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Biomedical subjects

G A Granger

Publications and source records attributed to G A Granger.

At least 73 records · Page 4Linked to original sources
PubMed1984

Lymphotoxins - a multicomponent system of growth inhibitory and cell-lytic glycoproteins.

Activated lymphocytes from experimental animals and man can release materials, termed lymphotoxins , which cause growth inhibition and cell lysis in vitro. These molecules, from human lymphocytes, are glycoproteins which can be divided into five molecular weight classes. These forms are heterogeneous, for each MW class can be further subdivided into multiple charge subclasses. It is now clear certain MW classes are interrelated and form a system of cell toxins. The larger classes (greater than 140,000 d) are associated with nonclassical antigen-binding receptors (R), which can be of T cell origin. The smaller forms (less than 90,000 d) do not express R function and are derived from the larger forms, possibly by enzymatic action. Two MW classes, one receptor-associated and one non-receptor associated, have been purified to homogeneity and their peptide composition is being studied. Functional studies reveal the larger MW forms derived from alloimmune cell populations; can induce selective and nonselective destruction of cells in vitro. Antibodies which inhibit the in vitro cell lytic ability of various human LT forms can block different classes of human lymphocyte cell killing reactions in vitro.

Animals↗
PubMed1984

Lymphotoxin detected in the blister fluid of bullous pemphigoid patients.

The role of lymphocytes in the pathogenesis of bullous pemphigoid was examined by assaying the blister fluid obtained from bullous pemphigoid patients for the presence of the lymphokine, lymphotoxin. Blister fluids from six bullous pemphigoid were assayed on L-929 target cells for the presence of cytolytic molecules in the standard lymphotoxin assay. Three of six blister fluids obtained from bullous pemphigoid patients and one linear IgA bullous dermatosis patient contained significant levels of cytolytic activity. Control blister fluids from suction blisters, herpes, pemphigus, and toxic epidermal necrolysis patients did not contain cytolytic activity. Serum from five bullous pemphigoid patients also had no cytolytic activity. Neutralization studies using rabbit anti-alpha-lymphotoxin demonstrated that 54 to 88% of the cytolytic activity found in bullous pemphigoid blister fluid was due to alpha-lymphotoxin. These results indicate that lymphotoxin is locally released in the skin of bullous pemphigoid lesions and is detectable in blister fluids.

Antibodies↗
PubMed1984

Soluble suppressor factors elaborated in experimental malignant ascites.

Soluble suppressor factors in the sera of cancer patients inhibit lectin-stimulated lymphocyte proliferation. These factors, derived from human material, preclude easy corroboration by other investigators. To gain a general understanding of soluble suppressor factors and to avoid the necessary restrictions of human experimentation, an animal model was devised. Sprague-Dawley rats were injected ip with the Walker 256 carcinoma. The resultant ascites proved to be a stable, reproducible source of soluble suppressor factors. Ascites inhibited phytohemagglutinin (PHA)-induced blastogenesis of normal splenocytes by 98%. The possibility of a toxic effect was eliminated by vital staining of splenocytes and by examination in a specific lymphotoxin assay. Suppressor activity persisted after heating at 100 degrees C for 40 min. Extraction by lipid solvents revealed that the bulk of suppressor activity resides in the lipid phase. The active fraction of heat-treated ascites passed through an Amicon PM-10 filter. Thin-layer chromatography revealed the presence of prostaglandins E2 and F2 alpha. Tissue culture supernatants from short-term cultures derived from tumor-bearing animals revealed suppressor activity from thymus, spleen, and liver cultures (97, 91, and 71%, respectively). No suppressor activity was detected in cultures of cancer cells. This study has demonstrated in this animal model that prostaglandins play a major role in suppression of lectin-induced blastogenesis. All suppressor factors appear to be host derived. An understanding of the mechanism of release of these suppressor substances may open new avenues in the immunotherapy of cancer.

Animals↗
PubMed1984

Phorbol myristate acetate induction of lymphotoxins from continuous human B lymphoid cell lines in vitro.

