Ini. Archs Allergy appl. Immun. 60: 249-257 (1979)
Depression of Contact Hypersensitivity to Oxazolone in Mice Exposed to Newcastle Disease Virus A. Bellavia, J. Ure, P. Ammatuna, C. Caruso and A. Salerno Immunobiology Unit, Department of Zoology, University of Edinburgh, Edinburgh; Istituto di Microbiologia, and Istituto di Patologia generale, Universita di Palermo, Palermo
Abstract. The effect of Newcastle disease virus (NDV) on delayed hypersensitivity to oxazolone in CBA mice was studied. There was a significant impairment of the ability of mice to develop cutaneous hypersensitivity shortly after injection of the virus. The effect was evident when NDV was administered up to 2 days before or within 24 h after sensitization, suggesting that NDV interferes with the process of sensitization. The degree of depression was related to the dose of virus inoculated. NDV inactivated by UV irra diation or heat did not depress contact sensitivity to oxazolone. These data are considered to support the hypothesis that the depression is mediated by a direct interaction between lymphocytes and NDV.
Prior exposure to Newcastle Disease Vi rus (NDV), an interferon inducer [4], pro tects mice against Semliki Forest Vims, but not against the arbovirus Tho-Ar-126 [1, 24, 25], It has been suggested that the growth of Tho-Ar-126 may be due to an NDV-induced depression of cell-mediated immunity [24], Several observations indi rectly support this possibility. (1) Injection of NDV to mice is followed by a transitory lymphocytopenia lasting 24-48 h [29] and a decrease in the proportion of Thy-1 (theta)-positive cells in the lymph nodes and spleen [24], (2) NDV produces a change in the pattern of lymphocyte localization in an tigen-stimulated [7, 20] or non-stimulated [20, 30, 31] animals. (3) A modest but sig
nificant prolongation of skin allograft sur vival in NDV-treated mice has been report ed [22, 32]. (4) Under certain temporal con ditions, NDV can depress delayed hypersen sitivity [11, 12], (5) NDV abrogates the protective immunity and delayed hypersen sitivity transferred by lymphocytes from rats infected with Listeria monocytogenes after brief exposure of cells in vitro to vims [20], We now report experiments on the ef fects of NDV on the hypersensitivity reac tion to the contact-sensitizing agent oxazo lone in CBA mice. Materials and Methods A nimals Male and female CBA mice were used at 3-4 months of age. Mice of one sex only were includ ed in any individual experiment.
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 3:38:34 PM
Introduction
Virus NDV (Hickman strain) was grown for 72 h in the allantoic cavity of 11-day-old cmbryonated eggs (Meridalma, Brescia, Italy). Infected allantoic fluid (AF) was clarified by centrifugation (500 g 30 min). Three types of NDV preparations were used in these experiments: (a) infectious virus in AF (the suspension contained 109 50% egg-infec tive doscs/ml); (b) virus in AF which was inacti vated by ultraviolet (UV) light, or (c) by heat. In preparation (b) 15-ml aliquots of NDV were dis pensed into glass Petri dishes; the dishes were sup ported on a bed of crushed ice, placed on a rotat ing platform, and exposed for 15 min to UV irra diation (30 cm from the light source, a Philips 30 W germicidal lamp). Heated virus (c) was pre pared by incubating 5 ml of NDV in a 56 °C wa ter bath for 30 min. The embryo infectivity dose (E1D50) and the hemagglutinating titer of these preparations were performed as described by Woodruff and Woodruff [30], Virus-inoculated mice were injected with 0.4 ml of each virus preparation; control mice were injected with 0.4 ml of AF from 14-day-old embryonated non-infected eggs.
Bellavia/Ure/Ammatuna/Caruso/Salerno
sides of the left ears and 16 fil of olive oil to the right ears. 10 h after challenge each mouse received 2 /tCi of 125I-labelcd 5-iodo-2deoxyuridine (lUdR: Radiochemical Centre, Amersham, England, specific activity 5 mCi/mg) by tail vein injection. Mice were sacrificed 26 h after challenge; their ears were cut off at the hair line and counted in a Nuclear Chicago /-counter. Results were expressed as a ratio of the radioac tivity in the left ear to that in the right ear, this being designated as L/R125IUdR uptake [27].
