Relationship of CIF, LT, and PIF released in vitro by activated human lymphocytes. II. A further functional comparison of LT and PIF activities on HeLa and L-929 target cells.

Lymphotoxin (LT), and proliferation inhibition factor (PIF) activities found in 5-day supernatants of mitogen-activated human lymphocytes (SAL) were further compared. In agreement with previous results, the activities could not be distinguished functionally. Quantitative differences in the amount of activity detected in the SAL could be accounted for on the basis of target cell differences, concentration of the lymphocyte effector molecules in the supernatant, and the parameter employed to assess cell function. Growth inhibitory activity detected at high supernatant dilutions was completely reversible, whereas the cytotoxic activity detected at low supernatant dilutions was irreversible. When the active medium was fractionated on DEAE, two peaks of inhibitory activity were detected. Depending upon the amount of activity and target cell, both peaks of activity were growth inhibitory or cytotoxic. Since both peaks of material affected HeLa and L-929 cells, the materials were not species specific. Thus, it appears that cloning inhibitor factor, LT, and PIF activities may actually be measures of the same stable materials found in 5-day activated lymphocyte supernatants.     

The human LT system. I. Physical-chemical heterogeneity of LT molecules released by mitogen activated human lymphocytes in vitro.

Molecules with LT activity which can be identified in supernatants from PHA stimulated human lymphocytes by lysis of murine L-929 cell in vitro are heterogeneous. They can be separated by MW on sephadex or ultrogel columns into four separate classes: (a) complex (>200,000 daltons); (b) α (70–90,000 d); (c) β (25–50,000 d); and (d) γ (10–20,000 d). The amount of activity in a supernatant due to each class varies but is approximately: Cx—5 to 20%, α—40 to 60%, β—20 to 40%, and γ—0 to 10%. These classes differ one from another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. Each class, except γ, can be further separated into subclasses by ion exchange chromatography and polyacrylamide gel electrophoresis. Alpha class can be separated on DEAE into the three subclasses, termed, α₁ (rf 0.25), α₂ (rf 0.37), and α₃ (rf 0.50). Alpha₂ subclasses can be further separated on phosphocellulose at pH 6.6 into α_2a (rf 0.37) and α_2b (rf 0.30). However, α₂ contains additional subclasses, which were resolved on PC columns at pH 5.5. Beta class activity can be resolved by DEAE and PAGE into two subclasses, termed β₁ (rf 0.28) and β₂ (rf 0.49). Gamma class activity was not studied, because of its instability. The complex class of LT activity is a macromolecular aggregate greater than 200,000 daltons which appears to contain smaller MW LT class(es). This study demonstrates that materials with LT activity in supernatants from PHA activated human lymphocytes in vitro are very heterogeneous.     

The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

Comparative behaviour of L-929 fibroblastic and human endothelial cells onto crosslinked protein substrates

Studies were carried out to compare the behaviour of human umbilical vein endothelial cells (HUVEC) and L-929 fibroblastic cells towards proteins crosslinked by glutaraldehyde (GTA) or carbodiimide (CDI) proposed for coating of vascular prostheses. CDI crosslinking of bovine serum albumin used alone, or mixed with gelatin, allowed higher rates of cell growth and DNA synthesis than GTA crosslinking independent of cells. Assessment of the plating efficiency revealed a similar behaviour of both cells towards membranes and reference plastic surface in terms of percentages of bound cells. HUVEC proliferation onto CDI crosslinked gelatin and/or albumin membranes did not differ significantly whereas the growth of L-929 was enhanced onto gelatin albumin membranes in comparison with both gelatin membranes and the reference surface. The analysis of DNA synthesis corroborated the results of the growth curves and elicited a delay of the growth phases in HUVEC cultured onto CDI crosslinked membranes, unlike the L-929 fibroblast.     