The phorbol ester 4 beta-phorbol 12-myristate 13-acetate (PMA) was studied for its ability to induce increased lymphotoxin (LT) production from a number of continuous human B lymphoblastoid cell lines that spontaneously release low levels of LT in vitro. A 5-20-fold increase in LT production was seen when all seven of these cell lines were cocultured with 20 ng/ml PMA for 72 h under serum-free conditions in 0.1% lactalbumin hydrolysate. One cell line, GM3104A, was cloned and repeatedly selected to obtain a high-level LT producer in the presence of PMA. This subline, termed IR 3.4, spontaneously released the alpha-LT molecular weight (MW) class; however, upon PMA stimulation it released high levels of alpha and an unrelated beta-LT MW class form. The increased amounts of LT released in the presence of PMA by IR 3.4 lymphoid cells provides a new method for the production of large amounts of alpha-LT for purification from a single cell source for biochemical and functional studies, and indicates that the type of induction signal may influence the type of LT released.

Antibodies↗
PubMed1983

Identification of human lymphocyte-derived lymphotoxins with binding and cell-lytic activity on NK-sensitive cell lines in vitro.

Supernatants obtained from lectin-restimulated, preactivated, human peripheral blood lymphocytes rapidly released (5-24 hr) high levels of lymphotoxin (LT) activity in vitro. Peripheral blood lymphocytes were preactivated by coculturing with either fetal calf serum or with allogeneic continuous B-cell lines (LCCL) which were treated with mitomycin C. These supernatants contained a population of L-929 cell-lytic LT forms which also selectively bind to the NK-sensitive K-562 cell. However, lytic LT forms for L-929 cells from cPBL and LCCL cultures did not bind to the NK-sensitive MOLT-4 or NK-resistant Raji cells. Additional studies reveal these supernatants contain a second set of LT forms which have cell-binding and cell-lytic activity detectable on MOLT-4 and K-562 cells in a 12 to 18 hr 51Cr-release assay. Cell-lytic form(s) for the MOLT-4 and K-562 cells were not stable for more than a week at -20 degrees C. These findings indicate that materials with LT activity are heterogeneous with respect to their capacity to recognize common and discrete cell-surface components on different types of target cells in vitro.

Animals↗
PubMed1983

Inhibition of human NK-induced cell lysis and soluble cell-lytic molecules with anti-human LT antisera and various saccharides.

The present study examines and compares the cytolysis of K-562 and MOLT-4 cells mediated by human natural killer (NK) cells from fresh peripheral blood and lymphotoxins (LT) derived from human lymphoid cell populations after lectin stimulation in vitro. Lymphotoxins were obtained from 5-hr concanavalin A (Con A)-restimulated human peripheral blood lymphocytes (PBL) which were precultured for 5 days in medium and fetal calf serum or with allogeneic human B-lymphoid cell lines. Two classes of probes were employed in both direct (cell) and indirect (supernatant) induced target-cell lysis: (a) various saccharides and (b) antibodies reactive with human LT forms. Two sugars, N-acetylglucosamine and alpha-methylmannoside, were able to inhibit direct cell lysis of both MOLT-4 and K-562 target cells. However, saccharide inhibition was distinct for each type of target even when effector cells were obtained from the same donor. These same saccharides were also able to inhibit 20-30% of the total LT activity in a supernatant for L-929 cells and 50-90% of the lytic activity on MOLT-4 cells. Anti-human F(ab')2 (IgG) and rabbit anti-alpha 2 LT sera blocked direct cell lysis of MOLT-4 and K-562 targets in 50% of the experiments. The anti-alpha 2 LT serum only recognizes a portion of the LT forms in these supernatants. These results reveal that, while both direct and indirect cell lysis are complex phenomena, they may both occur in some cases by a common mechanism(s).

Animals↗
PubMed1983

Purification to electrophoretic homogeneity of human alpha lymphotoxin from a cloned continuous lymphoblastoid cell line IR 3.4.

The 70-90,000 molecular weight (MW) alpha (alpha) component of the human lymphotoxin (LT) system has been purified to electrophoretic homogeneity. The alpha LT containing supernatants were obtained from a phorbol myristate (PMA) stimulated cloned continuous human B lymphoblastoid cell line IR 3.4. Supernatants were subjected to a biochemical separation scheme that consisted of chromatography on control pore glass beads, DEAE ion-exchange chromatography, lentil-lectin affinity chromatography, and electrophoresis on 7% native preparative polyacrylamide gels. The specific activity of the alpha LT in the final fractions was from 10(7) to 5 X 10(7) units of LT activity/mg protein. Approximately 3 to 5% of the initial alpha LT was recovered in the final fractions and a purification factor of 25,000 to 30,000 fold was required to achieve homogeneity. The alpha LT preparation from preparative PAGE exhibited concident migration of bioactivity and radioactivity on 5 and 7% native PAGE tube gels. Only a single protein peak was observed when the radiolabeled alpha LT was subjected to a two-dimensional SDS-reducing slab gel.

