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 (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). 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-GN(N) was markedly distinct from that labeled by anti-GN(C), 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.

Distinct distributions of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits and a related 53,000 M(R) antigen (GR53) in brain tissue.

Polyclonal antibodies against specific carboxy-terminal sequences of known alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunits (GluR-4) were used to screen regional homogenates and subcellular fractions from rat brain. Affinity purified anti-GluR1 (against amino acids 877-899), anti-GluR2/3 (850-862), and anti-GluR4a and anti-GluR4b (868-881) labeled distinct subunits with the expected molecular weight of approximately 105,000. These antigens were shown to have distinct distributions in the brain. While GluR2/3 epitopes had a distribution profile similar to that of the presynaptic marker synaptophysin, GluR1 was notable for its abundance in the hippocampus and its relatively low density in neocortical areas, and GluR4 was highly enriched in cerebellar tissue. An additional antigen (glutamate receptor-related, GR53) of lower molecular weight (50,000-59,000) was recognized in rat, human, frog, chick and goldfish brain samples by anti-GluR4a as well as by anti-GluR1 at, an antibody that specifically recognizes the extracellular aminoterminal domain of GluR1 (amino acids 163-188). Both antibodies also labeled antigens of approximately 105,000 mol. wt in brain tissue from all species tested. The approximately 53,000 mol. wt antigen was concentrated 10-20-fold in synaptic membranes vs homogenates across rat brain regions. Both the 105,000 and the 53,000 mol. wt proteins were also concentrated in postsynaptic densities, and neither of the two antigens were evident in seven non-brain tissue samples. These data indicate that AMPA receptors have regionally different subunit combinations and that some AMPA receptor composites include proteins other than the conventional 105,000 mol. wt GluR subunits.

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 tumor in 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 safe in vivo and more effective than IL-2 triggering enhanced effector function in vitro.

Blocking factors (soluble membrane receptors) for tumor necrosis factor and lymphotoxin detected in ascites and released in short-term cultures obtained from ascites and solid tumors in women with gynecologic malignancy.

Studies were conducted to identify and establish the cell and tissue source of blocking factors (BF), materials that inhibit the bioactivity of human TNF and LT in vitro. Ascites and samples of solid tumors were collected from women with various gynecologic malignancies. Supernatants were collected from cultures of tumor and ascites cells after 24, 48, and 72 h. Cell-free ascites (CFA) and culture supernatants were tested for their ability to block human recombinant TNF and LT-induced lysis of L929 cells in vitro. Levels of soluble forms of the 55- and 75-kDa TNF/LT receptors were measured by ELISA assay in the same samples. CFA and culture supernatants contained TNF/LT blocking factors and high levels of one or both soluble 55- and 75-kDa TNF/LT membrane receptors. Levels of BF bioactivity and receptors appeared rapidly, peaked at 24 h, and declined thereafter. Soluble TNF/LT receptors may be the active BF in these samples, and tumor tissues and ascitic cells may be a source of these receptors in the ascites fluid of these patients.

Trafficking of syngeneic murine lymphokine activated killer T cells following intraperitoneal administration in normal and tumor bearing mice.

Nongenetically restricted T cells may be important host effector cells in women with ovarian cancer receiving intraperitoneal (ip) IL-2 therapy. We developed an in vitro technique to produce murine lymphokine-activated killer T cells. Murine splenocytes were cultured in the presence of 1000 U/ml IL-2 for 10 to 15 days. Phenotypical analysis showed 95% of total cells to express the pan T phenotype Thy 1.2 and no NK cell phenotypes by Day 7 in culture. These cells were labeled with 51Cr and their trafficking pattern after ip administration into normal and M5067 tumor bearing mice was examined. Various organs and tissues were collected at different timepoints and monitored for radioactivity. Within 4 hr., about 60% of the counts were associated with the bowel, peritoneum, and omentum of both normal and tumor bearing mice. About 15% of counts were associated with the blood, lung, kidney, spleen, and liver of both normal and tumor bearing mice.