Interferon Assays Mice were injected in the tail vein with various preparations of NDV in 0.4 ml; blood was obtained by cardiac puncture 9 h after injection since serum interferon concentrations are maximal at this time [10]. Mouse interferon was assayed on mouse CCL1 cells grown in prescription bottles in TC 199 (Gibco, New York, N.Y.) supplemented to fi nal concentrations of 5% fetal calf serum (Gibco). The plaque reduction technique employing ves icular stomatitis virus as the challenge virus was used for interferon assays [8, 9, 15, 28], An international reference mouse interferon obtained from the National Institutes of Allergy Delay ed-Type-Hypersensitivity and Infectious Diseases of Bethesda, Md., was in 1 or 2 mg of oxazolone (4-ethoxymethy-cluded with each set of assays. The international lcne-2phenyl oxazolone: BDH, Poole, England), interferon standard (designated titer of 12,000 U) dissolved in 0.1 ml absolute ethanol at 60 °C, was titered 14,933 + 2,919 in our assay system applied to a previously shaved area, 2 cm in diam (correction factor, 0.80). eter, on the left of the thorax. Sometimes, 0.1 ml The titers of interferon were expressed as absolute ethanol was applied to controls, referred logl0 of the reciprocal of the dilution of the sam to as ‘sham-sensitized’. 7 days after sensitization, ple which reduced the plaque count to 50% of that the thickness of each ear was measured with a dial of the controls. gauge (Thomas Mercer Ltd., London, England) as described by Askerson and Ptak [3]. The gauge Statistical Methods had contact surfaces of 9 mm in diameter. The T h e results w ere a n aly sed by th e S tu d en t’s test. mean of two rapidly executed readings of pinna thickness was taken, to minimize errors due to lo cal differences in thickness and to compression of Results the tissue. A total of 32 /il of 2% oxazolone in ol ive oil was then applied evenly to both sides of Neither exposure to NDV nor sensitiza both ears. Sometimes control mice were ‘shamchallenged’ with olive oil only. The thickness was tion with 1 mg oxazolone significantly af measured again in the same way 6, 24 and 48 h fected the thickness of the recipients ears later. before challenge (table I). Sham challenge Measurement of reaction was also adapted with olive oil had no effect on ear thickness from Vadas et al. [27], Mice were challenged al ways 7 days after sensitization: a total of 16
Depression of Contact Hypersensitivity to Oxazolone in Mice Exposed to Newcastle Disease Virus A. Bellavia, J. Ure, P. Ammatuna, C. Caruso and A. Salerno Immunobiology Unit, Department of Zoology, University of Edinburgh, Edinburgh; Istituto di Microbiologia, and Istituto di Patologia generale, Universita di Palermo, Palermo
Abstract. The effect of Newcastle disease virus (NDV) on delayed hypersensitivity to oxazolone in CBA mice was studied. There was a significant impairment of the ability of mice to develop cutaneous hypersensitivity shortly after injection of the virus. The effect was evident when NDV was administered up to 2 days before or within 24 h after sensitization, suggesting that NDV interferes with the process of sensitization. The degree of depression was related to the dose of virus inoculated. NDV inactivated by UV irra diation or heat did not depress contact sensitivity to oxazolone. These data are considered to support the hypothesis that the depression is mediated by a direct interaction between lymphocytes and NDV.
Prior exposure to Newcastle Disease Vi rus (NDV), an interferon inducer [4], pro tects mice against Semliki Forest Vims, but not against the arbovirus Tho-Ar-126 [1, 24, 25], It has been suggested that the growth of Tho-Ar-126 may be due to an NDV-induced depression of cell-mediated immunity [24], Several observations indi rectly support this possibility. (1) Injection of NDV to mice is followed by a transitory lymphocytopenia lasting 24-48 h [29] and a decrease in the proportion of Thy-1 (theta)-positive cells in the lymph nodes and spleen [24], (2) NDV produces a change in the pattern of lymphocyte localization in an tigen-stimulated [7, 20] or non-stimulated [20, 30, 31] animals. (3) A modest but sig
nificant prolongation of skin allograft sur vival in NDV-treated mice has been report ed [22, 32]. (4) Under certain temporal con ditions, NDV can depress delayed hypersen sitivity [11, 12], (5) NDV abrogates the protective immunity and delayed hypersen sitivity transferred by lymphocytes from rats infected with Listeria monocytogenes after brief exposure of cells in vitro to vims [20], We now report experiments on the ef fects of NDV on the hypersensitivity reac tion to the contact-sensitizing agent oxazo lone in CBA mice. Materials and Methods A nimals Male and female CBA mice were used at 3-4 months of age. Mice of one sex only were includ ed in any individual experiment.
