Recombinant Human Lymphotoxin Research Articles

Activated T-lymphocytes induce growth inhibition and prostaglandin E2 release from syngeneic glomerular mesangial cells.

The concept of an active role of T lymphocytes in the initiation and development of autoimmune glomerulonephritis has gradually evolved from recent investigations. In the present study we started in a murine coculture system to directly examine cellular interactions of intrinsic glomerular mesangial cells (MC) and syngeneic T lymphocytes. Lymph node lymphocytes and, moreover, cloned T helper cells specifically affected syngeneic proliferating MC, causing growth inhibition and prostaglandin E2 (PGE2) release. The T cell specificity of mesangial cell responses was confirmed by demonstrating (i) that MC cocultured with other cell types showed no reaction and (ii) that additional activation of T lymphocytes by IL-2 or concanavalin A significantly enhanced the MC responses. Subsequently, we confirmed the presence of T cell factors in the supernatants responsible for the observed effects: interferon-gamma (IFN-gamma) and tumour necrosis factor (TNF). Experiments with combinations of recombinant mouse IFN-gamma and human lymphotoxin or TNF-alpha showed that these lymphokines could substitute for the direct T lymphocyte effects causing a synergistic growth inhibition and PGE2 release from mouse MC. The observed lymphokine activities were not due to mesangiolysis as shown by neutral red uptake of MC. Pointing to the essential role of T helper cell-specific products, IFN-gamma antibodies abolished both the IFN-gamma and the combined IFN-gamma/TNF-alpha effect. Thus, our investigations with syngeneic MC-lymphocyte cocultures demonstrated that cultured MC specifically responded to T lymphocytes and their products.

Indomethacin enhances the cytotoxicity of recombinant human lymphotoxin α on tumor cells by suppressing NF-κB signaling

Indomethacin is widely used to treat inflammatory and prevent adverse events which are frequently accrues in biotherapy. In phase I clinical trial of recombinant human lymphotoxin α (rhLTα), indomethacin was premedicated to alleviate chill and fever. However, it is unknown whether indomethacin influences the therapeutic efficacy of rhLTα. In this study we found that pre-treatment with low dose of indomethacin enhanced the cytotoxicity of rhLTα and/or cisplatin/adriamycin on human tumor cells. Further investigation demonstrated that indomethacin dose-dependently suppressed the activation of nuclear factor-kappa B (NF-κB) by inhibiting phosphorylation and degradation of IκB-α. In addition, indomethacin decreased the expression of NF-κB-regulated gene products involved in rhLTα-induced anti-apoptosis (XIAP, cFLIP and cIAP-1), which may explain its sensitization of tumor cells to rhLTα and/or cisplatin/adriamycin.

Epitope identification by polyclonal antibody from phage-displayed random peptide library.

Screening of bioactive peptides from random peptide libraries using monoclonal antibodies as ligates is an effective method to define epitopes of protein antigens. However, it is thought that polyclonal antibodies might also serve as promising ligates for screening. We illustrate this approach by using recombinant human lymphotoxin (rhLT) polyclonal antibody as a model. The procedure consists in (a) affinity purification of polyclonal antibody to obtain the "monospecific" antibody, (b) screening against a phage-displayed random peptide library using the affinity-purified antibody, (c) plating the enriched phage on agar plates, randomly picking clones, and selecting the positive ones by dot blotting, (d) DNA sequencing of the positive clones and conducting a homology search against the protein sequence databank, and (e) confirming the epitopes by chemical peptide synthesis. By employing this procedure, we identified a dominant epitope RQHPKM, located at residues 15-20 of the human lymphotoxin amino acid sequence. The usefulness of this general procedure is discussed.

Antibody against branched epitope as an affinity ligand to separate the parent protein

A peptide that contained one of the continuous epitopes of recombinant human lymphotoxin (rhLT) (amino acid residues 139–154) has been located by epitope mapping. The branched form of this peptide was synthesized by the multiple antigen peptide procedure with an octameric branched resin and was subsequently used to elicit anti-epitope antibody in rabbits. The resulting anti-epitope was then used as an immunoaffinity ligand in affinity chromatography to purify the parent protein, rhLT, from the host cell lysate directly. It is suggested that this approach would be a general way to create novel biospecific ligands for affinity separations.

In vivo effects of recombinant human lymphotoxin on human medulloblastoma xenograft: enhancement of antitumor activity of etoposide.