Immunotherapy of ovarian cancer. II. In vitro generation and characterization of lymphokine-activated killer T cells from the peripheral blood of recurrent ovarian cancer patients.

We examined the in vitro sensitivity of continuous ovarian cancer cells to lymphokine-activated killer T cells (T-LAK) alone or in combination with cytokines. Lymphocyte viability in T-LAK cultures generated from normal donors and ovarian cancer patients declined in the first 2 to 4 days; however, the remaining cells in these cultures maintained a constant rate of proliferation for long periods in vitro. These cells became 90-95% CD3+ TCR+ -alpha/beta T-cells after 7-10 days in culture. The T-LAK cells from normal donors and cancer patients expressed an equal ability to induce lysis of a panel of human target cells (NK-sensitive K562, NK-insensitive RAJI, and two human ovarian tumor lines, SKOV-3 and OVCAR-3), demonstrating that they are nongenetically restricted killers. Preincubation of either the effector or target cells with tumor necrosis factor or interferon-gamma or addition of these cytokines directly to cytolytic assays did not alter the degree of cell lysis in vitro. This is a method for generating large numbers of autologous, cytolytically active T-LAK cells from the blood of ovarian cancer patients that could be employed in adoptive intraperitoneal immunotherapy.

Growth of the endometrial adenocarcinoma cell line AN3 CA is modulated by tumor necrosis factor and its receptor is up-regulated by estrogen in vitro.

We demonstrate that tumor necrosis factor (TNF) has a biphasic effect on the growth of the human endometrial adenocarcinoma cell line AN3 CA in vitro. Low levels (0.2-5 pg/ml) of TNF were moderately growth stimulatory (up to 20% enhancement), while levels over 100 pg were growth inhibitory (up to 45% inhibition). Northern blot analysis showed expression of the 75-kilodalton (kDa) TNF receptor mRNA, but no expression of the 55-kDa TNF receptor mRNA or TNF mRNA. The growth of these cells was not directly affected by physiological concentrations (10(-7)-10(-9) M) of 17 beta-estradiol (E2). However, [125I]TNF binding studies and Scatchard analysis showed that 18-h coculture with 10(-8) M E2 increased the number of TNF receptors expressed on these cells 3-fold. Quantitative mRNA analysis confirmed that 75-kDa TNF receptor mRNA expression increased within 4 h of incubation with E2. These observations suggest an interaction between the endocrine and the immune systems, with an important implication for the homeostasis of endometrial tissues.

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.

A 20 amino acid synthetic peptide of a region from the 55 kDa human TNF receptor inhibits cytolytic and binding activities of recombinant human tumour necrosis factor in vitro.

Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) can exert a wide range of effects on cells and tissues and they are important effector molecules in cell mediated immunity. All these effects are induced subsequent to the binding of these cytokines to specific membrane receptors. Recently, two of these membrane receptors of 55 and 75 kDa, have been identified which share some amino acid (AA) homology in their N-terminal extracellular domains but differ in their intracellular domains. We synthesized two synthetic 20 AA peptides from hydrophilic regions of the N-terminal extracellular domains of the 55 kDa receptor; peptide A shares homology with both 55 and 75 kDa receptors, peptide B is unique. We found peptide B inhibits both the binding and cytolytic activity of recombinant human TNF when tested on murine L929 cells in vitro. Polyclonal antiserum generated against peptide B will block binding of 125I-labelled TNF to these cells in vitro. However, peptide A and antiserum prepared against peptide A are without effect in these same assay systems. These data suggest that the 20 AA sequences from AA 175 to 194 in the N-terminal extracellular domain of the 55 kDa TNF receptor are expressed on the cell surface and are involved in the binding of TNF.

Identification of tumor necrosis factor and lymphotoxin blocking factor(s) in the ascites of patients with advanced and recurrent ovarian cancer.