The human LT system. XII. Purification and functional studies of LT and "TNF-like" LT forms from a continuous human T cell line

A clone of the continuous human T cell line HUT-102, termed YM 1.2, can spontaneously release alpha-LT in vitro. However, when stimulated with phorbol myristic acetate, these cells release other LT forms. These LT forms were purified to homogeneity by DEAE chromatography, column isoelectric focusing, and polyacrylamide gel electrophoresis. One LT form, termed LT-2, is a 79,000 m.w. component in aqueous solution and composed of 21,000 m.w. subunits. This form is immunologically related to macrophage-derived TNF and has a lytic capacity in vitro on K-562, Molt-4F, and Raji cells similar to that described for cytotoxins derived from NK effector cells, termed NK-CF. A second LT form, termed LT-3, is a single 69,000 m.w. peptide which could not be reduced into the smaller subunits. This form expresses antigens in common with both alpha-LT and TNF, because both anti-LT and anti-TNF were required to completely neutralize cell lytic activity in vitro. Functional testing revealed that the LT-3 form is lytic on all continuous cells tested in vitro, including NK-resistant target cells. The LT-3 component appears similar by immunologic, biochemical, and functional criteria to the LT form derived from primary human cytolytic T cells in vitro. At the levels tested, none of these LT-TNF forms had measurable effects on primary fibroblasts in vitro.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

Inhibition of human lymphocyte-mediated mitogen-induced cytotoxicity of murine L-929 cells by heterologous anti-human lymphotoxin antisera in vitro.

Heterologous anti-human lymphotoxin (LT) antisera have been employed to investigate the role of LT in mitogen-(Con-A, PHA) induced destruction of murine L-929 cells by human lymphocytes in vitro. These various antisera will effectively neutralize human LT molecules associated with the stable (70 to 90,000 dalton) alpha-LT class of cytotoxin (anti-alpha-LT), the more unstable (35 to 50,000 dalton) beta-LT class of cytotoxins (anti-beta-LT), and antisera which will neutralize all classes of these cytotoxins in vitro, anti-whole supernatant (anti-W.S.). These anti-LT sera will greatly inhibit lysis of L-929 cells by using mitogen-activated human effector lymphocytes in vitro. This blocking was shown to be mediated by whole serum, purified IgG, or IgG-Fab fragments, which had been extensively absorbed with bovine serum, human serum, mitogens, and normal human lymphocytes. Inhibition of lysis was not apparently due to interference with either lymphocyte-target cell contact or lymphocyte activation step(s). The blocking effects of these sera were also shown to occur during the lymphocyte-independent phase of the lytic reaction. These data support the concept that the lymphocyte deposits an LT-like effector molecule on the target-L cell surface during the lymphocyte-dependent phase, which mediates cell lysis at a later time during the lymphocyte-independent phase.     

Role of de novo protein synthesis in target cells recognized by cytotoxic T lymphocytes specific for vesicular stomatitis virus.

The requirements for viral and host protein synthesis in the generation of target antigens for cytotoxic T lymphocytes (CTL) was evaluated by using vesicular stomatitis virus (VSV) inactivated by UV irradiation (UV-VSV). EL4 target cells incubated with UV-VSV were recognized and lysed by anti-VSV CTL, indicating that de novo synthesis of viral proteins was not required for the generation of antigens recognized by antiviral CTL. Anti-VSV CTL from H-2b mice primarily recognize determinants derived from the VSV N protein bound to the class I major histocompatibility complex (MHC) antigen H-2Kb. Comparison of a cloned CTL line representing this specificity and a heterogeneous population of anti-VSV CTL showed that determinants other than that recognized by the cloned CTL were generated more efficiently from UV-VSV. By using vaccinia virus recombinants that express deletion fragments of the N protein, it was shown that these additional determinants were probably derived from VSV proteins other than the N protein. The protein synthesis inhibitor emetine was used to determine whether newly synthesized host proteins were required for antigen generation. The addition of emetine to target cells prior to or at the time of the addition of UV-VSV inhibited lysis by anti-VSV CTL. This inhibition could be due to depletion of newly synthesized MHC molecules from intracellular membranes. This hypothesis was supported by using brefeldin A to delay membrane protein transport in target cells during the time of incubation with emetine and UV-VSV, which resulted in partial reversal of the effect of emetine. These results suggest that newly synthesized class I MHC molecules are required for the generation of antigens recognized by anti-VSV CTL.     