The authors investigated the antitumor activities of rHuLT alone and in combination with etoposide on human meduloblastoma xenografts growing subcutaneously in nude mice. Intravenous administration of rHuLT (1.0 x 10(5) U/kg, 5.0 x 10(5)U/kg, 2.5 x 10(6)U/kg, three times a week for three weeks) suppressed medulloblastoma growth depending on the dose. However, the highest dosage caused serious side effects. Combining rHuLT (intravenously, 5.0 x 10(5)U/kg, three times a week for three weeks) with etoposide (intraperitoneally, 20mg/kg, once a week for three weeks) increased the antitumor activity without causing serious toxicity. Microscopically, tumor specimen showed thrombosed tumor vessels and massive necrosis 3 weeks after rHuLT treatment. Ultrastructural examination revealed that 120 minutes after the administration of rHuLT alone, disruption of interendothelial junctions was evident, and that the endothelial cells were destroyed at 240 minutes. Concentration of etoposide in tumor tissue peaked 30 minutes after intraperitoneal administration, and then decreased with time. When etoposide was administered in combination with rHuLT, the concentration of etoposide in tumor tissue after 60 to 240 minutes was significantly higher than when etoposide was given alone, and the area under the concentration versus time curve was also greater for the tumors of mice with combination treatment. The findings suggest that the proper combination of rHuLT and etoposide may have synergistic antitumor activities. Histological changes suggest that increased concentrations of etoposide within the tumor after combination therapy may occur due to increased vascular permeability and/or decreased etoposide clearance which is the result of blood statis in the tumor vasculature.

Purification and renaturation of recombinant human lymphotoxin (tumour necrosis factor beta) expressed in Escherichia coli as inclusion bodies

High level expression of recombinant human tumour necrosis factor beta (rhTNF-beta) in Escherichia coli results in the formation of two portions of protein, namely soluble active protein and insoluble protein which is inactive and aggregates in the form of inclusion bodies (IBs). In this study, a procedure for purification and renaturation of rhTNF-beta from inclusion bodies has been designed and verified experimentally with a product purity of more than 90% and a recovery of about 30%. The procedure includes washing of IBs with specific wash buffer (Triton X-100/EDTA/lysozyme/PMSF), their solubilization with 8 mol dm-3 alkaline urea, purification with ion-exchange columns, refolding with renaturation buffer and finally concentration and desalination with an ultrafiltration membrane. The characteristics of the renatured protein were identical with those of purified protein from the soluble fraction as demonstrated by (1) SDS-PAGE, (2) cytotoxic activity on mouse L929 cells, (3) N-terminal amino acid sequence, and (4) gel filtration chromatography.

Prevention of Type I Diabetes with Lymphotoxin in BB Rats

We previously reported that nonspecific immunomodulations with a streptococcal preparation (OK-432), an inducer of tumor necrosis factor (TNF), or with recombinant TNF prevented development of insulin-dependent diabetes mellitus (IDDM) in animal models (NOD mice and BB rats). Recently we have further reported that lymphotoxin (LT), a cytokine with functional and structural characteristics similar to those of TNF, also protected NOD mice from diabetes. In this study, we have extended our observation on the LT to BB rats. Male and female BB/Wor rats were treated intraperitoneally with recombinant human LT three times a week from 4 to 11 weeks of age. The cumulative incidence of diabetes by 14 weeks of age was 24/30 (80.0%) in nontreated control rats, whereas it was 10/26 (38.5% vs control, P < 0.01) and 4/29 (13.8% vs control, P < 0.0001) in the rats treated with 1 × 103 and 1 × 101 of LT, respectively. There was no significant difference in nonfasting blood glucose levels and body weights between nontreated control and LT-treated rats, which were nondiabetic. In the LT-treated rats, intensity of insulitis was significantly reduced in comparison with the nontreated rats. Concanavalin A-stimulated TNF/LT productivity of spleen cells was significantly lower in BB/Wor and BB/Sendai rats than in Wistar rats or other normal rat strains. On the other hand, there was no difference between BB/Sendai and Wistar rats in the in vivo TNF/ LT productivity induced with LPS or with IFN-γ plus LPS, and the TNF/LT productivity of these rats was lower on stimulation with LPS alone, but higher with IFN-γ plus LPS than the other normal rats. These results indicate that treatment with LT, as well as TNF, modulated autoimmunity and prevented development of IDDM in BB/Wor rats which may be low producers of TNF/LT.