Ascites obtained from human ovarian cancer patients contains material(s) that inhibit the cytolytic activity of tumor necrosis factor (TNF) and lymphotoxin (LT) in vitro. These inhibitor(s) are found in ascites from ovarian cancer patients and are detected in very low amounts in the ascites from patients with nonmalignant hepatic disease. These ascites TNF/LT blocking factors are heat sensitive and heterogeneous with respect to molecular weight. Kinetic studies indicate these factors inhibited cytolysis at the stage of TNF/LT interaction with membrane receptors on L929 cells. Because TNF and LT are key cytokines in host cell-mediated antitumor mechanisms, factor(s) that inhibit these cytokines could have a profound effect on the tumor host interaction and their presence in the ascitic fluid, should be considered before designing clinical trials that employ intraperitoneal administration of TNF or LT for immunotherapy of ovarian cancer.

Therapy of recurrent high-grade gliomas with surgery, autologous mitogen-activated IL-2-stimulated (MAK) killer lymphocytes, and rIL-2: II. Correlation of survival with MAK cell tumor necrosis factor production in vitro.

Seven patients with recurrent high-grade glioma were treated in a Phase I/II trial with surgical debulking, after which mitogen-activated IL-2-stimulated killer (MAK) lymphocytes and 10(5) units rIL-2 were implanted in the surgical defect. The therapy was well tolerated, and the mean survival of this group of patients was 29 weeks. Tumor necrosis factor (TNF) production by MAK lymphocytes stimulated with IL-2 in vitro was measured. A significant (r = .78, p = .04) correlation between survival of patients after therapy and the ability of the MAK lymphocytes to produce TNF in vitro was noted. A significant negative correlation (r = -.82, p = .02) was found when comparing TNF production and increasing tumor size measured on MRI. No correlation was found between TNF production in vitro and MAK lymphocytes lytic activity on K562 and U373 target cells. No correlation was found between survival and MAK cell lytic activity measured on K562 and U373 target cells. We conclude that TNF production in vitro and cytotoxic activity measured in vitro are measures of different antitumor activity in vivo and in vitro. TNF production during IL-2-stimulated proliferation may be an important in vitro assay in terms of predicting length of survival of recurrent high-grade gliomas.

Generation of stimulated, lymphokine activated T killer (T-LAK) cells from the peripheral blood of normal donors and adult patients with recurrent glioblastoma.

Peripheral blood mononuclear cells (PBM) from normal donors and patients with recurrent glioma were activated initially for 48-72 h with phytohemagglutinin-P (PHA) and recombinant human interleukin-2 (IL-2), and then proliferated in vitro for up to 5 months with IL-2. These cells are termed mitogen-stimulated lymphokine-activated T killer (T-LAK) cells. We measured patterns of T-LAK cell growth, in vitro cytolytic activity on a panel of continuous and primary tumor cells, and the phenotypes of the cells in these cultures. Lymphocyte viability declined dramatically over the first 3-5 days; and then the remaining cells in these cultures began to divide and maintained a constant 30-36 h doubling time for long periods in vitro. Phenotyping revealed that cells in the initial few days of culture were heterogeneous, but became almost totally CD3 T cells after 7-10 days in culture. The T-LAK cells from individual normal donors and cancer patients demonstrated a non-genetically restricted cytolytic ability against a panel of both continuous cell lines and primary autologous and allogeneic glioblastoma cells in vitro. This technique provides a method of generating large numbers of autologous cytolytic T cells with non-restricted anti-tumor activity that can be derived from peripheral blood mononuclear cells.

Enhancement of lymphokine-activated T killer cell tumor necrosis factor receptor mRNA transcription, tumor necrosis factor receptor membrane expression, and tumor necrosis factor/lymphotoxin release by IL-1 beta, IL-4, and IL-6 in vitro.

