The LT system in experimental animals. I. Rapid release of high levels of lymphotoxin (LT) activity from murine lymphocytes during the interaction with lectin-treated allogeneic or xenogeneic target cells in vitro.

High levels of cytotoxic activity (lymphotoxin-like (LT) detectable on L-929 cells was obtained in serum-free culture supernatants when non-adherent murine splenic lymphocytes or nylon wool-enriched T cells were cultured on monolayers of mitogen- (PHA) coated allogeneic or xenogeneic stimulator cells in vitro. Levels of lytic activity were lower in supernatants obtained from splenocyte cultures containing glass-adherent cell populations. Although release of lytic activity was very rapid, reaching maximal levels by 6 to 10 hr, this activity was very unstable. The levels of toxic activity in 8-hr supernatants were 20 to 50 times the levels obtained when lymphocytes were cultured with various dosages of mitogen (PHA-P, Con A) alone, even after 5 days of incubation. This phenomenon was not unique to murine lymphoid cells, for similarly high levels of LT activity were found in supernatants from lymphoid cells obtained from several animal species activated in a similar fashion. These results indicate that lymphoid cells from several animal species are capable of rapidly releasing high levels of cell-lytic activity in vitro not previously noted, and provide a means for obtaining highly active supernatants for biochemical studies. Furthermore, the data suggest that rapid release of LT may depend upon the nature of the cellular activating stimulus involving interaction of the lymphocyte with both lectin and target cell surface

The LT system in experimental animals. II. Physical and immunologic characteristics of molecules with LT activity rapidly released by murine lymphoid cells activated on lectin-coated allogeneic monolayers in vitro.

The present studies investigate the physicochemical and immunologic properties of cell-lytic molecules released in vitro by nonadherent C57/BL/6 splenocytes or nylon wool-enriched T cell populations activated on monolayers of PHA coated L-929 cells. The findings reveal that cell-lytic molecules released by these lymphoid cells are physically heterogeneous. These molecules can be separated by gel filtration into similar m.w. classes previously observed for human lymphotoxin (LT) molecules. Three major classes, termed complex (Cx) (>200,000 d), α heavy [α(H)] (110 to 140,000 d), α light [α(L)] (60 to 90,000 d), and two minor classes, β at 40 to 50,000 d and γ at 10 to 20,000 d, were observed. Chromatography of supernatants in high ionic strength buffers dissociated Cx and α(H) to the smaller m.w. α(L) form. This evidence suggests that Cx and α(H) MW classes are physically related to the smaller m.w. α(L) class. Fractionation of the α(H) m.w. LT class by DEAE or PAGE resolved these molecules into additional distinct subclasses. Antisera were made against fresh serum-free whole supernatants (anti-WS) or rechromatographed Ultrogel fractions containing α(H) molecules [anti-α(H)]. Anti-α(H) and anti-WS react with all m.w. classes of murine LT molecules, indicating these various forms are immunologically related. These antisera do not react with LT molecules obtained from several other animal species or with 'nonspecific' intracellular toxins, e.g., lysosomal enzymes, present in normal PMN or phagocytic cells. These data indicate that materials with cell-lytic activity present in these culture supernatants are LT molecules, because: a) certain m.w. forms observed are similar to those reported previously, and b) these various m.w. forms are all physically and immunologically interrelated. These studies also indicate that murine LT molecules like human LT molecules are heterogeneous, but appear to comprise a system of subunits, in which the large m.w. form may dissociate into the smaller m.w. forms

The LT system in experimental animals. IV. Rapid specific lysis of 51CR-labeled allogeneic target cells by highly unstable high m.w. lymphotoxin-receptor complex(es) released in vitro by activated alloimmune murine T lymphocytes.

Lymphocytes or purified T cells obtained from the spleens of alloimmune C57BL/6, DBA/2, or C3H/DiSn mice, when placed on monolayers of lectin-coated allogeneic (L-929 or 3T3) fibroblasts, release into the supernatant various forms of cell lytic material. One form appears to be a high m.w. complex containing an antigen-binding receptor(s) that is highly labile and capable of causing rapid and specific lysis of allogeneic target cells in vitro. Material(s) that could mediate these cell lytic effects were detected in culture supernatants as early as 3 hr after stimulation, peaked at 6 to 9 hr, and declined thereafter. The specific cell lytic activity appeared to be due to high m.w. LT-receptor complexes for the following reasons: (a) antisera that could neutralize murine LT activity in vitro could inhibit this effect; (b) absorption of supernatants on the specific target cells at 4°C removed both the specific lytic activity and nonspecific LT activity detectable on L-929 cells in vitro; (c) this material(s) was highly unstable, as are LT complex forms; and (d) fractionation by molecular sieving of these supernatants revealed that cell lysis was mediated by material(s) in the high (>200,000) m.w. complex form(s). The lytic effect did not appear to be due to Ab + C because supernatants that had lost their specific lytic activity could not be reconstituted with fresh sources of C. Since purified alloimmune T lymphocytes yielded more active supernatants than unseparated nonadherent spleen cells, and polyspecific goat anti-mouse Ig sera had virtually no effect on this lytic activity, the authors feel the receptor(s) in these complexes originates from the T cell(s). The data support the concept that the short-lived specific cell lytic material in these supernatants is a high m.w. complex containing αH m.w. LT subunits in functional association with specific (T cell?) antigen-binding receptor(s) molecules. These findings strongly corroborate analogous findings in the human and support the concept that the smaller m.w. LT molecules represent a system of weakly lytic but related subunits released by cells that can associate together and functionally associate with antigen-binding receptor(s) to form highly effective cell lytic complexes. Furthermore, these lytic LT-receptor complexes can be directed by the specificity of the receptor with which they are associated