Downloaded by: Kings's College London 137.73.144.138 - 11/10/2017 3:38:34 PM
Introduction
Virus NDV (Hickman strain) was grown for 72 h in the allantoic cavity of 11-day-old cmbryonated eggs (Meridalma, Brescia, Italy). Infected allantoic fluid (AF) was clarified by centrifugation (500 g 30 min). Three types of NDV preparations were used in these experiments: (a) infectious virus in AF (the suspension contained 109 50% egg-infec tive doscs/ml); (b) virus in AF which was inacti vated by ultraviolet (UV) light, or (c) by heat. In preparation (b) 15-ml aliquots of NDV were dis pensed into glass Petri dishes; the dishes were sup ported on a bed of crushed ice, placed on a rotat ing platform, and exposed for 15 min to UV irra diation (30 cm from the light source, a Philips 30 W germicidal lamp). Heated virus (c) was pre pared by incubating 5 ml of NDV in a 56 °C wa ter bath for 30 min. The embryo infectivity dose (E1D50) and the hemagglutinating titer of these preparations were performed as described by Woodruff and Woodruff [30], Virus-inoculated mice were injected with 0.4 ml of each virus preparation; control mice were injected with 0.4 ml of AF from 14-day-old embryonated non-infected eggs.
Bellavia/Ure/Ammatuna/Caruso/Salerno
sides of the left ears and 16 fil of olive oil to the right ears. 10 h after challenge each mouse received 2 /tCi of 125I-labelcd 5-iodo-2deoxyuridine (lUdR: Radiochemical Centre, Amersham, England, specific activity 5 mCi/mg) by tail vein injection. Mice were sacrificed 26 h after challenge; their ears were cut off at the hair line and counted in a Nuclear Chicago /-counter. Results were expressed as a ratio of the radioac tivity in the left ear to that in the right ear, this being designated as L/R125IUdR uptake [27].
Interferon Assays Mice were injected in the tail vein with various preparations of NDV in 0.4 ml; blood was obtained by cardiac puncture 9 h after injection since serum interferon concentrations are maximal at this time [10]. Mouse interferon was assayed on mouse CCL1 cells grown in prescription bottles in TC 199 (Gibco, New York, N.Y.) supplemented to fi nal concentrations of 5% fetal calf serum (Gibco). The plaque reduction technique employing ves icular stomatitis virus as the challenge virus was used for interferon assays [8, 9, 15, 28], An international reference mouse interferon obtained from the National Institutes of Allergy Delay ed-Type-Hypersensitivity and Infectious Diseases of Bethesda, Md., was in 1 or 2 mg of oxazolone (4-ethoxymethy-cluded with each set of assays. The international lcne-2phenyl oxazolone: BDH, Poole, England), interferon standard (designated titer of 12,000 U) dissolved in 0.1 ml absolute ethanol at 60 °C, was titered 14,933 + 2,919 in our assay system applied to a previously shaved area, 2 cm in diam (correction factor, 0.80). eter, on the left of the thorax. Sometimes, 0.1 ml The titers of interferon were expressed as absolute ethanol was applied to controls, referred logl0 of the reciprocal of the dilution of the sam to as ‘sham-sensitized’. 7 days after sensitization, ple which reduced the plaque count to 50% of that the thickness of each ear was measured with a dial of the controls. gauge (Thomas Mercer Ltd., London, England) as described by Askerson and Ptak [3]. The gauge Statistical Methods had contact surfaces of 9 mm in diameter. The T h e results w ere a n aly sed by th e S tu d en t’s test. mean of two rapidly executed readings of pinna thickness was taken, to minimize errors due to lo cal differences in thickness and to compression of Results the tissue. A total of 32 /il of 2% oxazolone in ol ive oil was then applied evenly to both sides of Neither exposure to NDV nor sensitiza both ears. Sometimes control mice were ‘shamchallenged’ with olive oil only. The thickness was tion with 1 mg oxazolone significantly af measured again in the same way 6, 24 and 48 h fected the thickness of the recipients ears later. before challenge (table I). Sham challenge Measurement of reaction was also adapted with olive oil had no effect on ear thickness from Vadas et al. [27], Mice were challenged al ways 7 days after sensitization: a total of 16