Co-culture with IL-2 can induce human T lymphocytes to proliferate and become nongenetically restricted, lymphokine-activated killer (LAK) cells in vitro. Our studies were conducted with long term cultured, human T-LAK cells from peripheral blood, which are 95 to 99% CD3+. We found that proliferating 7 to 10-day human T-LAK cells express TNFR, by using a 125I-TNF binding assay. Additional treatment of these cells with the cytokines IL-1 beta, IL-4, or IL-6 rapidly up-regulated 55-kDa TNFR mRNA transcription and doubled TNFR membrane expression. Further studies revealed that these cytokines also increased the release of TNF and lymphotoxin (LT). Antibody neutralization studies indicated that IL-1 induces release of both TNF and LT; however, IL-4 and IL-6 induce primarily LT release. These results further support the concept that these cytokines are involved in the regulation of TNF/LT release, TNFR synthesis, and TNFR membrane expression. It is apparent that cytokines and their membrane receptors are involved in the autocrine/paracrine control of T cell proliferation, differentiation, and expression of functional activity after IL-2 stimulation in vitro.

Purification and characterization of an inhibitor (soluble tumor necrosis factor receptor) for tumor necrosis factor and lymphotoxin obtained from the serum ultrafiltrates of human cancer patients.

Serum ultrafiltrates (SUF) from human patients with different types of cancer contain a blocking factor (BF) that inhibits the cytolytic activity of human tumor necrosis factor alpha (TNF-alpha) in vitro. BF is a protein with a molecular mass of 28 kDa on reducing sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE). The active material was purified to homogeneity by a combination of affinity chromatography, PAGE, and high-pressure liquid chromatography. Amino acid sequence analysis revealed that BF is derived from the membrane TNF receptor. Purified BF blocks the lytic activity of recombinant human and mouse TNF-alpha and recombinant human lymphotoxin on murine L929 cells in vitro. However, BF inhibits the lytic activity of TNF-alpha more effectively than it does that of lymphotoxin. The BF also inhibits the necrotizing activity of recombinant human TNF-alpha when coinjected into established cutaneous Meth A tumors in BALB/c mice. The BF may have an important role in (i) the regulation and control of TNF-alpha and lymphotoxin activity in cancer patients, (ii) interaction between the tumor and the host antitumor mechanisms, and (iii) use of systemically administered TNF-alpha in clinical trials with human cancer patients.

Mechanism of human lymphotoxin and tumor necrosis factor induced destruction of cells in vitro: phospholipase activation and deacylation of specific-membrane phospholipids.

The role of phospholipase (PLase) activation and lipid metabolism in lymphotoxin (LT)- and tumor necrosis factor (TNF)-mediated destruction of murine L929 cells was examined. At the levels of LT and TNF employed, cell destruction began at 4-6 h and was 99% complete by 30 h. Cell membrane phospholipids (PL), labelled in situ at the C2 position with 14C arachidonic acid, were analyzed by two-dimensional thin-layer chromatography and quantitated over a 30 h time course after LT or TNF treatment. The ratio of radiolabel incorporation relative to the actual amount of each PL present was determined by inorganic phosphate analysis. Radiolabelled arachidonic acid, eicosanoids, and neutral lipids were released into the medium prior to the onset of cell death (4-6 h) and continued to accumulate linearly throughout the destructive reaction. There was a quantitative relationship between the appearance of radiolabelled metabolites in the media and the loss of radiolabelled cellular PL. Cellular phosphatidylethanolamine was the primary PL deacylated by PLase action, showing a 75% reduction in radiolabel. The PLase inhibitors--quinacrine, hydrocortisone, dexamethasone, and indomethacin--were potent inhibitors of LT- and TNF-mediated cell destruction, suggesting that selective deacylation of specific membrane PL by PLase activation is an important step in the events that lead to LT- and TNF-mediated cellular destruction in vitro.

The presence of antibodies to lymphotoxin and tumor necrosis factor in normal serum.

