Vol. 23, No. 2
INFECTION AND IMMUNITY, Feb. 1979, p. 244-248 0019-9567/79/02-0244/05$02.oo/o
Antigenic Properties of Human Lymphoblastoid Interferons BARBARA J. DALTON AND KURT PAUCKER* Microbiology Department, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129
Received for publication 26 July 1978
Most virus-induced human lymphoblastoid interferons examined contained variable proportions of the Le and F antigenic species described for human leukocyte interferon. The F species was not detectable in interferons liberated spontaneously from human lymphoblastoid cells in culture. Lymphoblastoid interferons differed considerably in their interaction with the same anti-interferon serum. Spontaneous interferons required approximately ten times less antibody for neutralization than interferon induced by virus in the same cultures or in Namalva cells. The findings suggest that either spontaneous interferons contain fewer inactive antibody-binding molecules than virus-induced lymphoblastoid interferons or the number and distribution of antibody-combining sites, and possibly other surface properties of the interferon molecule, may be influenced by the manner in which spontaneous and induced interferons egress from the cells.
Human lymphoblastoid interferon produced in Namalva cells (16) was previously shown to resemble human leukocyte interferon in antigenic composition (26). This is supported by the observation that Namalva interferon interacted quantitatively with sheep antileukocyte interferon gamma globulins coupled to a solid matrix (3). However, according to other reports, Namalva interferon was only incompletely neutralized by an excess of antibodies against leukocyte interferon (30), and a mixture of antisera against leukocyte and fibroblast interferons was in fact required for full neutralization of the Namalva interferon (6). On the other hand, leukocyte interferon was entirely neutralizable by antileukocyte interferon serum alone (2). These seemingly diverse findings can be explained in part by differences in the relative distribution of the two major antigenic species, Le and F, first identified in leukocyte interferon preparations (2, 12). Whereas more than 99% of the biological activity of leukocyte interferon has been attributed to the dominant Le antigenic species, characteristic of lymphoid interferons, and less than 1% to F, specific for nonlymphoid interferons (13, 22), the proportion of F in Namalva interferon was found to be much higher (15, 21). Since the amount of antibodies against F in individual antileukocyte interferon sera is extremely variable, or anti-F may be absent altogether (2, 23), neutralization of Namalva interferons can be subject to considerable fluctuations depending on the particular antiserum used for study. The present work was undertaken, first, to reassess the relative distribution of the dominant Le and F antigenic determinants in different
preparations of Namalva interferons by means of antisera of defined specificities. Second, we compared the antigenic composition of Namalva interferons with that of interferons liberated either spontaneously or after viral stimulation from other human lymphoblastoid cell lines. Certain differences among these interferons became apparent and are discussed herein. MATERIALS AND METHODS Interferons. Human lymphoblastoid interferons from Namalva cells were either prepared in our own laboratory (The Medical College of Pennsylvania [MCP]) according to Strander et al. (26) or obtained from K. H. Fantes and M. D. Johnston, Wellcome Research Laboratories, Beckenham, England. The latter interferon, designated as Namalva (Eng), consisted primarily of lot 397-12 as well as of samples of lots 423, 431, 432, 441, and 442. Spontaneously liberated lymphoblastoid interferons were obtained from cell lines derived from Epstein-Barr virus-seronegative individuals whose lymphocytes were superinfected with Epstein-Barr virus (J.H., A.G., and J.P.) and from a case of acute infectious mononucleosis (A.F.). These lines were developed and made available to us by J. F. Hewetson, The Medical College of Pennsylvania, Philadelphia. The interferon preparations consisted of 7day-old spent culture media concentrated about 10fold by means of Aquacide (Calbiochem, La Jolla, Calif.). Induced interferons were prepared in J.P. and A.F. cells by exposure to blue tongue virus at a multiplicity of infection of 2.5. The interferon was harvested 24 h later, dialyzed for 4 days in the cold against pH 2, and then returned to neutrality. Blue tongue virus, strain BT-8, was a gift from P. Jameson, The Medical College of Wisconsin, Milwaukee. Human leukocyte interferon (4, 25), partially purified from an acid ethanolic solution (5, 9), was donated by Kari Cantell, State Serum Institute, Helsinki, Finland. Human fibroblast interferon was prepared in 244