The migration of labeled phosphatidylcholine from the nuclear-associated endoplasmic reticulum to plasma membranes in L-929 cells

Summary The nuclear-associated endoplasmic reticulum of L-929 cells was found to contain the highest amount of labeled phosphatidylcholine after a 60 min incubation with¹⁴C-choline. Radioactivity was otherwise distributed relatively evenly among other membrane-containing organelles (nuclei, mitochondria, plasma membranes and endoplasmic reticulum membranes). During a 120 min chase following removal of isotope and addition of cold choline chloride, there was a considerable reduction in labeled phosphatidylcholine in the NER and nuclei. The decrease in radioactivity in these fractions was matched by an almost identical increase in the fraction containing mitochondria and plasma membranes. Separation of mitochondria and plasma membranes by centrifugation on discontinuous gradients showed that¹⁴C-choline labeled phosphatidylcholine appeared most rapidly in the plasma membranes. The results indicate that phospholipid molecules migrate within a short period of time from their site of synthesis in the NER to plasma membranes.     

Spontaneous human lymphocyte-mediated cytotoxicity against tumor target cells. VII. The effect of immunodeficiency disease.

Spontaneous lymphocyte-mediated cytotoxicity (SLMC) and antibody-dependent cellular cytotoxicity (ADCC) was assessed in 13 patients with immunodeficiency diseases—immunodeficiency-thymoma syndrome (1), Bruton type agammaglobulinemia (3), and common variable hypogammaglobulinemia (9). SLMC and ADCC function were intact (and possibly enhanced) in the patient with immunodeficiency thymoma. Both ADCC and SLMC were detectable in the three patients with X-linked agammaglobulinemia, one of whom had lower than expected SLMC. In all of the immunodeficient patients, the relative inability of B lymphocytes to produce immunoglobulin in vivo or in vitro did not consistently affect the ability of (presumably) other lymphocytes to mediate SLMC and ADCC, although in three of the CVH patients this was lower than normal. In every case, removal of Fc receptor-bearing cells from the patients' lymphocyte preparations severely depleted SLMC (and ADCC when tested), but cytotoxicity was either unchanged or enhanced by depletion of E rosette forming T cells. The effects of Fc receptor-positive cell depletion, T-cell depletion, culture serum variation, or the addition of antibody-coated erythrocytes to the assay were similar on both SLMC and ADCC effector cells (“NK” and “K” cells), and whether patients' or normal lymphocytes were tested. The possible significance of the results with respect to surveillance against cancer is discussed.     

Release of soluble resident as well as secretory proteins from HepG2 cells by partial permeabilization of rough-endoplasmic-reticulum membranes.

Secretory proteins migrate from the rough endoplasmic reticulum (ER) to the Golgi complex at different rates. Selective retention of specific proteins to rough-ER membrane constituents could explain this phenomenon. We have permeabilized HepG2 cells with low concentrations of saponin. Release of newly synthesized proteins was studied after brief labelling in the presence of [35S]methionine. The efflux of several secretory proteins was studied at various saponin concentrations; a 2-fold higher saponin concentration was required to release transferrin compared with that required to release albumin and orosomucoid. Glucosidase II, a soluble resident protein of the ER, is released at the same saponin concentration as albumin. Saponin did not destroy the membrane skeleton structure; at the concentrations used, the integral membrane protein G of vesicular-stomatitis virus remained fully associated with the cells.     

NS35 and not vp7 is the soluble rotavirus protein which binds to target cells.

