M., 1997. Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens. Avian Dis. 41, 638–645. Winterfield, R.W., Fadly, A.M., Bickford, A., 1972. Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis. 16, 623–632.