VOL. 23, 1979
HUMAN LYMPHOBLASTOID INTERFERON ANTIGENS
245
leukocyte interferon sera, obtained in rabbits or mice, tended to be monospecific; i.e., they neutralized leukocyte but not fibroblast interferon. In addition, whereas the antileukocyte interferon sera exhibited similar neutralizing titers against the immunizing leukocyte interferon and one of the lymphoblastoid interferons, Namalva (Eng), the titer against the second lymphoblastoid interferon, Namalva (MCP), was either less or absent. The rabbit antifibroblast interferon control serum reacted, as expected, only with its homologous antigen. These observations suggested that (i) the Namalva interferon used for injection contained more F antigen than leukocyte interferon, and (ii) minor differences in antigenicity or antibody-binding capacity among human lymphoblastoid interferon preparations may exist. In subsequent experiments presented in Table 2, several Namalva interferons were examined for the presence of directly measurable quantities of an F-specific component, recognizable by its interaction with anti-interferon sera that specifically neutralized either the Le or the F antigenic species. The results indicate that all five Namalva interferons tested in the presence of a monospecific antileukocyte interferon serum (specific only for Le) harbored varying amounts of residual interferon activity, ranging from 3 to 50% of the initial titer (mean, 17%). Preincubation with antifibroblast interferon serum (specific only for F) appeared to neutralize some of the interferon activity in three of the preparations, whereas the titers of the other two were comparable to those recorded with normal rabbit serum. Ostensibly, neutralization of F in the RESULTS presence of predominant amounts of Le cannot When two different preparations of Namalva be reliably demonstrated in this test. However, interferons, as well as leukocyte and fibroblast when the monospecific anti-interferon sera were interferon controls, were examined in direct neu- mixed in equal proportions, or when the Natralization assays against serial dilutions of an- malva interferons were preincubated with antitileukocyte and anti-Namalva interferon sera, sera that already contained antibodies against two distinct patterns of neutralization emerged Le and F, all of the initial interferon activity was (see Table 1). Rabbit anti-Namalva interferon neutralized. Comparable results not tabulated serum neutralized all interferon antigens to aphere were obtained with five other Namalva proximately the same degree. In contrast, anti- (Eng) preparations listed in Materials and Methforeskin fibroblasts (FS-4 strain) by the superinduction scheme of Havell and Vilcek (14). Anti-interferon sera. Monospecific antileukocyte and antifibroblast interferon sera were produced in rabbits as described earlier (2). Antileukocyte interferon sera and ascitic fluids were also obtained in mice according to an earlier protocol (8). Sheep antileukocyte interferon serum was provided by Kari Cantell, and rabbit anti-Namalva interferon serum was provided by K. H. Fantes and M. D. Johnston. Neutralizing antibody titers of these sera are listed in the relevant tables. Assays. Interferon activity was titrated in the presence of an excess of antibodies directed against either one or both antigenic species of leukocyte interferon (Le and F). Titrations were performed in microplates essentially according to Havell and Vilcek (14), except for minor modifications and the use of encephalomyocarditis virus for challenge. Samples of 0.1 ml of individual interferon preparations adjusted to contain about 200 (internal) U were preincubated for 1 h at 37°C with an equal volume of excess antibody as specified in the text. To determine residual non-neutralized interferon, the mixtures were then diluted out in twofold steps, and 30,000 FS-4 cells were added to each well. Eighteen hours later, the cultures were challenged with encephalomyocarditis virus at a multiplicity of infection of 0.25. On day 3, the extent of cytopathic damage was scored. Titers were expressed as the highest dilution of interferon which protected half of the cells against encephalomyocarditis virus, corrected for 1-ml volumes. In this assay, 10 of our units are equivalent to 1 unit of human leukocyte interferon reference standard G023-901-527 distributed by the Reference Reagents Branch of the National Institutes of Health, Bethesda, Md. Serum titers of interferon-neutralizing antibodies were measured as described in full detail elsewhere (22).