Lymphotoxin (LT) and tumor necrosis factor (TNF) are cytokines that probably play a role in several autoimmune diseases. In this report, we describe the detection, in normal nonimmune serum, of naturally occurring antibodies that bind to recombinant human LT and TNF. The naturally occurring antibodies that bind to LT and TNF could explain why normal immunoglobulin is effective therapy in idiopathic thrombocytopenic purpura and Kawasaki syndrome.

Pharmacokinetics and tissue distribution of parenterally administered human alpha-lymphotoxin in normal and meth-A tumor-bearing BALB/c mice.

These in vivo studies examine the pharmacokinetics of parenterally administered purified, native human alpha-lymphotoxin (LT) in normal and Meth-A bearing BALB/c mice. We found that the lytic activity of alpha-LT was inactivated within 5 h in the blood of both normal and tumor-bearing mice in vivo. However, LT bioactivity in vitro was not affected by incubation with fresh serum. Radioiodinated LT was rapidly sequestered in the kidneys of both normal and tumor-bearing animals. Systemically administered, radioiodinated LT did not selectively localize in tumor tissues.

Purified native and recombinant human alpha lymphotoxin [tumor necrosis factor (TNF)-beta] induces inflammatory reactions in normal skin.

These studies report findings that demonstrate that human alpha lymphotoxin (LT) induces local, visible, and microscopic inflammatory reactions in normal skin. Skin sites in rabbits, when inoculated with a single injection of native or recombinant human alpha lymphotoxin, demonstrated erythema, swelling, and warmth within 5 hr. Erythema peaked between 24 and 48 hr had resolved by 72 hr. Histologic studies of skin sites injected with native LT revealed polymorphonuclear neutrophil (PMN) infiltration and edema beginning as early as 3 hr posttreatment. Individual skin sites that received three daily injections of native LT exhibited persistent erythema and swelling. Palpable induration was evident 24 hr after the second injection in the series. Histologic examination revealed the presence of many PMNs with associated focal dermal destruction, in the form of microabscesses, and scattered mononuclear cells. In contrast, control materials and recombinant human tumor necrosis factor (TNF-alpha) did not induce visible skin reactions in the rabbit. Several additional controls excluded endotoxin as being the agent responsible for the inflammatory skin reactions observed. The ability of LT to induce inflammation may have a role in its antitumor activity and it may be an important endogenous mediator in other immunologic reactions.

The human LT system. XI. Identification of LT and "TNF-like" forms from stimulated natural killers, specific and nonspecific cytotoxic human T cells in vitro.

These studies demonstrate that specific and nonspecific cytolytic human T cells can release LT forms in vitro. Nonspecific cytolytic T cells were derived from IL 2-dependent cultures initiated by allogeneic mixed lymphocyte reaction (AMLR). Specific cytolytic T cells (CTL) were derived from IL 2-dependent T cell clones, initiated by mixed lymphocyte reaction and specific for class II antigens. alpha-LT was the major lytic component released by these cells in IL 2-dependent cultures. However, on Con A stimulation or contact with target cells, both AMLR and CTL effectors release a new LT form. The new LT form released by AMLR and CTL effectors appear similar, for they both elute from gel filtration at 60,000 to 70,000 m.w. and migrate as a single peak with an Rf of 0.4 on 7% native PAGE tube gels. Moreover, testing these materials on a panel of target cells in vitro indicates that they are both nonspecific, and lyses NK-resistant target cells in vitro. Additional studies revealed that in vitro lytic activity of this form(s) is not affected by either anti-alpha-LT serum or a monoclonal reagent which inactivates macrophage cell toxins (MCT) and tumor necrosis factor (TNF). However, when these two immunologic reagents are tested together, activity is totally neutralized. Thus, this LT form expresses antigens in common with alpha-LT, MCT, and TNF. Finally, studies with NK-CF and NK-LT forms revealed that they were also completely neutralized with a mixture of anti-LT and monoclonal anti-TNF antibody. These data suggest that certain macrophage- and lymphocyte-derived cell toxins are interrelated.

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.