N-Linked Sugar Chain Structure of Recombinant Human Lymphotoxin Produced by CHO Cells: The Functional Role of Carbohydrate as to Its Lectin-like Character and Clearance Velocity

Recombinant human lymphotoxin (rhLT) produced by CHO cells transfected with human LT genomic DNA was purified to homogeneity, but approximately 5% of the molecules were devoid of the last two amino terminal residues. A peptide N-glycosylated at Asn62 (Tr-45) and one partially O-glycosylated at Thr7 (Tr-14) on cleavage with trypsin were separated by reverse phase HPLC. The N-linked sugar chains of Tr-45 were released quantitatively as oligosaccharides on hydrazinolysis (100°C, 8 h), followed by N-acetylation. After being reduced with either NaB3H4, or NaB2H4, their structures were determined by a combination of serial lectin affinity chromatography, exoglycosidase digestion, and methylation analysis: 82.7% of the sugar chains occur as biantennary complex-type sugar chains, the remainder being C-2 and C-2,4/C-2,6 branched triantennary, and C-2,4 and C-2,6 branched tetraantennary complex-type sugar chains with a fucosylated mannose core. Their sialic acid residues occur only as the Neu5Acα2 → 3Gal group. The clearance velocity from the bloodstream dramatically increased with desialylation, and rhLT tends to have accumulated in the kidney, indicating that there may exist other mechanisms for clearance from the circulation besides the galactose-binding protein in hepatocytes and the filtration system of the kidney. Desialylated rhLT showed a lectin-like binding character to uromodulin similar to that of tumor necrosis factor, although intact rhLT did not. The interaction between desialylated rhLT and uromodulin was inhibited by N,N′-diacetylchitobiose and {Manα1 → 6(Manα1 → 3)Manα1 → 6}(Manα1 → 2Manα1 → 3)Manβ1 → 4GlcNAcβ1 → 4GlcNAc → Asn. These results indicate that the lectin-like domain of rhLT is exposed on its desialylation.

Increased lymphotoxin in human malarial serum, and the ability of this cytokine to increase plasma interleukin-6 and cause hypoglycaemia in mice: implications for malarial pathology

The origin of illness and pathology in malaria is now largely attributed to high levels of circulating tumour necrosis factor (TNF), released from cells of macrophage lineage after triggering by the products of malarial schizogony. The role of lymphocytes and their products in malarial pathology is not yet known. This paper reports the presence of a related cytokine, lymphotoxin, which is produced only by lymphocytes, in the serum of malarial patients. This is the first report of raised serum levels of lymphotoxin in a systemic disease state. When injected into mice, recombinant human lymphotoxin induced hypoglycaemia and increased serum levels of interleukin-6. These changes, which are seen in severe experimental and human malaria, were also provoked by TNF. Both of these cytokines acted synergistically with interleukin-1, which has also been reported to be raised in malaria, to produce these alterations. These observations imply that lympotoxin, as well as TNF, may contribute to the hypoglycaemia and raised serum interleukin-6 observed in malaria. This reduces the likelihood of effectively blocking the pathology of this disease by the use of neutralizing antibody directed against just one member of this family of functionally overlapping mediators.

Effect of local secondary structure of mRNA on expression of recombinant human lymphotoxin inEscherichia coli

We substituted a few alternative codons close to the N-terminal coding region of human lymphotoxin (LT) gene for natural ones to avoid stable local secondary structures of mRNA, predicted in the 5′-terminal 130 nucleotides of mRNA. With this modification, we have overexpressed a recombinant lymphotoxin (rLT) inEscherichia coli under the control of trc promoter. Most of the produced rLT was a soluble and active form.

The structure of human lymphotoxin (tumor necrosis factor-beta) at 1.9-A resolution.

The three-dimensional structure of recombinant human lymphotoxin (residues 24-171 of the mature protein) has been determined by x-ray crystallography at 1.9-A resolution (Rcryst = 0.215 for I greater than 3 sigma (I)). Phases were derived by molecular replacement using tumor necrosis factor (TNF-alpha) as a search model. Like TNF-alpha, lymphotoxin (LT) folds to form a "jellyroll" beta-sheet sandwich. Three-fold related LT subunits form a trimer stabilized primarily by hydrophobic interactions. A cluster of 6 basic residues around the 3-fold axis may account for the acid lability of the trimer. Although the structural cores of TNF-alpha and LT are similar, insertions and deletions relative to TNF-alpha occur in loops at the "top" of the LT trimer and significantly alter the local structure and the overall shape trimer is highly conserved. The sites of two mutations (Asp-50 and Tyr-108) that abolish the cytotoxicity of LT are contained within poorly ordered loops of polypeptide chain that flank the cleft between neighboring subunits at the base of the molecule, suggesting that the receptor recognizes an intersubunit binding site.