B-Lymphocytes↗
PubMed1983

Differences in cytotoxic effects of activated murine peritoneal macrophages and J774 monocytic cells on metastatic variants of B16 melanoma.

The cytotoxic effects of activated peritoneal macrophages and the J774 reticulum cell sarcoma cell line on B16 melanoma cells of differing metastatic potential were investigated in vitro. The melanoma target cells were sublines of low (B16-F1) or high (B16-F10) lung colonization potential as well as a subline of high (B16-B14b) brain colonization ability. Thioglycolate-elicited peritoneal macrophages from syngeneic C57BL/6 mice and J774 cells were activated in vitro with polyinosinic-polycytidylic acid (poly I:C) and used as effector cells. Macrophage-mediated cytolysis was determined by means of 24- to 72-hour radioactivity release assays with [125I]5-iodo-2'-deoxyuridine-labeled melanoma cells; the results indicated that the more metastatic sublines B16-F10 and B16-B14b were less susceptible to cytolysis by activated macrophages and J774 cells than was the poorly metastatic B16-F1 subline. The poly I:C-activated effector cells also released soluble cytotoxin(s), which resulted in melanoma cell lysis and growth inhibition. Cytotoxin-mediated melanoma cell cytolysis was determined by counting the number of viable mitomycin-treated target cells remaining after a 40-hour incubation period in the absence or presence of various concentrations of cell-released factors, and cytotoxin-mediated cytostasis was performed with the use of the same procedures without mitomycin pretreatment of targets. The factors released from both activated macrophages and J774 cells were more effective against the poorly metastatic B16-F1 cells than against the highly metastatic B16-F10 or B16-F14b cells. In addition, the activity of the factors from both activated effector cells was inhibited by fetal bovine serum. The J774 cells and the activated peritoneal macrophages demonstrated similar activities against B16 melanoma variants, indicating that the J774 cell line may be suitable as a model for the study of macrophage cytotoxicity. The results suggested that the potential of the highly metastatic melanoma cells to implant, survive, and grow at secondary sites may be due, in part, to their ability to circumvent host antitumor mechanisms.

Animals↗
PubMed1983

Studies on the mechanism of natural killer cytotoxicity. II. coculture of human PBL with NK-sensitive or resistant cell lines stimulates release of natural killer cytotoxic factors (NKCF) selectively cytotoxic to NK-sensitive target cells.

This investigation has employed the "innocent bystander" type of experimental design to determine whether soluble cytotoxic factor(s) are released during interactions between human peripheral blood lymphocytes (PBL) and NK-sensitive target cells. PBL cocultured with NK-sensitive Molt-4 or K562 target cells in the lower well of a miniaturized Marbrook culture released natural killer cytotoxic factors (NKCF), which diffused across a 0.2-mu Nucleopore membrane and lysed Molt-4 or K562 target cells cultured in the upper chamber. Coculture of PBL with the NK-resistant Raji or WI-L2 cell lines also induced release of NKCF. These factors were selectively cytotoxic to NK-sensitive targets and lysed Molt-4 and, to a lesser extent, K562 cells. However, Raji, WI-L2, and RPMI 1788 cells were all resistant to lysis. In addition, low density fractions from Percoll density gradients that were enriched for NK effector cells also released increased levels of NKCF during coculture with Molt-4 cells. Lysis of Molt-4 and K562 targets was observed after exposure to NKCF for 48 hr and 60 to 70 hr, respectively. Cellfree supernatants containing NKCF were obtained after a short time of incubation (i.e., within 5 hr of coculture of PBL with NK target cells). The factors were nondialyzable, stable at 56 degrees C for 3 hr, and showed partial loss of activity on storage at 4 degrees C or -20 degrees C for 7 days. These data suggest that NKCF may be involved in the lytic mechanism of human NK cell-mediated cytotoxicity.

Cell Line↗
PubMed1982

Regulation of growth of human diploid fibroblasts by factors elaborated by activated lymphoid cells.