TABLE 1. Neutralization of Namalva interferons by antibodies against human leukocyte and lymphoblastoid interferons raised in rabbits and mice Antibodies Neutralizing titer/mla against interferons from: Namalva cells
Prepared in:
Against interferons from:
Leukocytes
MCP
Fibroblasts
Eng
Rabbit (serum) Namalva (Eng) cells 200 300 150 350 Rabbit (serum) Leukocytes 200 1,000 1,500
INFECTION AND IMMUNITY, Feb. 1979, p. 244-248 0019-9567/79/02-0244/05$02.oo/o
Antigenic Properties of Human Lymphoblastoid Interferons BARBARA J. DALTON AND KURT PAUCKER* Microbiology Department, The Medical College of Pennsylvania, Philadelphia, Pennsylvania 19129
Received for publication 26 July 1978
Most virus-induced human lymphoblastoid interferons examined contained variable proportions of the Le and F antigenic species described for human leukocyte interferon. The F species was not detectable in interferons liberated spontaneously from human lymphoblastoid cells in culture. Lymphoblastoid interferons differed considerably in their interaction with the same anti-interferon serum. Spontaneous interferons required approximately ten times less antibody for neutralization than interferon induced by virus in the same cultures or in Namalva cells. The findings suggest that either spontaneous interferons contain fewer inactive antibody-binding molecules than virus-induced lymphoblastoid interferons or the number and distribution of antibody-combining sites, and possibly other surface properties of the interferon molecule, may be influenced by the manner in which spontaneous and induced interferons egress from the cells.
Human lymphoblastoid interferon produced in Namalva cells (16) was previously shown to resemble human leukocyte interferon in antigenic composition (26). This is supported by the observation that Namalva interferon interacted quantitatively with sheep antileukocyte interferon gamma globulins coupled to a solid matrix (3). However, according to other reports, Namalva interferon was only incompletely neutralized by an excess of antibodies against leukocyte interferon (30), and a mixture of antisera against leukocyte and fibroblast interferons was in fact required for full neutralization of the Namalva interferon (6). On the other hand, leukocyte interferon was entirely neutralizable by antileukocyte interferon serum alone (2). These seemingly diverse findings can be explained in part by differences in the relative distribution of the two major antigenic species, Le and F, first identified in leukocyte interferon preparations (2, 12). Whereas more than 99% of the biological activity of leukocyte interferon has been attributed to the dominant Le antigenic species, characteristic of lymphoid interferons, and less than 1% to F, specific for nonlymphoid interferons (13, 22), the proportion of F in Namalva interferon was found to be much higher (15, 21). Since the amount of antibodies against F in individual antileukocyte interferon sera is extremely variable, or anti-F may be absent altogether (2, 23), neutralization of Namalva interferons can be subject to considerable fluctuations depending on the particular antiserum used for study. The present work was undertaken, first, to reassess the relative distribution of the dominant Le and F antigenic determinants in different
preparations of Namalva interferons by means of antisera of defined specificities. Second, we compared the antigenic composition of Namalva interferons with that of interferons liberated either spontaneously or after viral stimulation from other human lymphoblastoid cell lines. Certain differences among these interferons became apparent and are discussed herein. MATERIALS AND METHODS Interferons. Human lymphoblastoid interferons from Namalva cells were either prepared in our own laboratory (The Medical College of Pennsylvania [MCP]) according to Strander et al. (26) or obtained from K. H. Fantes and M. D. Johnston, Wellcome Research Laboratories, Beckenham, England. The latter interferon, designated as Namalva (Eng), consisted primarily of lot 397-12 as well as of samples of lots 423, 431, 432, 441, and 442. Spontaneously liberated lymphoblastoid interferons were obtained from cell lines derived from Epstein-Barr virus-seronegative individuals whose lymphocytes were superinfected with Epstein-Barr virus (J.H., A.G., and J.P.) and from a case of acute infectious mononucleosis (A.F.). These lines were developed and made available to us by J. F. Hewetson, The Medical College of Pennsylvania, Philadelphia. The interferon preparations consisted of 7day-old spent culture media concentrated about 10fold by means of Aquacide (Calbiochem, La Jolla, Calif.). Induced interferons were prepared in J.P. and A.F. cells by exposure to blue tongue virus at a multiplicity of infection of 2.5. The interferon was harvested 24 h later, dialyzed for 4 days in the cold against pH 2, and then returned to neutrality. Blue tongue virus, strain BT-8, was a gift from P. Jameson, The Medical College of Wisconsin, Milwaukee. Human leukocyte interferon (4, 25), partially purified from an acid ethanolic solution (5, 9), was donated by Kari Cantell, State Serum Institute, Helsinki, Finland. Human fibroblast interferon was prepared in 244
