Robert S. Yamamoto

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

Abstract 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, α 1 (rf 0.25), α 2 (rf 0.37), and α 3 (rf 0.50). Alpha 2 subclasses can be further separated on phosphocellulose at pH 6.6 into α 2a (rf 0.37) and α 2b (rf 0.30). However, α 2 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 β 1 (rf 0.28) and β 2 (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

Abstract 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.

The regulation of TNF receptor mRNA synthesis, membrane expression, and release by PMA- and LPS-stimulated human monocytic THP-1 cells in vitro

The regulation of the 55-kDa TNF receptor (TNF-R) mRNA synthesis, membrane expression, and TNF binding factor (BF) release was examined in resting and activated human monocytic THP-1 and human promyelocytic leukemia HL-60 cells in vitro. Cells were activated with phorbol myristate acetate (PMA) and bacterial lipopolysaccharide (LPS). TNF alpha cytolytic activity in the supernatant of THP-1 cells stimulated by PMA began to appear at 4 hr, reached a peak at 8 hr, and declined by 12 hr. For THP-1 cells stimulated with LPS, the peak of TNF alpha activity appeared at 4 hr and then declined. TNF alpha-binding sites on the cell membrane were down-regulated within 1 hr after PMA and LPS treatment and then reappeared 12 hr later. Fifty-five-kilodalton TNF-R mRNA expression during this time period did not correlate with the level of membrane TNF-binding site expression. Additional studies indicated the presence of a 30-kDa TNF-BF in the supernatants which appeared after 24 hr. These data suggest that activated THP-1 and HL-60 cells are capable of releasing TNF-BF into the supernatant and this material may be involved in the control of secreted TNF alpha activities.

Therapy of recurrent high grade gliomas with surgery, and autologous mitogen activated IL-2 stimulated killer (MAK) Lymphocytes: I. Enhancement of MAK lytic activity and cytokine production by PHA and clinical use of PHA

Nineteen patients with recurrent high grade gliomas were treated in a phase I/II trial with aggressive debulking of the tumor, mitogen activated IL-2 stimulated peripheral blood lymphocytes (MAK cells), and rIL-2. Phytohemagglutin (PHA) was introduced into the tumor site in 16 patients prior to implanting MAK cells and IL-2 in an attempt to trigger more effective lysis of the tumorin vivo. In vitro both TNF bioactivity and cytolytic activity of long term cultured MAK (LMAK) cells were dramatically enhanced by adding PHA to the cultures of these activated PBL. Three of eleven patients (27%) had a decrease in size of the enhancing lesion on CT and/or MRI. Seven (37%) patients clinically improved. Median survival after therapy was 30 weeks. PHA was shown to be safein vivo and more effective than IL-2 triggering enhanced effector functionin vitro.

Variants of the receptor/channel clustering molecule gephyrin in brain: distinct distribution patterns, developmental profiles, and proteolytic cleavage by calpain.

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino‐terminal domain of gephyrin (GNN) and 2) its invariant carboxy‐terminus (GNC). Both antibodies recognized an antigen with the expected molecular weight of 93–95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti‐GNN was markedly distinct from that labeled by anti‐GNC, a difference consistent with carboxy‐terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy‐terminal gephyrin fragments of 48–50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis. J. Neurosci. Res. 49:381–388, 1997.

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)☆

Abstract 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 α 2 -LT subclass (anti- α 2 ) and partially neutralized with anti-human Fab 2 ′ 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).

The human LT system: III. Characterization of a high molecular weight LT class (complex) composed of the various smaller MW LT classes and subclasses in association with Ig-like molecules