Fibroblasts growth synthesis activities appear to be under exquisite control. This control is mediated in part by substances present in blood plasma or released by other cells. We have studied the role of peripheral blood mononuclear cells (PBM) activated with phytohemagglutinin-P (PHA) on DNA synthesis, proliferation, and the cell cycle of human diploid fibroblasts. Culture medium from activated but not from unactivated PBM cultures inhibited fibroblast DNA synthesis and growth in a dose-dependent manner. The activity, which was designated as lymphocyte factor (LF), was very potent; it inhibited 50% of the DNA synthesis and cell growth at a dilution of 1:160. It has a molecular weight between 50,000 and 100,000 daltons and it is destroyed by trypsin digestion or by heating at 80 degrees C for 30 minutes. The activity was not due to the presence of prostaglandin. Furthermore, using immunoprecipitation and affinity chromatography, it was shown conclusively to to be distinctly different from alpha lymphotoxin (alpha-LT). It was not cytotoxic, as shown by the 51chromium release technique. Using flow microfluorimetry it was shown that the activity regulates fibroblast growth by preventing quiescent cells in the G0 or G1 stage of the cell cycle from entering the S phase. Cells already in S at the time of exposure complete DNA synthesis but cannot divide, and they accumulate in G2. The activity also has marked effects on protein synthesis. Activated mononuclear cells may play a major role in regulating fibroblast growth and synthesis in normally healing wounds and in acute and chronic inflammatory processes.

Cell Cycle↗
PubMed1982

Inducible macrophage cytotoxins. II. Tumor lysis mechanism involving target cell-binding proteases.

Thioglycollate-elicited C57BL/6 peritoneal exudate macrophage monolayers (PEMM) stimulated with poly I . poly C or LPS released a macrophage cytotoxin (MCT) that rapidly bound to syngeneic (EL 4) or allogeneic (NS-1, YAC-1) tumor cells but did not bind to normal splenocytes. No binding to human (K562) tumor cells was observed. PEMM stimulated with poly I . poly C destroyed allogeneic tumor cells (NS-1) when separated by cell-impermeable Millipore filters in vitro; in contrast, PEMM not stimulated with poly I . poly C were incapable of lysing targets when separated by membranes. The reversible inhibitors N alpha-p-tosyl-L-arginine methyl ester and soybean trypsin inhibitor and the irreversible inhibitors N alpha-p-tosyl-L-lysine chloromethyl ketone and phenylmethylsulfonyl fluoride, of trypsin-like proteases, significantly or totally inhibited MCT cell-lytic activity for L-929 cells in vitro. Furthermore, modification of MCT-associated arginine residues by 1,2-cyclohexanedione completely blocked lytic activity. MCT was concluded to be an inducible nonspecific cell-lytic effector molecule elaborated by activated macrophages, which could bind to potential target cells, and was itself or was associated with a protease.

Animals↗
PubMed1982

Inducible macrophage cytotoxins. I. Biokinetics of activation and release in vitro.

Peritoneal exudate macrophage monolayers (PEMM) from C57BL/6 and DBA/2 mice inoculated ip with tumor allografts were induced to release in vitro labile cell toxin(s), herein called "macrophage cytotoxin(s)" (MCT). Macrophages released MCT spontaneously for a short interval when initially established as monolayers, and they were reinduced to secrete MCT by exposure to allogeneic and syngeneic tumor cells (but not to normal cells) and by exposure to polyinosinic-polycytidylic acid (poly I . poly C) and lipopolysaccharide (LPS). PEMM from normal mice treated ip 3 days previously with thioglycollate were also induced to release toxins in vitro. These cells did not release MCT spontaneously before or after treatment with neoplastic cells but were induced to release MCT by exposure to poly I . poly C or LPS. Resident peritoneal macrophages did not release MCT either spontaneously or after treatment with tumor cells, poly I . poly C, or LPS. MCT released from alloimmune mice stimulated with syngeneic or allogeneic tumor cells were resolved by molecular sieving into a major peak at 140,000--160,000 daltons, called "alpha-MCT," and into a minor peak at 60,000 daltons, called "beta-MCT." However, supernatants from thioglycollate-induced PEMM, stimulated with poly I . poly C or LPS, appeared to be composed entirely of the alpha-class. alpha-MCT from poly I . poly C-stimulated PEMM caused 31--56% lysis of syngeneic EL-4 and allogeneic L-929, NS-1, and YAC-1 tumor cells in vitro but was not cytotoxic for normal cells. Secretion of the MCT by PEMM derived from thioglycollate-treated animals stimulated with poly I . poly C was inhibited by colchicine, emetine, iodoacetic acid, trypan blue, and cytochalasin B.

Animals↗