Lymphotoxin in Human Middle Ear Cholesteatoma

The progressive growth of epidermis appears to be a crucial factor in the pathogenesis of cholesteatoma. How this growth is encouraged is still not well understood. In the present study, lymphotoxin was found in human middle ear cholesteatoma tissues by the immunoperoxidase method using rabbit anti-human lymphotoxin immunoglobulin G (IgG). Lymphotoxin was localized in the epithelium and connective tissues of the cholesteatomas studied. The epithelium of normal external ear canal skin was lightly stained, but the stain was much weaker than that of the cholesteatomas. In vitro, recombinant human lymphotoxin stimulated proliferation, protein synthesis, and terminal differentiation of basal keratinocytes. These findings suggest that lymphotoxin may be one of the mediators involved in the development of cholesteatoma.

Usefulness of Glycosylated Recombinant Human Lymphotoxin for Growth Inhibition of Human and Murine Solid Tumors and Experimental Metastasis in Mice

We have examined the antitumor and antimetastatic effects of native-type, glycosylated recombinant lymphotoxin (LT) on human and murine tumors transplanted in mice. The results reported here are as follows: (a) The in vivo antitumor spectrum of LT is not coincident with the in vitro study, and it has a wide antitumor spectrum and substantially inhibits the growth of human solid tumors, (b) When both syngeneic and nude mice are transplanted with Meth A tumor, the significant growth-inhibitory effect of LT is obtained in syngeneic mice, but the effect is quite small in nude mice regardless of the routes; LT attains the same degree of effectiveness as that in syngeneic mice, but at an 8 to 16 times higher dose. Furthermore, the pretreatment with anti-asialo-GM1 antibody inhibits the antitumor effects of LT in syngeneic mice, (c) In the pulmonary metastasis model induced by i.v. injection of Meth A cells, a high preventive effect of LT is obtained by systemic administration in syngeneic mice, but not in nude mice. In addition, the pretreatment with anti-asialo-GM1 antibody completely prevents the antimetastatic effect of LT, but also blocks that effect of control mice without LT treatment. In conclusion, LT appears to be a potent cytokine against tumor growth and metastasis in vivo. The differences between nude and syngeneic mice suggest the involvement of host immunity in the expression of LT function.

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.

Highly sensitive enzyme immunoassay for human lymphotoxin (tumor necrosis factor β) in serum

We have developed a rapid, simple and highly sensitive ‘sandwich’ enzyme immunoassay (ELISA) for the detection and quantification of human lymphotoxin (= tumor necrosis factor β) in serum. The assay performed in microtiter plates, employs two monoclonal murine antibodies able to neutralize the cytotoxic activity of lymphotoxin. In a one-step procedure, antibody LTX-21 (IgG2b) coated on to the solid phase captures antigen present in the sample; subsequently antibody LTX-22 (IgG1), covalently coupled to horseradish peroxidase, labels the bound antigen. The assay is able to detect lymphotoxin spiked into human serum in concentrations as low as 7 pg/ml, whereas human tumor necrosis factor α does not cross-react even at 10 7-fold higher concentrations. Only biologically active protein is recognized by the antibodies, since inactivation of lymphotoxin measured by bioassay results in a parallel decrease in immunoreactivity. Natural, glycosylated lymphotoxin shows the same reactivity as recombinant, unglycosylated protein. The assay will be useful for the qunatification of endogenous human lymphotoxin in serum, other body fluids, and culture supernatants of human cells, and can also be used to monitor levels of recombinant human lymphotoxin in animal studies and clinical trials.

Synergistic Antitumor Effect of Glycosylated Recombinant Human Lymphotoxin with Human Interferon-γ on Lymphotoxin-Sensitive Human Tumor

We examined the antitumor effect of glycosylated recombinant lymphotoxin (LT) in combination with human interferon-gamma (IFN-gamma) on human tumors transplanted into nude mice and compared it with that of tumor necrosis factor (TNF). The results were as follows: (i) The systemic administration of glycosylated LT combined with IFN-gamma produced a significant antitumor activity against HT-1080 fibrosarcoma, G-361 malignant melanoma, KB nasopharyngeal carcinoma, and ZR-75-1 breast carcinoma, all of which are relatively resistant to a single treatment with LT or IFN-gamma. The synergistic effect was also seen in LT-sensitive HeLa S3 tumors. The effect was observed after either i.v. or s.c. injection. (ii) In contrast, no synergistic or additive effect on HeLa S3 tumors was observed in the case of TNF combined with IFN-gamma. (iii) The serum half-life of glycosylated LT in tumor-bearing mice was about 22-fold longer than that of TNF. In conclusion, glycosylated LT, especially in combination with IFN-gamma, appears to be a potent cytokine against tumor growth in vivo compared with TNF. Its long serum half-life can result in a strong antitumor effect in combination with IFN-gamma in vivo.