VOL. 23, 1979
HUMAN LYMPHOBLASTOID INTERFERON ANTIGENS
245
leukocyte interferon sera, obtained in rabbits or mice, tended to be monospecific; i.e., they neutralized leukocyte but not fibroblast interferon. In addition, whereas the antileukocyte interferon sera exhibited similar neutralizing titers against the immunizing leukocyte interferon and one of the lymphoblastoid interferons, Namalva (Eng), the titer against the second lymphoblastoid interferon, Namalva (MCP), was either less or absent. The rabbit antifibroblast interferon control serum reacted, as expected, only with its homologous antigen. These observations suggested that (i) the Namalva interferon used for injection contained more F antigen than leukocyte interferon, and (ii) minor differences in antigenicity or antibody-binding capacity among human lymphoblastoid interferon preparations may exist. In subsequent experiments presented in Table 2, several Namalva interferons were examined for the presence of directly measurable quantities of an F-specific component, recognizable by its interaction with anti-interferon sera that specifically neutralized either the Le or the F antigenic species. The results indicate that all five Namalva interferons tested in the presence of a monospecific antileukocyte interferon serum (specific only for Le) harbored varying amounts of residual interferon activity, ranging from 3 to 50% of the initial titer (mean, 17%). Preincubation with antifibroblast interferon serum (specific only for F) appeared to neutralize some of the interferon activity in three of the preparations, whereas the titers of the other two were comparable to those recorded with normal rabbit serum. Ostensibly, neutralization of F in the RESULTS presence of predominant amounts of Le cannot When two different preparations of Namalva be reliably demonstrated in this test. However, interferons, as well as leukocyte and fibroblast when the monospecific anti-interferon sera were interferon controls, were examined in direct neu- mixed in equal proportions, or when the Natralization assays against serial dilutions of an- malva interferons were preincubated with antitileukocyte and anti-Namalva interferon sera, sera that already contained antibodies against two distinct patterns of neutralization emerged Le and F, all of the initial interferon activity was (see Table 1). Rabbit anti-Namalva interferon neutralized. Comparable results not tabulated serum neutralized all interferon antigens to aphere were obtained with five other Namalva proximately the same degree. In contrast, anti- (Eng) preparations listed in Materials and Methforeskin fibroblasts (FS-4 strain) by the superinduction scheme of Havell and Vilcek (14). Anti-interferon sera. Monospecific antileukocyte and antifibroblast interferon sera were produced in rabbits as described earlier (2). Antileukocyte interferon sera and ascitic fluids were also obtained in mice according to an earlier protocol (8). Sheep antileukocyte interferon serum was provided by Kari Cantell, and rabbit anti-Namalva interferon serum was provided by K. H. Fantes and M. D. Johnston. Neutralizing antibody titers of these sera are listed in the relevant tables. Assays. Interferon activity was titrated in the presence of an excess of antibodies directed against either one or both antigenic species of leukocyte interferon (Le and F). Titrations were performed in microplates essentially according to Havell and Vilcek (14), except for minor modifications and the use of encephalomyocarditis virus for challenge. Samples of 0.1 ml of individual interferon preparations adjusted to contain about 200 (internal) U were preincubated for 1 h at 37°C with an equal volume of excess antibody as specified in the text. To determine residual non-neutralized interferon, the mixtures were then diluted out in twofold steps, and 30,000 FS-4 cells were added to each well. Eighteen hours later, the cultures were challenged with encephalomyocarditis virus at a multiplicity of infection of 0.25. On day 3, the extent of cytopathic damage was scored. Titers were expressed as the highest dilution of interferon which protected half of the cells against encephalomyocarditis virus, corrected for 1-ml volumes. In this assay, 10 of our units are equivalent to 1 unit of human leukocyte interferon reference standard G023-901-527 distributed by the Reference Reagents Branch of the National Institutes of Health, Bethesda, Md. Serum titers of interferon-neutralizing antibodies were measured as described in full detail elsewhere (22).
TABLE 1. Neutralization of Namalva interferons by antibodies against human leukocyte and lymphoblastoid interferons raised in rabbits and mice Antibodies Neutralizing titer/mla against interferons from: Namalva cells
Prepared in:
Against interferons from:
Leukocytes
MCP
Fibroblasts
Eng
Rabbit (serum) Namalva (Eng) cells 200 300 150 350 Rabbit (serum) Leukocytes 200 1,000 1,500