The LT system in experimental animals. II. Physical and immunologic characteristics of molecules with LT activity rapidly released by murine lymphoid cells activated on lectin-coated allogeneic monolayers in vitro.

The present studies investigate the physicochemical and immunologic properties of cell-lytic molecules released in vitro by nonadherent C57/BL/6 splenocytes or nylon wool-enriched T cell populations activated on monolayers of PHA coated L-929 cells. The findings reveal that cell-lytic molecules released by these lymphoid cells are physically heterogeneous. These molecules can be separated by gel filtration into similar m.w. classes previously observed for human lymphotoxin (LT) molecules. Three major classes, termed complex (Cx) (>200,000 d), α heavy [α(H)] (110 to 140,000 d), α light [α(L)] (60 to 90,000 d), and two minor classes, β at 40 to 50,000 d and γ at 10 to 20,000 d, were observed. Chromatography of supernatants in high ionic strength buffers dissociated Cx and α(H) to the smaller m.w. α(L) form. This evidence suggests that Cx and α(H) MW classes are physically related to the smaller m.w. α(L) class. Fractionation of the α(H) m.w. LT class by DEAE or PAGE resolved these molecules into additional distinct subclasses. Antisera were made against fresh serum-free whole supernatants (anti-WS) or rechromatographed Ultrogel fractions containing α(H) molecules [anti-α(H)]. Anti-α(H) and anti-WS react with all m.w. classes of murine LT molecules, indicating these various forms are immunologically related. These antisera do not react with LT molecules obtained from several other animal species or with 'nonspecific' intracellular toxins, e.g., lysosomal enzymes, present in normal PMN or phagocytic cells. These data indicate that materials with cell-lytic activity present in these culture supernatants are LT molecules, because: a) certain m.w. forms observed are similar to those reported previously, and b) these various m.w. forms are all physically and immunologically interrelated. These studies also indicate that murine LT molecules like human LT molecules are heterogeneous, but appear to comprise a system of subunits, in which the large m.w. form may dissociate into the smaller m.w. forms

The LT system in experimental animals. IV. Rapid specific lysis of 51CR-labeled allogeneic target cells by highly unstable high m.w. lymphotoxin-receptor complex(es) released in vitro by activated alloimmune murine T lymphocytes.

Lymphocytes or purified T cells obtained from the spleens of alloimmune C57BL/6, DBA/2, or C3H/DiSn mice, when placed on monolayers of lectin-coated allogeneic (L-929 or 3T3) fibroblasts, release into the supernatant various forms of cell lytic material. One form appears to be a high m.w. complex containing an antigen-binding receptor(s) that is highly labile and capable of causing rapid and specific lysis of allogeneic target cells in vitro. Material(s) that could mediate these cell lytic effects were detected in culture supernatants as early as 3 hr after stimulation, peaked at 6 to 9 hr, and declined thereafter. The specific cell lytic activity appeared to be due to high m.w. LT-receptor complexes for the following reasons: (a) antisera that could neutralize murine LT activity in vitro could inhibit this effect; (b) absorption of supernatants on the specific target cells at 4°C removed both the specific lytic activity and nonspecific LT activity detectable on L-929 cells in vitro; (c) this material(s) was highly unstable, as are LT complex forms; and (d) fractionation by molecular sieving of these supernatants revealed that cell lysis was mediated by material(s) in the high (>200,000) m.w. complex form(s). The lytic effect did not appear to be due to Ab + C because supernatants that had lost their specific lytic activity could not be reconstituted with fresh sources of C. Since purified alloimmune T lymphocytes yielded more active supernatants than unseparated nonadherent spleen cells, and polyspecific goat anti-mouse Ig sera had virtually no effect on this lytic activity, the authors feel the receptor(s) in these complexes originates from the T cell(s). The data support the concept that the short-lived specific cell lytic material in these supernatants is a high m.w. complex containing αH m.w. LT subunits in functional association with specific (T cell?) antigen-binding receptor(s) molecules. These findings strongly corroborate analogous findings in the human and support the concept that the smaller m.w. LT molecules represent a system of weakly lytic but related subunits released by cells that can associate together and functionally associate with antigen-binding receptor(s) to form highly effective cell lytic complexes. Furthermore, these lytic LT-receptor complexes can be directed by the specificity of the receptor with which they are associated