The pharmacokinetic pattern of glycosylated human recombinant lymphotoxin(LT) in rats after intravenous administration.

We have examined the pharmacokinetics of glycosylated recombinant human lymphotoxin (LT) after intravenous bolus injection in rats and compared them with those of tumor necrosis factor (TNF) or LT species. The results are as follows. 1) The mean half-life of glycosylated LT in serum increases for each increase in dose, and the distribution volume (V) and total body clearance [(Cl (total)] tend to decrease for increase in dose. On the other hand, the half-life of TNF also increases for increase in dose, but the V tends to increase for increase in dose and Cl (total) does not change. 2) The glycosylated LT distributes to all organs so far tested except brain, and tends to accumulate to kidney more than other tissues at 6 h after the injection. 3) Nonglycosylated LT produced by E. coli and the glycosylated LT species carrying both N-type and mucin-type sugar moieties (25 kDa LT) have shorter half-lives and higher Cl (total)s than 23 kDa LT carrying N-type sugar moieties alone. The 21 kDa LT, the same species as 23 kDa LT except that it lacks 15 amino acid residues at the N-terminus, disappears much faster than 23 kDa LT and shows higher V and Cl (total). Thus, glycosylated LT shows nonlinear pharmacokinetics like TNF, but the deposition is quite different from that of TNF. The high serum concentration of glycosylated LT depends upon the presence of N-type sugar moieties, but not mucin-type sugar moieties. The N-terminal protein chain of LT also correlates with the serum concentration.

Characterization of recombinant human lymphotoxin (tumor necrosis factor-β) produced by a mammalian cell line

Lymphotoxin (LT) was purified from serum-free conditioned media of a recombinant mammalian cell line transfected with human lymphotoxin cDNA. The purification scheme consisted of controlled pore glass chromatography, Q-Sepharose ion-exchange chromatography, and concanavalin A-Sepharose chromatography. The purified protein was found to be homogeneous by reverse-phase high-performance liquid chromatography and had an approximate specific activity of 130 × 10 6 units per milligram protein as determined by the L-929 cytotoxicity assay. Purified LT had an isoelectric point of approximately 6.85 and an apparent molecular weight of 50,000 by gel permeation high-pressure liquid chromatography. However, when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, two distinct bands at approximate molecular sizes of 25 and 20 kDa were observed. Both the bands were immunoreactive by Western blot analysis and found to be associated with biological activity. The two forms of lymphotoxin differed from each other with respect to protein structure. Amino-terminal amino acid sequence analysis revealed that the 25-kDa LT sequence starts with LeuProGly-residues whereas that of the 20-kDa LT begins with HisLeuAla; thus the latter form is truncated by 20 amino acid residues from the amino terminal. Two species of LT also differed from each other with respect to carbohydrate structure. Enzymatic removal of sialic acid reduced the molecular weight of 25 kDa by approximately 5 kDa whereas that of the 20-kDa LT was unchanged. A reduction in an apparent molecular size by ~4 kDa of both species of LT was observed on removal of N-linked oligosaccharides. Treatment with O-Glycanase had minimal effect on either form of LT. The recombinant lymphotoxin described here was found superior in its solubility behavior as compared to bacterial cell derived LT. Overall, mammalian cell line derived recombinant LT appears closer in its properties to natural LT than does bacterial cell derived recombinant LT.

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.

Increase in permeability of human endothelial cell monolayer by recombinant human lymphotoxin

The effect of lymphotoxin(LT) on transendothelial permeability was examined using in vitro human endothelial cell (EC) culture system. To assess permeability, we measured the movement of human IgG labeled with horseradish peroxidase(HRP-huIgG) across an EC monolayer cultured on a fibronectin and collagen-coated membrane. LT increased the permeability dose dependently within 16 hours. Similar results were obtained with tumor necrosis factor(TNF)-α. The results suggest that increase in vascular permeability by LT and TNF-α plays an important role in initiating hemorrhagic necrosis of tumors in vivo.