Abstract Cytotoxic activity (lymphotoxin (LT)) present in supernatants from lectin stimulated human lymphocytes in vitro is composed of a heterogeneous system of biological macromolecules which can be separated into multiple classes and subclasses on the basis of their molecular weight and charge. These studies further characterize a large molecular weight human LT class, termed complex (MW >200,000 d), which elutes in the void volume off Sephadex G-150 or Ultrogel AcA 44. Immunological studies on the complex, employing various rabbit anti-LT class and subclass antisera, revealed this material is a macromolecular assemblage of the smaller MW α, β, γ LT classes and subclasses. Furthermore, the reactivity of this material with anti-human Fab′ 2 (IgG) indicates these smaller molecular weight LT components can associate with immunoglobulin or Ig-like molecules. The materials present in the LT complex class appear to be noncovalently associated, since conditions of high ionic strength dissociate certain small MW LT components, while low ionic strength buffers may cause these components to reaggregate with the complex. When subjected to velocity sedimentation on sucrose gradients or gel filtration on Ultrogel AcA 22, LT complex activity elutes as several discrete peaks of activity in the 200,000 to 1,000,000 MW range. These findings suggest the concept that LT molecules can form discrete and specific macromolecular structures which contain the smaller MW LT classes. Moreover, these structures can also associate with immunoglobulin-like molecules to form secondary LT-Ig complexes. This may have important biological significance in explaining how nonspecific cell toxins could play a role in specific or nonspecific cell lytic reactions in vitro .

Antibodies against human lymphokines: I. Methods for induction of antibodies capable of neutralizing stable (α) and unstable (β) lymphotoxins released in vitro by activated human lymphocytes

Various methods were employed to induce antibodies in rabbits that were capable of neutralizing different families of lymphotoxins (LT). Both stable (alpha-LT) and unstable (beta-LT) molecules, released by activated human lymphocytes in vitro, were neutralized. The different LT families were first separated into their respective groups by physical-chemical methods. Immunization with small quantities of antigen yielded a high percentage of responder animals. Techniques were developed for eliciting alpha-LT antibodies using as little as 2--3 ml of a cell-free supernatant. The situation was more difficult, however, when the unstable beta-LT molecules were employed as antigens. We found that because of the low concentration and lability of beta-LT in supernatants, the immunizing dose had to be: a) handled rapidly, b) larger than that used with the alpha-LT, and c) injected at closer intervals and over a longer immunization protocol. Physical-chemical studies supperted the concept that the LT-neutralizing activity in the immune serum was immunoglobulin.

The human LT system: IV. Studies on the large MW LT complex class: Association of these molecules with specific antigen binding receptor(s) in vitro

Abstract The present studies demonstrate that a portion of lymphotoxin (LT) cell-lytic activity present in supernatants from: 1) lectin (Con A, PHA) stimulated nonimmune; or 2) antigen (soluble or cellular) stimulated immune human lymphocytes in vitro, is associated with immunoglobulin (Ig) or “Ig-like” receptor molecule(s). This concept was supported by three findings: 1) LT activity in these supernatants was partially inhibited by heterologous anti-human (IgG) Fab′2 antisera; 2) LT activity present in soluble antigen stimulated immune human lymphocyte supernatants could specifically bind to and be eluted from Sepharose 4B columns to which the specific stimulating antigen was covalently attached; and 3) LT activity present in primary one-way mixed lymphocyte culture (MLC) supernatants could be removed by absorption on the specific stimulator cells. The amount of total LT activity found to be associated with “Ig” in these supernatants was variable, but ranged from 5 to 20% in lectin stimulated cell supernatants to 20 to 50% in antigen or MLC stimulated supernatants. Physical-chemical studies on the molecular weight class of LT molecules having reactivity with anti-Fab′2 sera, as well as antigen binding capacity, revealed these properties reside in the large (>200,000) MW LT class, termed complex. The nature and biological significance of these “antigen specific” LT complexes, as they relate to mechanisms of cytotoxicity in vitro, will be discussed.

Lymphocyte effector molecules: An in vitro production method for obtaining liter volumes of supernatants from mitogen-stimulated human lymphocytes

An in vitro method has been developed utilizing phytohemagglutinin (PHA) activated lymphocytes obtained from human tonsils and adenoids which permit the accumulation of multi-liter quantities of cell-free supernatants containing lymphotoxin and other lymphocyte effector molecules (LEM). An enriched media is employed which contains a large molecular weight, heat stable bovine serum fraction which supports lymphoid cell activation and levels of LEM secretion equal to that of cultures maintained in medium supplemented with whole serum. Elimination of whole serum from the media greatly reduces overall protein concentrations and facilitates concentration and purification studies. Various technical aspects of these cultures have been examined, i.e.: 1) cell concentration, 2) kinetics of LT production over a ten-day period, 3) mitogen dosage, and 4) types of media. Supernatants can be harvested repeatedly from a single culture over the ten day period, thus doubling the yield of LEM collected from a single culture.