The LT system in experimental animals. III. Physicochemical characteristics and relationships of lymphotoxin (LT) molecules released in vitro by activated lymphoid cells from several animal species.

High levels of material with lymphotoxin- (LT) like activity, cytolytic for L-929 cells in vitro, were rapidly released by lymphoid cells obtained from rat, guinea pig, hamster, and rabbit after co-culture for 7 to 10 hr with lectin-coated L-929 cells in vitro. These supernatants were all rapidly fractionated under similar conditions by molecular sieving, ion exchange chromatography, and polyacrylamide gel electrophoresis, and the results were compared to those previously obtained for human and murine LT molecules. The activity in all supernatants was separated by gel filtration into multiple m.w. classes that were strikingly similar to those previously assigned to human and murine LT molecules, i.e. complex (>200,000 d), α (70 to 150,000 d), β (35 to 50,000 d), and γ (12 to 20,000 d). The α m.w. class was resolved on Ultrogel AcA 44 into two distinct m.w. forms, termed α heavy (α(H)) (110 to 150,000 d), and α light (α(L)) (70 to 100,000 d). All supernatants contained LT forms of the Cx, α(H) and α(L) m.w. classes. The smaller β and γ m.w. forms were not as evident in supernatants induced in this fashion. The percentage of activity in each m.w. class varied, but the majority of activity was generally due to α(H) and α(L) forms. However, the relative percentage of activity in any given m.w. class was dependent upon the ionic strength of the separating column buffer. The actual molecular dimensions of materials within the α m.w. class varied somewhat between species, however, they did not vary within the smaller m.w. β and γ forms when detected. The α(H) and α(L) m.w. forms could each be further resolved into multiple charge subclasses. Although the α(H) and α(L) forms in guinea pig and rabbit have been shown by immunologic and/or physical data to be related forms, they can be distinguished by charge from one another. This evidence supports the concept that LT molecules detected in supernatants from experimental animals, although heterogeneous, represent a system of related subunits similar to that defined for the human and mouse. Moreover, this system of cell toxins appears to be conserved in evolution and has common features in each of the animals examined in this study

The LT system in experimental animals. III. Physicochemical characteristics and relationships of lymphotoxin (LT) molecules released in vitro by activated lymphoid cells from several animal species.

High levels of material with lymphotoxin- (LT) like activity, cytolytic for L-929 cells in vitro, were rapidly released by lymphoid cells obtained from rat, guinea pig, hamster, and rabbit after co-culture for 7 to 10 hr with lectin-coated L-929 cells in vitro. These supernatants were all rapidly fractionated under similar conditions by molecular sieving, ion exchange chromatography, and polyacrylamide gel electrophoresis, and the results were compared to those previously obtained for human and murine LT molecules. The activity in all supernatants was separated by gel filtration into multiple m.w. classes that were strikingly similar to those previously assigned to human and murine LT molecules, i.e. complex (>200,000 d), α (70 to 150,000 d), β (35 to 50,000 d), and γ (12 to 20,000 d). The α m.w. class was resolved on Ultrogel AcA 44 into two distinct m.w. forms, termed α heavy (α(H)) (110 to 150,000 d), and α light (α(L)) (70 to 100,000 d). All supernatants contained LT forms of the Cx, α(H) and α(L) m.w. classes. The smaller β and γ m.w. forms were not as evident in supernatants induced in this fashion. The percentage of activity in each m.w. class varied, but the majority of activity was generally due to α(H) and α(L) forms. However, the relative percentage of activity in any given m.w. class was dependent upon the ionic strength of the separating column buffer. The actual molecular dimensions of materials within the α m.w. class varied somewhat between species, however, they did not vary within the smaller m.w. β and γ forms when detected. The α(H) and α(L) m.w. forms could each be further resolved into multiple charge subclasses. Although the α(H) and α(L) forms in guinea pig and rabbit have been shown by immunologic and/or physical data to be related forms, they can be distinguished by charge from one another. This evidence supports the concept that LT molecules detected in supernatants from experimental animals, although heterogeneous, represent a system of related subunits similar to that defined for the human and mouse. Moreover, this system of cell toxins appears to be conserved in evolution and has common features in each of the animals examined in this study