Recent studies using radiolabeled rotavirus lysates have demonstrated a 35-kilodalton viral protein that binds specifically to the surface of MA104 cells (N. Fukuhara, O. Yoshie, S. Kitakoa, and T. Konno, J. Virol. 62:2209-2218, 1988; M. Sabara, J. Gilchrist, G.R. Hudson, and L.A. Babiuk, J. Virol. 53:58-66, 1985). The binding protein was identified as vp7, an outer capsid glycoprotein and the product of rotavirus gene 9. These studies concluded that vp7 mediated viral attachment to MA104 cells and that the binding of a soluble viral protein to a cell monolayer mirrored the attachment of infectious rotavirus to permissive tissue culture cells. In the process of determining which viral protein adheres to the in vivo target cell in rotavirus infection, the mammalian enterocyte, we found that a similar 35-kilodalton rhesus rotavirus (RRV) protein bound to both MA104 cells and murine enterocytes. However, further analysis of this protein by immunoprecipitation, inhibition of glycosylation, and partial proteolysis showed that it was not the RRV gene 9 product, vp7, but the gene 8 product, NS35. Similar results were obtained by using porcine rotavirus (OSU) and bovine rotavirus (NCDV) strains. Binding studies using the in vitro-expressed products of RRV genes 8 and 9 confirmed these results. Since double-shelled virions inhibited the binding of NS35 to cells, we looked for the presence of this protein in preparations of purified virus. Examination of density gradient-purified virus preparations revealed biochemical and immunological evidence that NS35 copurifies in small amounts with double-shelled virions. Thus, these studies clearly demonstrated that when rotavirus proteins are prepared in a soluble form from infected cells, NS35, and not vp7, binds to the surfaces of MA104 cells and murine enterocytes. The observations do not confirm previous experimental results which supported the hypothesis that vp7 was the viral attachment protein. They are consistent with but do not prove the hypothesis that NS35 functions as the rotavirus attachment protein.     

The direct antiviral cytotoxicity by bovine lymphocytes is not restricted by genetic incompatibility of lymphocytes and target cells.

Experiments were designed to determine if in the ox a requirement for genetic compatibility between antiviral cytotoxic cells and target cells was needed for cytotoxicity to occur. Unrelated bovine animals were immunized with vaccinia or IBR virus (a herpesvirus), and PBL were collected at various times for measurement of their cytotoxicity against autologous or heterologous uninfected or virus-infected fibroblasts. In all instances, cytotoxicity was expressed against heterologous as well as autologous targets and in most cases there was no evidence of enhanced killing of autologous cells. The cytotoxicity was shown to be direct, presumably T cell mediated, and was not attributable to ADCC. The likelihood of the animals under investigation sharing histocompatibility antigens was considered extremely remote but was not formally excluded.     

MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes.

A CD8+ alpha beta TCR+ T cell clone (A35) was isolated from the synovial fluid of a patient with post-enteric reactive arthritis caused by Yersinia enterocolitica. This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B, or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, but did not lyse target cells preincubated with Staphylococcus epidermidis. MAb to CD2, CD3, and CD8 efficiently blocked A35, whereas the addition of mAb to HLA class II or to HLA class I did not. This clone apparently represents a novel effector mechanism against bacteria-infected or -modified cells that could be involved in the immunopathology of reactive arthritis.     

The secretory granule protein syncollin localizes to HL-60 cells and neutrophils.

The secretory granule protein syncollin was first identified in the exocrine pancreas where a population of the protein is associated with the luminal surface of the zymogen granule membrane. In this study we provide first morphological and biochemical evidence that, in addition to its pancreatic localization, syncollin is also present in neutrophilic granulocytes of rat and human origin. By immunohistological studies, syncollin was detected in neutrophilic granulocytes of the spleen. Furthermore, syncollin is expressed by the promyelocytic HL-60 cells, where it is stored in azurophilic granules and in a vesicular compartment. These findings were confirmed by fractionation experiments and immunoelectron microscopy. Treatment with a phorbol ester triggered the release of syncollin indicating that in HL-60 cells it is a secretory protein that can be mobilized upon stimulation. A putative role for syncollin in host defense is discussed.